WO2024155699A1 - Anaerobic bioproduction of compounds - Google Patents

Abstract

The present application relates to anaerobic bioproduction of certain compounds and intermediates. The present application also relates to genome-scale metabolic models (OEMs) (including novel OEMs), including methods of modifying OEMs for bioproduction of target compounds. The present application also relates to recombinant organisms for producing target compounds using anaerobic processes. The present application also relates to anaerobic and ethanol-producing systems and infrastructure (including retrofitted ethanol systems) for producing compounds in anaerobic processes.

Description

ANAEROBIC BIOPRODUCTION OF COMPOUNDS INCORPORATION BY REFERENCE OF RELATED PATENT APPLICATIONS This application is based upon and claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No.63/439,550, filed January 17, 2023, U.S. Provisional Application No. 63/582,133, filed September 12, 2023, and to U.S. Provisional Application No.63/589,530, filed October 11, 2023, the entire contents of all of which are incorporated herein by reference in their entirety. FIELD OF THE INVENTION The present application relates to anaerobic bioproduction of certain compounds, preferably 3- hydroxypropionic acid (i.e., 3-HP), and intermediates and derivatives thereof. The present application also relates to genome-scale metabolic models (GEMs) (including novel GEMs), including methods of modifying GEMs for bioproduction of target compounds. The present application also relates to engineered organisms for producing target compounds using anaerobic processes. The present application also relates to anaerobic and ethanol-producing systems and infrastructure (including retrofitted ethanol systems) for producing compounds in anaerobic processes. STATEMENT To comply with 37 C.F.R. § 1.831, this application contains sequence listings included in an XML file submitted via the Patent Center. The XML file has the following attributes: (1) Name: 444150- 0120-PCT_Sequence Listing, (2) Date of creation: January 8, 2024, (3) File size in kilobytes: 526 KB. Pursuant to MPEP § 2422.03(a), Applicant hereby incorporates by reference the foregoing XML file and all material disclosed therein into this application. BACKGROUND Bioproduction of commodities and chemicals currently produced from non-renewable sources -- such as petroleum, hydrocarbons, and natural gas substitutes -- provides an opportunity for significant impact on global sustainability. Sustainability through a bioeconomy here includes environmental and economic sustainability, but also social sustainability, including long term rural development. It is generally accepted in the scientific community that petroleum-based production of commodities and chemicals is a significant contributor to climate change. Bioproduction of commodities and chemicals offers an alternative with the potential to reduce climate impact. There is a strong demand for products and systems which are capable of transforming petroleum-based production to more sustainable production systems, such as bioproduction. Indeed, public demand for sustainable raw materials has been demonstrated by numerous public commitments from major retailers for sourcing these renewable materials as well as calls from the U.S. federal government to harness biomanufacturing to further societal goals. Indeed, in 2022, the White House issued Executive Order 14081 focusing on expanding biotechnology and bioproduction to address climate change, energy, and supply chain security; and, in 2023 the U.S. Dept. of Energy called for development of new bioproduction technology that would enable biomanufacturing of at least 20 commercially viable chemicals, noting bio-based feedstocks and bioprocessing routes can reduce net greenhouse gas emissions, stabilize commodity chemical prices, and avoid supply chain disruptions (U.S. Dept. of Energy, Biotechnology and Biomanufacturing R&D to Further Climate Change Solutions (2023)). Biobased production of chemicals and materials, rather than manufacturing from petroleum derivatives, provides an opportunity to have a significant impact on greenhouse gas (GHG) emissions – 7-9% reduction in GHG emissions compared to 2018 levels across all sectors evaluated. Bioproduction of certain chemicals and commodities can leverage existing industrial infrastructure and bolster regional economies in areas that have previously been reliant on other forms of bioproduction, which in recent years has waned. For example, as bioethanol and motor gasoline demand may decline further due to pressures from emerging electric vehicle penetration and improving fuel efficiency, economies in areas such as the midwestern region of the United States would benefit from new products to manufacture using existing capital assets and labor. Moreover, bioproduction of chemicals may enable future supply chains around these critical intermediates for the downstream manufacturing of adhesives, coatings, paints, and personal care products. There exists an acute need for new approaches and related technologies for designing microbes for anaerobic bioproduction of certain compounds with the final infrastructure in mind. In a preferred embodiment, the subject of the present application is the anaerobic bioproduction of 3-hydroxypropionic acid (CAS 503-66-2), which may be referred to as 3-hydroxy propionic acid, 3-HP, 3HP, 3-HPA or, when appropriate, the anionic form 3-hydroxypropionate (CAS 1190-23- 4).3-HP is a platform molecule of economic importance that can serve as a biologically-derived precursor to several products, including acrylic acid, acrylonitrile, acrylamide, acrylate esters (e.g., methacrylate, ethyl acrylate, butyl acrylate, among others), polyhydroxyalkanoates (PHA’s), poly- 3-hydroxypropionic acid, and their downstream derivative polymers and products. It was highlighted almost 20 years ago by Werpy and Petersen as a one of the top 12 building block molecules of critical national interest for bioproduction. (See T. Werpy and G. Petersen, eds., Top Value Added Chemicals from Biomass: Volume I—Results of Screening for Potential Candidates from Sugars and Synthesis Gas (U.S. Department of Energy, 2004).) There are compelling techno-economic reasons to target 3-HP as a molecule to produce in existing anaerobic (ethanol) fermentation infrastructure. First, it has the opportunity to have advantaged economics compared to ethanol - ethanol has a five-year average selling price since 2017 of approximately ~$0.58/kg (~$1.70 / gallon), whereas acrylate selling prices over the same period have been at least two- to three-fold higher (~$1.58+ / kg). On a mass yield basis from glucose as a feedstock, 3-HP has an approximately 100% increase in theoretical yield compared to ethanol (1.0 g/g vs.0.51 g/g), and accounting for the mass loss when dehydrating 3-HP to acrylic acid, the ceiling is still above ethanol at ~0.77 g acrylic acid per g glucose. Initial technoeconomic estimates suggest that under certain conservative assumptions for an existing ethanol facility retrofit to produce acrylic acid, there is a strong case for attractive economics that do not require a market premium. Finally, fundamental to the economic success of bio-based 3-HP and its derivative products reaching price parity with petroleum-derived incumbents is the anaerobic production utilizing existing infrastructure. Anaerobic production offers the biological path to achieving the maximum theoretical mass yield from substrate, provides up to 50% reduction in operational expenses attributed to reduced mixing and cooling costs, and allows for a brownfield retrofit to significantly reduce capital expenditure. However, despite the potential for this platform molecule, no commercial solutions exist for making 3-HP anaerobically. Due to the long-standing interest in 3-HP as a platform molecule, extensive work has been done over the past 20 years to develop microorganisms and processes capable of producing 3-HP from biological substrates. However, nearly all of these efforts have focused on the aerobic production of 3-HP, which are processes not suitable for deployment into existing ethanol (anaerobic) fermentation infrastructure. To date, there have been no efforts at homofermentative production of 3-HP in an anaerobic, substantially anaerobic, or facultatively anaerobic process. There have been at least 7 biochemical pathways for 3-HP production described theoretically (Vidra 2017, doi:10.3311/PPch.10861), however only a subset of these pathways meet the requirements for anaerobic bioproduction – overall thermodynamic feasibility, net cellular ATP production, and redox balance. The current best published academic titers in Saccharomyces are in an aerobic process using the Malonyl-CoA pathway, which is not suitable for anaerobic production. A subset of the biochemistry required for one of the feasible anaerobic routes has been described in detail, but was tested under aerobic conditions (Borodina 2015, doi:10.1016/j.ymben.2014.10.003), and was missing key components that would enable homofermentative anaerobic production of 3-HP. There are also reports from industry of microaerobic fungal production up to 80 g/L 3-HP, but these solutions cannot directly take advantage of existing ethanol infrastructure due to their requirement for oxygen. The solutions to enable homofermentative anaerobic production of 3-HP are described herein. First, the selected biochemical pathways must yield ATP. Second, but of equal importance, the pathway must be capable of recycling reducing equivalents produced from the glycolytic oxidation of glucose (redox balanced). Thus, despite the extensive commercial and academic efforts expended over the past 20 years and the relatively favorable economics involved in theoretical 3-HP bioproduction, none have succeeded to date in developing and commercializing cost-effective 3-HP bioproduction, underscoring the significant technical challenges that still remain in the art. SUMMARY The inventors of the present application have overcome these technical challenges and have invented certain systems, methods, and approaches for anaerobic bioproduction of 3- hydroxypropionic acid that solve the two fundamental requirements for anaerobic metabolism as described above. First, the inventors of the present application have developed pathways for bioproducing 3-HP which yield ATP. In preferred embodiments, these biochemical routes to yield ATP from 3-HP production rely on generating a critical intermediate, oxaloacetic acid (oxaloacetate or OAA) by the carboxylation of phosphoenolpyruvate (PEP) via the anapleurotic function of phosphoenolpyruvate carboxykinase (PEPCK). In preferred embodiments, in this manner, 1 mole of ATP can be generated per mole of 3-HP produced. In the absence of PEPCK such 3-HP is typically produced from OAA generated by PEP carboxylase or by pyruvate carboxylase. Both of these latter solutions result in no net ATP produced per 3-HP, and therefore these approaches are not preferred. The inventors have shown that PEPCK improves 3-HP production anaerobically, surprisingly, even in instances where ATP is not thought to be limiting (e.g., when ethanol production (also a net ATP-generating pathway) is still partially intact). This is embodied in Pathways 1 and 6 described herein. Next, OAA must be converted to malonate semialdehyde (MSA) either through a series of transaminations and decarboxylations (via the “ ^-alanine loop”), or directly decarboxylated on the ^ carboxyl group. The inventors have demonstrated anaerobic production of 3-HP via the ^-alanine loop. Second, the inventors of the present application have developed pathways for bioproducing 3-HP that are capable of recycling reducing equivalents produced from the glycolytic oxidation of glucose (redox balanced). In two preferred embodiments, the inventors have demonstrated unique combinations of enzymes in the ^-alanine loop capable of balancing the amino donors and acceptors for ideal anaerobic production of 3-HP as described in pathways 1 and 2. The final biochemical step is reduction of MSA to 3-HP. In silico constraint-based modeling suggests this reaction should use NADH as the electron donor, as it can be directly recycled in glycolysis (NAD+ reduction to NADH by glyceraldehyde 3-phosphate dehydrogenase). Surprisingly, the inventors have shown that either NADH-dependent or NADPH-dependent enzymes are capable of serving as the electron donor for malonate semialdehyde reduction during facultative anaerobiosis. In a preferred embodiment, 3-HP may be produced via Pathway 1. In this pathway, the glycolytic metabolite PEP is first carboxylated to oxaloacetate. In one embodiment this carboxylation is performed by PEP carboxylase. In a preferred embodiment, the carboxylation is linked with the production of ATP via the enzyme PEP carboxykinase (PEPCK). Next, oxaloacetate is converted to aspartate in a reaction catalyzed by an aspartate aminotransferase. In a preferred embodiment, the amino donor is L-glutamate. Aspartate is then decarboxylated to ^-alanine in a reaction catalyzed by an aspartate decarboxylase. Next, ^-alanine is converted to malonate semialdehyde, catalyzed by a ^-alanine aminotransferase. In one embodiment, the amino acceptor is pyruvate. In a preferred embodiment the amino acceptor is ɑ-ketoglutarate. Finally, malonate semialdehyde is reduced to 3-HP via a malonate semialdehyde reductase. In one embodiment, the reducing agent is NADH, where in another embodiment, the reducing agent is NADPH. In one embodiment, this pathway may be engineered into a microorganism, and in a preferred embodiment, this recombinant microorganism is a yeast. In one embodiment, the pathway is engineered into a yeast microorganism of the Saccharomyces genus. In another embodiment, the pathway is engineered into yeast microorganism selected from a Saccharomyces sensu stricto yeast, including S. cerevisiae, S. uvarum, S. bayanus, S. pastorianus, S. paradoxus, S. kudriavzevii, S. mikatae, or S. castellii, or hybrids thereof. In another embodiment, the pathway is engineered into a yeast microorganism selected from a post-whole genome duplication yeast, including yeasts from the Saccharomyces sensu stricto as well as certain Candida species (e.g. Candida glabrata, Candida castellii). In another embodiment, the pathway is engineered into a yeast microorganism selected from a pre-whole genome duplication yeast, including certain species from the genera including Saccharomyces, Kluyveromyces, Pichia, Issatchenkia, Zygosaccharomyces, Schizosaccharomyces, Dekkera (Brettanomyces), and Yarrowia. In another embodiment, the pathway is engineered into a Crabtree-positive yeast microorganism selected from certain species in the genera Saccharomyces, Kluyveromyces, Pichia, Zygosaccharomyces, Debaryomyces, Dekkera (Brettanomyces), or Schizosaccharomyces. In another embodiment, the pathway is engineered into a Crabtree-negative yeast microorganism selected from a species within genera including Kluyveromyces, Pichia, Hansenula, Candida, or Issatchenkia. In a preferred embodiment, the pathway is engineered into Saccharomyces cerevisiae. Further aspects and details of this pathway are discussed further below. The inventors of the present application have also invented systems, methods, and approaches to modify and customize genome-scale metabolic models (GEMs) involved in design processes for anaerobic bioproduction. The inventors of the application have also invented systems, methods, and approaches to modify and customize organisms capable of anaerobic metabolism to efficiently produce 3-HP. The inventors have also invented systems, methods, and approaches to adapt or retro-fit ethanol-producing infrastructure, including existing infrastructure, for anaerobic production of the target compounds. Thus, in one aspect, the target compound of the present application is 3-HP, as well as intermediate compounds produced by pathways for making these target compounds. Intermediate compounds are discussed in more detail in the detailed description. A person of ordinary skill in the art would recognize intermediate compound production can essentially be as important and commercially significant as producing a final target compound under certain circumstances. In one aspect, the present application provides methods and pathways for making and producing certain compounds. In preferred embodiments, the pathways are anaerobic biopathways for use in a novel recombinant organism or novel GEMs. In a preferred embodiment, the recombinant organism or GEM is adapted to produce a specific target compound from an initial feedstock. Embodiments of such organisms and GEMs are discussed elsewhere in this application. Another aspect of the application provides a genome-scale metabolic model or GEM. The GEM includes added metabolites associated with a cellular compartment with or without expected concentrations and novel reactions with predefined flux boundaries. Yet another aspect relates to a genetically modified organism. The organism may include a nucleic acid sequence encoding an enzyme associated with a metabolic step included in a novel GEM. Another aspect of the application relates to a method for genetically modifying an organism. The method can include using a vector to modify the organism to express a nucleic acid sequence encoding an enzyme associated with a metabolic step included in a novel GEM. In another aspect, the present application includes a recombinant vector. The recombinant vector can include a nucleic acid sequence encoding an enzyme included in a novel GEM. Another aspect of the present application provides a method for using ethanol fermentation equipment to produce a product. The method can include providing a source corn kernel, mechanically processing the corn kernel to produce a corn mash, hydrolyzing the corn mash to convert starch to simple sugars, and transferring the hydrolyzed corn mash or sugars derived thereof to a fermentor containing a genetically modified organism to produce a target compound other than ethanol. Further details, objects, features, and advantages of the present application will become apparent from the detailed description of preferred embodiments which is set forth below, when considered together with the figures of drawing. BRIEF DESCRIPTION OF THE DRAWINGS Fig.1 depicts data from Example 1 demonstrating strains containing some combination of overexpression of a subset of the tested enzymes successfully produce 3-HP in an aerobic plate model for Pathways 1 and 2. Fig.2 depicts data from Example 1 demonstrating strains containing some combination of overexpression of a subset of the tested enzymes produce 3-HP in an anaerobic plate model also for Pathway 1 and Pathway 2 of the present application. Fig.3 depicts data from Example 1 demonstrating the production of up to 1.6 g/L of 3-HP in an aerobic plate model. Fig.4 depicts data from Example 1 demonstrating the production of up to 350 mg/L of 3-HP in an anaerobic plate model. Fig.5 depicts data from Example 1 demonstrating production of up to 1.5 g/L 3- HP in an aerobic plate model in an alternative genetic context. Fig.6 depicts data from Example 1 providing further evidence for anaerobic production of 3-HP from Pathway 1. DEFINITIONS In order to ensure a clear and consistent understanding of the terms and phrases used in this patent application, the following definitions are provided. It is important to note that these definitions are specific to the context of this application and are provided to facilitate the interpretation and understanding of the invention described herein. Anaerobic: Description of a biological process and/or specific testing conditions that occur in the absence of intentionally added oxygen. As used herein, anaerobic conditions include processes for organisms considered facultatively anaerobic - meaning they are capable of growing and/or producing product in the presence or absence of oxygen, yet do not require the absence of oxygen (they are not obligate). Such processes do not intentionally control for the removal of oxygen, but rather any oxygen that is available is consumed by the production organism. Such processes can comprise distinct phases, where oxygen may be present or added during a growth phase and naturally consumed by the microorganism resulting in a substantially oxygen-free production environment. In some embodiments, a substantially anaerobic production phase can be considered as having an oxygen transfer rate (OTR) of 2 to 5 mmol/L/hr. In some embodiments anaerobic production can be considered as having an OTR of less than 2 mmol/L/hr. In some embodiments anaerobic production can be considered as having a measured dissolved oxygen content of less than 1%. In a preferred embodiment, the anaerobic production phase can be considered as having an OTR of less than 1 mmol/L/hr. Aspartate aminotransferase: An enzyme that catalyzes the reversible conversion of oxaloacetate and L-glutamate into L-aspartate and 2-oxoglutarate (sometimes referred to as glutamate- oxalacetate aminotransferase and aspartate transaminase). Aspartate decarboxylase: An enzyme that catalyzes the conversion of L-aspartate into β-alanine and carbon dioxide (CO₂) through a decarboxylation reaction. ^-Alanine aminotransferase: An enzyme that catalyzes the reversible conversion of β-alanine and 2-oxoglutarate (ɑ-ketoglutarate) into malonate semialdehyde and L-glutamate through a transfer of an amino group. Alternatively, this enzyme may utilize pyruvate as the amino acceptor from ^-alanine, forming L-alanine and malonate semialdehyde. It may also be referred to herein as ^-alanine-oxoglutarate transaminase, pyruvate aminotransferase, pyruvate transamination and alanine transaminase. Carbon molar yield: The actual moles of carbon atoms of desired product obtained from a provided number of carbon moles of substrate. Expression: The term “expression” as used herein refers to the process and/or magnitude by which genetic information or functional molecules (including DNA, RNA, and proteins) are utilized by an organism to produce the corresponding molecules for their functional roles. Feedstock-derived substrate: The term "feedstock-derived substrate" refers to any substance or compound that can be transformed or is intended for transformation into another compound through various processes, including enzymatic, mechanical, chemical, or catalytic. This definition encompasses not only individual or pure compounds but also compound combinations, including solutions, mixtures, and other materials that contain at least one substrate or its derivatives. Furthermore, the term "substrate" encompasses not just compounds that serve as a carbon source suitable for use as initial materials, such as sugars or carbohydrates derived from biomass, but also can include intermediate and end-product metabolites that may play a role within a metabolic pathway associated with a recombinant microorganism, as outlined herein. Examples of relevant feedstock-derived substrates include corn, corn stover, molasses, sugarcane, cellulosic biomass, lignocellulosic biomass, waste products, agricultural products/by-products and similar sources of carbohydrates which may undergo varying degrees of processing. Flux: In metabolic engineering, flux refers to the rate at which substrates and intermediates flow through a metabolic pathway, herein determined as moles (or millimoles) per gram of dry cell weight per unit time. Heterologous: The term “heterologous” as used herein indicates an enzyme, protein, functional molecule, or sequences that are expressed in an organism from which they did not originate. Moreover, the levels of expression or utilization may be unrelated to the native expression or organism of origin. Malonate semialdehyde reductase (MSA reductase): An enzyme that catalyzes the reduction of malonate semialdehyde to 3-HP using an external electron donor (otherwise known as a redox cofactor). The external electron donor may be NADH or NADPH, for example. Alternatively, this enzyme is sometimes referred to as 3-hydroxypropionic acid dehydrogenase. Mass yield: The actual mass (typically in units of grams or kilograms) of desired product obtained from a provided mass of substrate. Molar yield: The actual moles of desired product obtained from provided moles of substrate. Native: The term “native” as used herein indicates molecules, including DNA, RNA, proteins and enzymes, that are expressed or encoded by the organism in which they originated or as they are found in nature. The expression of native enzymes may be modified from the original state in recombinant organisms. OTR (Oxygen Transfer Rate): The rate of mass transfer of oxygen (O2) per volume fermentation broth per unit time. Herein most often described in units of millimoles O₂ per liter fermentation broth per hour. PEPC (phosphoenolpyruvate carboxylase): An enzyme that catalyzes the conversion of phosphoenolpyruvate and the fixation of carbon dioxide (CO₂) to produce inorganic phosphate and oxaloacetate. This reaction may additionally utilize bicarbonate (HCO3⁻) in place of, or in combination with, carbon dioxide (CO2). PEPCK (phosphoenolpyruvate carboxykinase): An enzyme that catalyzes the conversion of phosphoenolpyruvate (PEP) and a C1 substrate (e.g., carbon dioxide and/or bicarbonate) into oxaloacetate, concomitantly converting a nucleotide diphosphate (e.g., ADP) into a nucleotide triphosphate (e.g., ATP) using substrate level phosphorylation from cleaving the phosphate bond in PEP. PYC (pyruvate carboxylase): An enzyme that catalyzes the hydrolysis of ATP to transfer a carboxyl group from bicarbonate to pyruvate resulting in the generation of oxaloacetate. Recombinant microorganism: The terms “recombinant microorganism, “genetically engineered organism,” “engineered organism,” “engineered strain,” “recombinant cell”, or “recombinant host cell” are employed synonymously to refer to microorganisms that have undergone genetic modification(s). Such modifications may involve the introduction, expression, or over-expression of native or non-native (heterologous) polynucleotides, potentially carried by a vector. Additionally, these terms encompass alterations that result in the modified expression or deletion of native genes within the organism. Titer: The concentration of a substance in a solution, e.g. the quantity (typically the mass or molar concentration) of a product (such as a 3-HP) in a given volume of culture. Theoretical yield: The maximum amount of product that can be produced from a given amount of reactants under perfect conditions (e.g., no electrons or carbon attributed to biomass formation or maintenance energy), as predicted by stoichiometric and degree of reduction calculations without any loss or inefficiency. Yield can be expressed as a fraction or percent. Vector: A DNA molecule (polynucleotide) or genetic element used to transport and replicate foreign genetic material (often from one organism) into a host organism. Vectors can be plasmids, viruses, or other genetic constructs designed for introducing specific genes or DNA sequences into a target organism for various purposes, such as gene cloning, expression, or modification. Vectors include both polynucleotides intended to persist in the cell outside of the host genome (episomal), or intended to deliver polynucleotides into the host organism and be integrated into the host genome in directed (targeted) or undirected integration events. DETAILED DESCRIPTION The inventors of the present application have invented certain systems, methods, and approaches for anaerobic bioproduction of target compounds. The inventors of the present application have also invented systems, methods, and approaches to modify and customize genome-scale metabolic models (GEMs) involved in design processes for anaerobic bioproduction. The inventors of the application have also invented systems, methods, and approaches to modify and customize organisms capable of anaerobic metabolism to efficiently produce the target compounds. The inventors have also invented systems, methods, and approaches to adapt ethanol-producing infrastructure, including existing infrastructure, for anaerobic production of the target compounds. General The present application also provides methods and pathways for making and producing certain compounds. In preferred embodiments, the pathways are anaerobic biopathways for use in a novel engineered organism or novel GEMs. In a preferred embodiment, the engineered organism or GEM is adapted to produce a specific target compound from an initial feedstock. Embodiments of such organisms and GEMs are discussed elsewhere in this application. Generally, several preferred alternative pathways for producing each compound are provided. It should be understood that other pathways similar to those shown below having only minor changes (e.g., a substitution of similar enzymes or cofactors for those described herein) are also encompassed by the present application. It should also be understood that electron transfer (redox) cofactors may be substituted where an electron donor or acceptor is required and may include, but are not limited to, nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), quinone, or inorganic or organometallic cofactors. It should also be understood that enzyme may require other organic or inorganic cofactors, which may include, but are not limited to pyridoxal 5’- phosphate, thiamine pyrophosphate, and Coenzyme A. It should also be understood that steps in these pathways may be substituted for one another (where possible), and that such substitutions are within the scope of the present application even if not expressly shown below (for example to reduce redundancy in the application). It should also be understood by a person of ordinary skill in molecular biology and enzymology that any enzyme described or known to use a particular redox cofactor can be engineered to utilize a different cofactor. E.g., NADH-utilizing enzymes may be modified to utilize NADPH, or vice versa (Cahn 2017, doi: 10.1021/acssynbio.6b00188). 3-hydroxypropionate (or 3-hydroxypropionic acid) Production of 3-hydroxypropionate In one embodiment, the present application provides pathways for bio-producing 3- hydroxypropionate. Those skilled in the art would understand that 3-hydroxypropionate may also be referred to as 3-HP, 503-66-2, 3-hydroxy propanoic acid, 3-hydroxypropanoate, ^- hydroxypropionate, 3-hydroxypropanoic acid ion, 1190-23-4 and CHEBI:16510. Those skilled in the art would also understand that this compound may exist as the protonated form, 3- hydroxypropionic acid (CHEBI:33404), depending on the pH of the solution in which the compound is produced. Several alternative pathways are provided below: Pathway 1 Step 1.1 In this step, the metabolite phosphoenolpyruvate is reacted to obtain oxaloacetate.

phosphoenolpyruvate -> oxaloacetate -40.27 kJ/mol to 6.49 kJ/mo Gibbs Free Energy (ΔrG’°) of specific embodiments of foregoing reaction can range from -40.27 to 6.49 kJ/mol depending on co-reactants, under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, Ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 4.1.1.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 4.1.1.49 (encoded by one or more of SEQ ID NOS. 2, 25-31), 4.1.1.31 (encoded by one or more of SEQ ID NOS. 1, 32-56), or 4.1.1.32 (encoded by one or more of SEQ ID NOS. 18-24). In a particularly preferred embodiment, the above scheme is mediated by ATP:oxaloacetate carboxy-lyase, also known as phosphoenolpyruvate carboxykinase (ATP) or PEPCK. In a particularly preferred embodiment, the reaction is catalyzed by SEQ ID NO.1 or 2. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: ADP + phosphoenolpyruvate + CO2 [or HCO3-] <=> ATP + oxaloacetate or H2O + phosphoenolpyruvate + CO₂ <=> phosphate + oxaloacetate Step 1.2 In this step, oxaloacetate is reacted to obtain L-aspartate.

oxaloacetate -> L-aspartate -19.87 kJ/mol to -2.46 kJ/mo Gibbs Free Energy (ΔrG’°) of specific embodiments of foregoing reactions can range from -19.87 to -2.46 kJ/mol depending on co-reactants, under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, Ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 2.6.1.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 2.6.1.1 (encoded by one or more of SEQ ID NOS. 6-7, 57-81, 83-84, 383). In a particularly preferred embodiment, the above scheme is mediated by L-aspartate:2-oxoglutarate aminotransferase. In a particularly preferred embodiment, the aspartate aminotransferase reaction is catalyzed by SEQ ID NOS.6 or 7. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: oxaloacetate + L-glutamate <=> L-aspartate + 2-oxoglutarate Step 1.3 In this step, L-aspartate is reacted to obtain ^-alanine.

L-aspartate -> ^-alanine -34.01 kJ/mol Gibbs Free Energy (ΔrG’°) is approximately -34.01 kJ/mol under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, Ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 4.1.1.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 4.1.1.15 (encoded by one or more of SEQ ID NOS.8, 159-187, 189-193) and 4.1.1.11 (encoded by one or more of SEQ ID NOS.8-9, 384- 391, 393-400). In a particularly preferred embodiment, the above scheme is mediated by L- aspartate 1-carboxy-lyase ( ^-alanine-forming), also known as an aspartate decarboxylase. In a particularly preferred embodiment, the aspartate decarboxylase reaction is catalyzed by SEQ ID NOS.8 or 9. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: L-aspartate <=> ^-alanine + CO2 Step 1.4 In this step, ^-alanine is reacted to obtain malonate semialdehyde.

^-alanine -> malonate semialdehyde -3.72 kJ/mol to -3.56 kJ/mo Gibbs Free Energy (ΔrG’°) of specific embodiments of foregoing reaction can range from -3.72 to -3.56 kJ/mol depending on co-reactants, under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 2.6.1.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 2.6.1.18 (encoded by one or more of SEQ ID 10, 112, 115-116, 212), 2.6.1.19 (encoded by one or more of SEQ ID NOS.11, 194-213), or 2.6.1.120 (encoded by SEQ ID NO. 198). In a particularly preferred embodiment, the above scheme is mediated by ^-alanine:2-oxoglutarate aminotransferase. In a particularly preferred embodiment, the ^-alanine aminotransferase reaction is catalyzed by SEQ ID NOS.10 or 11. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: ^-alanine + 2-oxoglutarate <=> malonate semialdehyde + L-glutamate Step 1.5 In this step, malonate semialdehyde is reacted to obtain 3-hydroxypropionate.

malonate semialdehyde -> 3-hydroxypropionate -20.41 kJ/mol Gibbs Free Energy (ΔrG’°) is approximately -20.41 kJ/mol under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 1.1.1.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 1.1.1.35 (encoded by one or more of SEQ ID NOS. 218-227), 1.1.1.298 (encoded by one or more of SEQ ID NOS. 12-13, 214, 216-217), and 1.1.1.59 (encoded by one or more of SEQ ID NOS. 14-17, 243). In a particularly preferred embodiment, the above scheme is mediated by 3-hydroxypropionate:NAD+ oxidoreductase. In a particularly preferred embodiment, the malonate semialdehyde reduction is catalyzed by SEQ ID NOS.12, 13, 14, 15, 16, or 17. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: malonate semialdehyde + NADH + H+ <=> 3-hydroxypropionate + NAD+ or malonate semialdehyde + NADPH + H+ <=> 3-hydroxypropionate + NADP+ Pathway 2 Step 2.1 In this step, the metabolite pyruvate is reacted to obtain oxaloacetate.

pyruvate -> oxaloacetate -7.19 kJ/mol Gibbs Free Energy (ΔrG’°) is approximately -7.19 kJ/mol under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 6.4.1.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 6.4.1.1 (encoded by one or more of SEQ ID NOS.3-5, 188, 215, 392, 401-404). In a particularly preferred embodiment, the above scheme is mediated by 6.4.1.1 (pyruvate carboxylase). In a particularly preferred embodiment, the pyruvate carboxylation is catalyzed by SEQ ID NOS.3, 4, or 5. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: ATP + pyruvate + bicarbonate <=> ADP + phosphate + oxaloacetate Step 2.2 In this step, oxaloacetate is reacted to obtain L-aspartate.

oxaloacetate -> L-aspartate -2.84 kJ/mol Gibbs Free Energy (ΔrG’°) is approximately -2.84 kJ/mol under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 2.6.1.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 2.6.1.1 (encoded by one or more of SEQ ID NOS. 6-7, 57-81, 83-84, 383). In a particularly preferred embodiment, the above scheme is mediated by L-aspartate:2-oxoglutarate aminotransferase. In a particularly preferred embodiment, the aspartate aminotransferase reaction is catalyzed by SEQ ID NOS.6 or 7. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: oxaloacetate + L-glutamate <=> L-aspartate + 2-oxoglutarate Step 2.3 In this step, L-aspartate is reacted to obtain ^-alanine.

L-aspartate -> ^-alanine -34.01 kJ/mol Gibbs Free Energy (ΔrG’°) is approximately -34.01 kJ/mol under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, Ionic strength of 0.25 M, temperature of 25°C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 4.1.1.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 4.1.1.15 (encoded by one or more of SEQ ID NOS.8, 159-187, 189-193), 4.1.1.11 (encoded by one or more of SEQ ID NOS.8-9, 384-391, 393-400). In a particularly preferred embodiment, the above scheme is mediated by L-aspartate 1- carboxy-lyase ( ^-alanine-forming), also known as an aspartate decarboxylase. In a particularly preferred embodiment, the aspartate decarboxylase reaction is catalyzed by SEQ ID NOS.8 or 9. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: L-aspartate <=> ^-alanine + CO2 Step 2.4 In this step, ^-alanine is reacted to obtain malonate semialdehyde.

^-alanine -> malonate semialdehyde -3.72 kJ/mol to -3.56 kJ/mo Gibbs Free Energy (ΔrG’°) of specific embodiments of foregoing reaction can range from -3.72 to -3.56 kJ/mol depending on co-reactants, under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 2.6.1.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 2.6.1.18 (encoded by one or more of SEQ ID NOS.10, 112, 115-116, 212), 2.6.1.19 (encoded by one or more of SEQ ID NOS.11, 194-213). In a particularly preferred embodiment, the above scheme is mediated by ^-alanine:2-oxoglutarate aminotransferase. In a particularly preferred embodiment, the ^-alanine aminotransferase reaction is catalyzed by SEQ ID NOS.10 or 11. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: ^-alanine + 2-oxoglutarate <=> malonate semialdehyde + L-glutamate Step 2.5 In this step, malonate semialdehyde is reacted to obtain 3-hydroxypropionate.

malonate semialdehyde -> 3-hydroxypropionate -20.41 kJ/mol Gibbs Free Energy (ΔrG’°) is approximately -20.41 kJ/mol under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 1.1.1.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 1.1.1.298 (encoded by one or more of SEQ ID NOS.12-13, 214, 216-217), 1.1.1.59 (encoded by one or more of SEQ ID NOS.14-17, 243), and 1.1.1.35 (encoded by one or more of SEQ ID NOS. 218-227). In a particularly preferred embodiment, the above scheme is mediated by 1.1.1.59. In a particularly preferred embodiment, the malonate semialdehyde reduction is catalyzed by SEQ ID NOS.12, 13, 14, 15, 16 or 17. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: malonate semialdehyde + NADH + H+ <=> 3-hydroxypropionate + NAD+ or malonate semialdehyde + NADPH + H+ <=> 3-hydroxypropionate + NADP+ Pathway 3 Step 3.1 In this step, the metabolite pyruvate is reacted to obtain lactate.

pyruvate -> lactate -20.63 kJ/mol to 5.87 kJ/mo Gibbs Free Energy (ΔrG’°) of specific embodiments of foregoing reaction can range from -20.63 to 5.87 kJ/mol depending on co-reactants, under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 1.1.1.X, 1.1.99.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 1.1.1.272 (encoded by SEQ ID NO.358), 1.1.1.337 (encoded by one or more of SEQ ID NOS.269-272), 1.1.1.345 (encoded by one or more of SEQ ID NOS.273-276), 1.1.1.27 (encoded by one or more of SEQ ID NOS.265, 320-346), 1.1.1.28 (encoded by one or more of SEQ ID NOS. 248, 276, 347-357), 1.1.99.6 (encoded by one or more of SEQ ID NOS.244-248). In a particularly preferred embodiment, the above scheme is mediated by lactate dehydrogenase. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: pyruvate + NADH + H+ <=> lactate + NAD+ Step 3.2 In this step, lactate is reacted to obtain lactoyl-CoA.

lactate -> lactoyl-CoA -13.89 kJ/mol to 0.85 kJ/mo Gibbs Free Energy (ΔrG’°) of specific embodiments of foregoing reaction can range from -13.89 to 0.85 kJ/mol depending on co-reactants, under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 2.8.3.X and/or 6.2.1.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 2.8.3.27, 6.2.1.17 (encoded by one or more of SEQ ID NOS.374, 380-382), 2.8.3.1 (encoded by one or more of SEQ ID NOS. 359-360), 6.2.1.13 (encoded by one or more of SEQ ID NOS.361-366), 2.8.3.3, or 6.2.1.1. In a particularly preferred embodiment, the above scheme is mediated by acetate:CoA ligase (AMP- forming) or propionate CoA-transferase. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: ATP + lactate + coenzyme A <=> AMP + diphosphate + lactoyl-CoA or lactate + acetyl-CoA <=> lactoyl-CoA + acetate Step 3.3 In this step, lactoyl-CoA is reacted to obtain acrylyl CoA.

lactoyl-CoA -> acrylyl CoA 2.09 kJ/mol Gibbs Free Energy (ΔrG’°) is approximately 2.09 kJ/mol under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 4.2.1.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 4.2.1.17 (encoded by one or more of SEQ ID NOS.226, 367-371), 4.2.1.54 (encoded by SEQ ID NO.319), or 4.2.1.116 (encoded by SEQ ID NO.372). In a particularly preferred embodiment, the above scheme is mediated by 4.2.1.54 ( lactoyl-CoA dehydratase). In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: lactoyl-CoA <=> acrylyl CoA + H2O Step 3.4 In this step, acrylyl CoA is reacted to obtain 3-hydroxypropionyl-CoA.

a 2.3 kJ/mol Gibbs Free Energy (ΔrG’°) is approximately 2.3 kJ/mol under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 4.2.1.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 4.2.1.116 (encoded by SEQ ID NO.372), 4.2.1.54 (encoded by SEQ ID NO. 319), or 4.2.1.17 (encoded by one or more of SEQ ID NOS. 226, 367-371). In a particularly preferred embodiment, the above scheme is mediated by 3- hydroxypropionyl-CoA hydro-lyase. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: acrylyl CoA + H2O <=> 3-hydroxypropionyl-CoA Step 3.5 In this step, 3-hydroxypropionyl-CoA is reacted to obtain 3-hydroxypropionate.

3 -59.45 kJ/mol to 0.83 kJ/mo Gibbs Free Energy (ΔrG’°) of specific embodiments of foregoing reaction can range from -59.45 to 0.83 kJ/mol depending on co-reactants, under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 4.2.1.X, 2.8.3.X, 6.2.1.X, or 3.1.2.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 2.8.3.27, 6.2.1.23, 2.8.3.1 (encoded by one or more of SEQ ID NOS. 359-360), 6.2.1.36 (encoded by one or more of SEQ ID NOS.373-374), 6.2.1.76, 4.2.1.155, 2.8.3.8 (encoded by SEQ ID NO.375), or 3.1.2.4 (encoded by one or more of SEQ ID NOS. 376-379). In a particularly preferred embodiment, the above scheme is mediated by 3.1.2.4. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: 3-hydroxypropionyl-CoA + H2O <=> CoA + 3-hydroxypropionate Pathway 4 Step 4.1 In this step, the metabolite pyruvate is reacted to obtain oxaloacetate.

pyruvate -> oxaloacetate -7.19 kJ/mol Gibbs Free Energy (ΔrG’°) is approximately -7.19 kJ/mol under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 6.4.1.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 6.4.1.1 (encoded by one or more of SEQ ID NOS.3-5, 188, 215, 392, 401-404). In a particularly preferred embodiment, the above scheme is mediated by 6.4.1.1 (pyruvate carboxylase). In a particularly preferred embodiment, the pyruvate carboxylation is catalyzed by SEQ ID NOS.3, 4, or 5. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: ATP + pyruvate + bicarbonate <=> ADP + phosphate + oxaloacetate Step 4.2 In this step, oxaloacetate is reacted to obtain malonate semialdehyde.

oxaloacetate -> malonate semialdehyde -40.4 kJ/mol Gibbs Free Energy (ΔrG’°) is approximately -40.4 kJ/mol under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 4.1.1.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 4.1.1.82, 4.1.1.54 (encoded by SEQ ID NO.319), 4.1.1.79, 4.1.1.75, or 4.1.1.43. In a particularly preferred embodiment, the above scheme is mediated by an oxaloacetate decarboxylase. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: oxaloacetate <=> malonate semialdehyde + CO₂ Step 4.3 In this step, malonate semialdehyde is reacted to obtain 3-hydroxypropionate.

malonate semialdehyde -> 3-hydroxypropionate -20.41 kJ/mol Gibbs Free Energy (ΔrG’°) is approximately -20.41 kJ/mol under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 1.1.1.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 1.1.1.298 (encoded by one or more of SEQ ID NOS.12-13, 214, 216-217), 1.1.1.59 (encoded by one or more of SEQ ID NOS.14-17, 243), or 1.1.1.35 (encoded by one or more of SEQ ID NOS. 218-227). In a particularly preferred embodiment, the above scheme is mediated by 1.1.1.59. In a particularly preferred embodiment, the malonate semialdehyde reduction is catalyzed by SEQ ID NOS.12, 13, 14, 15, 16, or 17. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: malonate semialdehyde + NADH + H+ <=> 3-hydroxypropionate + NAD+ or malonate semialdehyde + NADPH + H+ <=> 3-hydroxypropionate + NADP+ Pathway 5 Step 5.1 In this step, the metabolite pyruvate is reacted to obtain alanine.

pyruvate -> L-alanine -16.46 kJ/mol to -0.16 kJ/mo Gibbs Free Energy (ΔrG’°) of specific embodiments of foregoing reaction can range from -16.46 to -0.16 kJ/mol depending on co-reactants, under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 2.6.1.X or 1.4.1.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 2.6.1.2 (encoded by one or more of SEQ ID NOS.198, 231-238), 1.4.1.1 (encoded by one or more of SEQ ID NOS.124-141). In a particularly preferred embodiment, the above scheme is mediated by alanine transaminase. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: pyruvate + L-glutamate <=> L-alanine + 2-oxoglutarate Step 5.2 In this step, alanine is reacted to obtain ^-alanine.

L-alanine -> ^-alanine -5.17 kJ/mol Gibbs Free Energy (ΔrG’°) is approximately -5.17 kJ/mol under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 5.4.3.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 5.4.3.3 (encoded by one or more of SEQ ID NOS. 239-242) or 5.4.3.2. In a particularly preferred embodiment, the above scheme is mediated by alanine aminomutase ( ^-alanine forming). In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: L-alanine <=> ^-alanine Step 5.3 In this step, ^-alanine is reacted to obtain malonate semialdehyde.

^-alanine -> malonate semialdehyde -3.72 kJ/mol to -3.56 kJ/mo Gibbs Free Energy (ΔrG’°) of specific embodiments of foregoing reaction can range from -3.72 to -3.56 kJ/mol depending on co-reactants, under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 2.6.1.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 2.6.1.18 (encoded by one or more of SEQ ID NOS.10, 112, 115-116, 212), 2.6.1.120 (encoded by SEQ ID NO.198), or 2.6.1.19 (encoded by one or more of SEQ ID NOS.11, 194-213) or 2.6.1.55. In a particularly preferred embodiment, the above scheme is mediated by 2.6.1.19. In a particularly preferred embodiment, the ^-alanine aminotransferase reaction is catalyzed by SEQ ID NOS.10 or 11. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: ^-alanine + 2-oxoglutarate <=> malonate semialdehyde + L-glutamate Step 5.4 In this step, malonate semialdehyde is reacted to obtain 3-hydroxypropionate.

malonate semialdehyde -> 3-hydroxypropionate -20.41 kJ/mol Gibbs Free Energy (ΔrG’°) is approximately -20.41 kJ/mol under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 1.1.1.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 1.1.1.298 (encoded by one or more of SEQ ID NOS.12-13, 214, 216-217), 1.1.1.59 (encoded by one or more of SEQ ID NOS.14-17, 243) or 1.1.1.35 (encoded by one or more of SEQ ID NOS. 218-227). In a particularly preferred embodiment, the above scheme is mediated by 1.1.1.59. In a particularly preferred embodiment, the malonate semialdehyde reduction is catalyzed by SEQ ID NOS.12, 13, 14, 15, 16, or 17. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: malonate semialdehyde + NADH + H+ <=> 3-hydroxypropionate + NAD+ or malonate semialdehyde + NADPH + H+ <=> 3-hydroxypropionate + NADP+ Pathway 6 Step 6.1 In this step, the metabolite phosphoenolpyruvate is reacted to obtain oxaloacetate.

phosphoenolpyruvate -> oxaloacetate -40.27 kJ/mol to 6.49 kJ/mo Gibbs Free Energy (ΔrG’°) of specific embodiments of foregoing reaction can range from -40.27 to 6.49 kJ/mol depending on co-reactants, under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 4.1.1.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 4.1.1.49 (encoded by one or more of SEQ ID NOS.2, 25-31) or 4.1.1.31 (encoded by one or more of SEQ ID NOS.1, 32-56). In a preferred embodiment, the above scheme is mediated by 4.1.1.31 (phosphoenolpyruvate carboxylase, or PEPC), and in a particularly preferred embodiment, the above scheme is mediated by ATP:oxaloacetate carboxy-lyase, also known as phosphoenolpyruvate carboxykinase (ATP) or PEPCK. In a particularly preferred embodiment, the reaction is catalyzed by SEQ ID NO.1 or 2. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: ADP + phosphoenolpyruvate + CO2 [or HCO3-] <=> ATP + oxaloacetate or H2O + phosphoenolpyruvate + CO2 <=> phosphate + oxaloacetate Step 6.2 In this step, oxaloacetate is reacted to obtain malonate semialdehyde.

oxaloacetate -> malonate semialdehyde -40.4 kJ/mol Gibbs Free Energy (ΔrG’°) is approximately -40.4 kJ/mol under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 4.1.1.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 4.1.1.82, 4.1.1.54, 4.1.1.79, 4.1.1.75 or 4.1.1.43. In a particularly preferred embodiment, the above scheme is mediated by an oxaloacetate decarboxylase. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: oxaloacetate <=> malonate semialdehyde + CO₂ Step 6.3 In this step, malonate semialdehyde is reacted to obtain 3-hydroxypropionate.

malonate semialdehyde -> 3-hydroxypropionate -20.41 kJ/mol Gibbs Free Energy (ΔrG’°) is approximately -20.41 kJ/mol under 1 molar concentrations, pH of 7.5, magnesium concentration (pMg) of 3.0, ionic strength of 0.25 M, temperature of 25 °C, and 1 bar of pressure. In some embodiments, the above scheme is mediated by an enzyme having an EC number selected from the group consisting of 1.1.1.X. In a preferred embodiment, the above scheme is mediated by an enzyme selected from the EC group consisting of 1.1.1.298 (encoded by one or more of SEQ ID NOS.12-13, 214, 216-217), 1.1.1.59 (encoded by one or more of SEQ ID NOS.14-17, 243) or 1.1.1.35 (encoded by one or more of SEQ ID NOS. 218-227). In a particularly preferred embodiment, the above scheme is mediated by 1.1.1.59. In a particularly preferred embodiment, the malonate semialdehyde reduction is catalyzed by SEQ ID NOS.12, 13, 14, or 15, 16, or 17. In some embodiments, the above scheme includes cofactors selected from the aforementioned list. Thus, in a preferred embodiment of this step the reaction is: malonate semialdehyde + NADH + H+ <=> 3-hydroxypropionate + NAD+ or malonate semialdehyde + NADPH + H+ <=> 3-hydroxypropionate + NADP+ Metabolic Modifications The present application also provides methods, pathways, and GEMs for improving the flux, efficiency, and/or product output when making the compounds of the present application. In a preferred embodiment, the engineered organism or GEM adapted to produce a specific target compound from an initial feedstock-derived substrate using a pathway provided herein is further modified to include additional reactions and/or environmental constraints. These reactions and constraints indirectly improve yield or increase the rate of product formation (i.e., they do not alter the steps in the pathway, but impact other parts of cellular metabolism that ultimately improves an aspect of product formation). In a preferred embodiment, the reactions and/or constraints are engineered to improve the end product flux, efficiency, output, and/or yield. In embodiments of the present application, the engineered organism or GEM is derived from a species within one of the following genera or hybrids thereof: Saccharomyces, Kluyveromyces, Pichia, Hansenula, Issatchenkia, Candida, Zygosaccharomyces, Debaryomyces, Dekkera (Brettanomyces), and Schizosaccharomyces. In a preferred embodiment, the species is Saccharomyces cerevisiae. The engineered organism or GEM includes modifications for introducing the pathway for making the compound. In preferred embodiments, the engineered organism or GEM also includes modifications for introducing the additional reactions discussed herein designed to improve the end product flux, efficiency, output, and/or yield. With respect to production of the compounds herein, several preferred alternative modifications of an organism or GEM are provided, which include additional reactions and/or constraints. It should be understood that other models similar to those shown below having only minor changes (e.g., a substitution of similar reactions or model constraints for those described herein) are also encompassed by the present application. It should also be understood that aspects of the models may be substituted for one another (where possible), and that such substitutions are within the scope of the present application even if not expressly shown below (for example to reduce redundancy in the application). Within the context of this section, “flux” is defined as the specific rate of formation or consumption in millimoles per gram of dry biomass per hour, “molar yield” is defined as the molar flux of product divided by molar flux of substrate, “carbon molar yield” is defined as the molar flux of product multiplied by the number of carbon atoms in the product divided by molar flux of substrate multiplied by the number of carbon atoms in substrate, “mass yield” is defined as the molar flux of product multiplied by the molecular weight of product divided by molar flux of substrate multiplied by molecular weight of substrate. Yields: 3-hydroxypropionate Production of 3-hydroxypropionate In some embodiments, the present application provides models associated with the production of 3-hydroxypropionate. In a preferred embodiment, the modified organism or GEM for producing 3-hydroxypropionate is derived from Chalmers Yeast GEM. However, it should be understood that it remains possible to engineer an organism/GEM derived from another genus/species selection from the aforementioned list to bioproduce this product with the use of pathways and reactions discussed below. As discussed previously, 3-hydroxypropionate may be produced according to any of several pathways set forth elsewhere in this application. In addition to these pathways, the present application also provides further potential modifications for improving product formation. The table below summarizes the various modifications to pathways for producing 3- Hydroxypropionate. The following table provides summary results for various modifications discussed in more detail below. Some of the models enumerated in the table are described in further detail; however, it should be understood each of the models and pathway combinations provided below is within the scope of the application. Pathway Model Mass Carbon 6, 6,

Pathway Model Mass Carbon 6, 6, 6, 3, 3,

Pathway Model Mass Carbon 2, 4, 7, 0, 2, 4, 7, 0, 2, 4, 7, 0,

Pathway Model Mass Carbon 2, 4, 7, 0, 5 2, 4, 7, 0, 5

Pathway Model Mass Carbon 2, 4, 7, 0, 5 2, 4, 7, 0, 5 3,

Pathway Model Mass Carbon 3, 3, 2, 4, 7, 0, 3, 3,

Pathway Model Mass Carbon 3, 3, 3, 5, 5 3,

Pathway Model Mass Carbon 3, 5, 5 3, 3, 3, 3, 5, 5

Pathway Model Mass Carbon 3, 3, 3, 5, 5 3, 3,

Pathway Model Mass Carbon 3, 5, 5 3, 3, 3, 3, 5, 5

Pathway Model Mass Carbon 3, 3, 5, 5 3, 3, 3, 5, 5

Pathway Model Mass Carbon 3, 3, 3, 3, 3, 3, 3,

Pathway Model Mass Carbon 3, 3, 3, 3, 3, 3,

Pathway Model Mass Carbon 3, 3, 3, 3, 3, 3, 3,

Pathway Model Mass Carbon 3, 3, 3, 3, 3,

Pathway Model Mass Carbon 3, 3, 3, 3, 3, 3,

Pathway Model Mass Carbon 3, 3, 3, 3, 3,

Pathway Model Mass Carbon 3, T

able : escribes various genome scale models with different combinations of the enzymes described in the pathway(s) above as well as off-pathway modifications notated by a model modification id, which are further described below in the modifications dictionary

Modifications Dictionary Reaction ID Reaction Description Constraint Native vs.

Reaction ID Reaction Description Constraint Native vs.

Reaction ID Reaction Description Constraint Native vs.

Reaction ID Reaction Description Constraint Native vs.

Reaction ID Reaction Description Constraint Native vs.

Table 2: The table above describes, in more detail, the modifications that are proposed for the various models in Table 1 and the model details below. It should be understood that all metabolites are present in the cytosol of the GEM (S. cerevisiae) unless otherwise specified (m = mitochondrial, e = extracellular, c = cytosol) Model 555 In one embodiment, the application provides a modified organism or GEM adapted to produce 3- hydroxypropionate via pathway 1. In one embodiment (and as shown in the table above), the organism or GEM is further adapted to incorporate the following additional metabolic reactions, denoted in the Applied Model Modifications list below. Applied Model Modifications: r_0226, r_1110, r_0446, r_2111_constrained_4083 and r_1992_constrained_4078 In this embodiment, the following enzymes and reaction stoichiometries were specifically added to the Yeast 8 Saccharomyces cerevisiae GEM (Lu 2019 doi.org/10.1038/s41467-019-11581-3) along with the Applied Model Modifications listed above, representing a particular embodiment of pathway 1 as “Model 555”. Pathway EC Enzyme Name Modeled Reaction >

A GEM incorporating the additional reactions and constraints set forth above was analyzed in silico using stoichiometric Flux Balance Analysis to assess uptake and secretion (boundary) fluxes as well as mass yield, molar yield, carbon molar yield, and overall specific rate of product formation. The results are set forth in the tables below: Overall Yield / Flux for 3-hydroxypropionate Mass yield 95.4%

Uptake Flux (boundary reactions and metabolites that are entering the model) Flux (uptake in millimole / Reaction Metabolite

Secreting Flux (boundary reactions and metabolites that are exiting the model) Flux (secretion in millimole / Reaction Metabolite

Flux (secretion in millimole / Reaction Metabolite

Model 557 In one embodiment, the application provides a modified organism or GEM adapted to produce 3- hydroxypropionate via pathway 1. In one embodiment (and as shown in the table above), the organism or GEM is further adapted to incorporate the following additional metabolic reactions, denoted in the Applied Model Modifications list below. Applied Model Modifications: r_0226, r_1110, r_0446, r_2111_constrained_4083 and r_1992_constrained_4078 In this embodiment, the following enzymes and reaction stoichiometries were specifically added to the Yeast 8 Saccharomyces cerevisiae GEM along with the Applied Model Modifications listed above, representing a particular embodiment of pathway 1 as “Model 557”. Pathway EC +

A GEM incorporating the additional reactions and constraints set forth above was analyzed in silico using stoichiometric Flux Balance Analysis to assess uptake and secretion (boundary) fluxes as well as mass yield, molar yield, carbon molar yield, and overall specific rate of product formation. The results are set forth in the tables below: Overall Yield / Flux for 3-hydroxypropionate Mass yield 95.4%

Uptake Flux (boundary reactions and metabolites that are entering the model) Flux (uptake in millimole / Reaction Metabolite

Secreting Flux (boundary reactions and metabolites that are exiting the model) Flux (secretion in millimole / Reaction Metabolite

Flux (secretion in millimole / Reaction Metabolite

Model 559 In one embodiment, the application provides a modified organism or GEM adapted to produce 3- hydroxypropionate via pathway 1. In one embodiment (and as shown in the table above), the organism or GEM is further adapted to incorporate the following additional metabolic reactions, denoted in the Applied Model Modifications list below. Applied Model Modifications: r_0226, r_1110, r_0446, r_2111_constrained_4083 and r_1992_constrained_4078 In this embodiment, the following enzymes and reaction stoichiometries were specifically added to the Yeast 8 Saccharomyces cerevisiae GEM along with the Applied Model Modifications listed above, representing a particular embodiment of pathway 1 as “Model 559”. Pathway EC E^{nzyme Name Modeled Reaction} > t^e

A GEM incorporating the additional reactions and constraints set forth above was analyzed in silico using stoichiometric Flux Balance Analysis to assess uptake and secretion (boundary) fluxes as well as mass yield, molar yield, carbon molar yield, and overall specific rate of product formation. The results are set forth in the tables below: Overall Yield / Flux for 3-hydroxypropionate Mass yield 47.7%

Uptake Flux (boundary reactions and metabolites that are entering the model) Flux (uptake in millimole / Reaction Metabolite

Secreting Flux (boundary reactions and metabolites that are exiting the model) Flux (secretion in millimole / Reaction Metabolite

Flux (secretion in millimole / Reaction Metabolite

Model 11322 In one embodiment, the application provides a modified organism or GEM adapted to produce 3- hydroxypropionate via pathway 2. In one embodiment (and as shown in the table above), the organism or GEM is further adapted to incorporate the following additional metabolic reactions, denoted in the Applied Model Modifications list below. Applied Model Modifications: r_0226, r_1110, r_2111_constrained_4083, r_1992_constrained_4084 and r_1714_constrained_4140 In this embodiment, the following enzymes and reaction stoichiometries were specifically added to the Yeast 8 Saccharomyces cerevisiae GEM along with the Applied Model Modifications listed above, representing a particular embodiment of pathway 2 as “Model 11322”. Pathway EC E^{nzyme Name Modeled Reaction} e >

A GEM incorporating the additional reactions and constraints set forth above was analyzed in silico using stoichiometric Flux Balance Analysis to assess uptake and secretion (boundary) fluxes as well as mass yield, molar yield, carbon molar yield, and overall specific rate of product formation. The results are set forth in the tables below: Overall Yield / Flux for 3-hydroxypropionate Mass yield 47.7%

Uptake Flux (boundary reactions and metabolites that are entering the model) Flux (uptake in millimole / Reaction Metabolite

Secreting Flux (boundary reactions and metabolites that are exiting the model) Flux (secretion in millimole / Reaction Metabolite

Flux (secretion in millimole / Reaction Metabolite

Model 11342 In one embodiment, the application provides a modified organism or GEM adapted to produce 3- hydroxypropionate via pathway 2. In one embodiment (and as shown in the table above), the organism or GEM is further adapted to incorporate the following additional metabolic reactions, denoted in the Applied Model Modifications list below. Applied Model Modifications: ec111157nadph_4131, ec111157nadh_4132, ec42155_4133, ec13144_4134, ec13144nadph_4135, ada_coa_cleavage_4136, butanal_adh_4137, butanal_adh_4138, r_0226, r_1110, r_2111_constrained_4083, r_1992_constrained_4084, r_1714_constrained_4140, phosphoketolase_fpk_4141, phosphoketolase_xpk_4142, phosphotransacetylase_pta_4144 and ada_4145 In this embodiment, the following enzymes and reaction stoichiometries were specifically added to the Yeast 8 Saccharomyces cerevisiae GEM along with the Applied Model Modifications listed above, representing a particular embodiment of pathway 2 as “Model 11342”. Pathway EC E^{nz me Name Modeled Reaction P}

_{2.4 2.6.1.19 ^-alanine aminotransferase} ^{^-alanine + 2-oxoglutarate <=> malonate} s_{emialdehyde + L-glutamate}

A GEM incorporating the additional reactions and constraints set forth above was analyzed in silico using stoichiometric Flux Balance Analysis to assess uptake and secretion (boundary) fluxes as well as mass yield, molar yield, carbon molar yield, and overall specific rate of product formation. The results are set forth in the tables below: Overall Yield / Flux for 3-hydroxypropionate Mass yield 47.7%

Uptake Flux (boundary reactions and metabolites that are entering the model) Flux (uptake in millimole / Reaction Metabolite

Secreting Flux (boundary reactions and metabolites that are exiting the model) Flux (secretion in millimole / Reaction Metabolite

Model 10990 In one embodiment, the application provides a modified organism or GEM adapted to produce 3- hydroxypropionate via pathway 4. In one embodiment (and as shown in the table above), the organism or GEM is further adapted to incorporate the following additional metabolic reactions, denoted in the Applied Model Modifications list below. Applied Model Modifications: r_0226, r_1110, r_2111_constrained_4083, r_1992_constrained_4084 and r_1714_constrained_4140 In this embodiment, the following enzymes and reaction stoichiometries were specifically added to the Yeast 8 Saccharomyces cerevisiae GEM along with the Applied Model Modifications listed above, representing a particular embodiment of pathway 4 as “Model 10990”. Pathway EC E^{nz me Name Modeled Reaction} +

A GEM incorporating the additional reactions and constraints set forth above was analyzed in silico using stoichiometric Flux Balance Analysis to assess uptake and secretion (boundary) fluxes as well as mass yield, molar yield, carbon molar yield, and overall specific rate of product formation. The results are set forth in the tables below: Overall Yield / Flux for 3-hydroxypropionate Mass yield 31.8%

Uptake Flux (boundary reactions and metabolites that are entering the model) Flux (uptake in millimole / Reaction Metabolite

Secreting Flux (boundary reactions and metabolites that are exiting the model) Flux (secretion in millimole / Reaction Metabolite

Model 11131 In one embodiment, the application provides a modified organism or GEM adapted to produce 3- hydroxypropionate via pathway 4. In one embodiment (and as shown in the table above), the organism or GEM is further adapted to incorporate the following additional metabolic reactions, denoted in the Applied Model Modifications list below. Applied Model Modifications: r_0226, r_1110, r_2111_constrained_4083, r_1992_constrained_4084 and r_1714_constrained_4140 In this embodiment, the following enzymes and reaction stoichiometries were specifically added to the Yeast 8 Saccharomyces cerevisiae GEM along with the Applied Model Modifications listed above, representing a particular embodiment of pathway 4 as “Model 11131”. Pathway EC E^{nz me Name Modeled Reaction P de} +

A GEM incorporating the additional reactions and constraints set forth above was analyzed in silico using stoichiometric Flux Balance Analysis to assess uptake and secretion (boundary) fluxes as well as mass yield, molar yield, carbon molar yield, and overall specific rate of product formation. The results are set forth in the tables below: Overall Yield / Flux for 3-hydroxypropionate Mass yield 31.8%

Uptake Flux (boundary reactions and metabolites that are entering the model) Flux (uptake in millimole / Reaction Metabolite

Secreting Flux (boundary reactions and metabolites that are exiting the model) Flux (secretion in millimole / Reaction Metabolite

Model 11106 In one embodiment, the application provides a modified organism or GEM adapted to produce 3- hydroxypropionate via pathway 5. In one embodiment (and as shown in the table above), the organism or GEM is further adapted to incorporate the following additional metabolic reactions, denoted in the Applied Model Modifications list below. Applied Model Modifications: r_0226, r_1110, r_2111_constrained_4083, r_1992_constrained_4084 and r_1714_constrained_4140 In this embodiment, the following enzymes and reaction stoichiometries were specifically added to the Yeast 8 Saccharomyces cerevisiae GEM along with the Applied Model Modifications listed above, representing a particular embodiment of pathway 5 as “Model 11106”. Pathway EC E^{nz me Name Modeled Reaction} > e

_{5.4 1.1.1.59} ^{malonate semialdehyde} malonate semialdehyde + NADH + H+ r_{eductase (NADH)} <=> 3-hydroxypropanoate + NAD+

A GEM incorporating the additional reactions and constraints set forth above was analyzed in silico using stoichiometric Flux Balance Analysis to assess uptake and secretion (boundary) fluxes as well as mass yield, molar yield, carbon molar yield, and overall specific rate of product formation. The results are set forth in the tables below: Overall Yield / Flux for 3-hydroxypropionate Mass yield 95.4%

Uptake Flux (boundary reactions and metabolites that are entering the model) Flux (uptake in millimole / Reaction Metabolite

Secreting Flux (boundary reactions and metabolites that are exiting the model) Flux (secretion in millimole / Reaction Metabolite

Model 11140 In one embodiment, the application provides a modified organism or GEM adapted to produce 3- hydroxypropionate via pathway 5. In one embodiment (and as shown in the table above), the organism or GEM is further adapted to incorporate the following additional metabolic reactions, denoted in the Applied Model Modifications list below. Applied Model Modifications: r_0226, r_1110, r_2111_constrained_4083, r_1992_constrained_4084 and r_1714_constrained_4140 In this embodiment, the following enzymes and reaction stoichiometries were specifically added to the Yeast 8 Saccharomyces cerevisiae GEM along with the Applied Model Modifications listed above, representing a particular embodiment of pathway 5 as “Model 11140”. Pathway EC E^{nz me Name Modeled Reaction e}

_{5.4 1.1.1.298} ^{malonate semialdehyde} malonate semialdehyde + NADPH + H+ r_{eductase (NADPH)} <=> 3-hydroxypropanoate + NADP+

A GEM incorporating the additional reactions and constraints set forth above was analyzed in silico using stoichiometric Flux Balance Analysis to assess uptake and secretion (boundary) fluxes as well as mass yield, molar yield, carbon molar yield, and overall specific rate of product formation. The results are set forth in the tables below: Overall Yield / Flux for 3-hydroxypropionate Mass yield 47.7%

Uptake Flux (boundary reactions and metabolites that are entering the model) Flux (uptake in millimole / Reaction Metabolite

Secreting Flux (boundary reactions and metabolites that are exiting the model) Flux (secretion in millimole / Reaction Metabolite

Model 11308 In one embodiment, the application provides a modified organism or GEM adapted to produce 3- hydroxypropionate via pathway 6. In one embodiment (and as shown in the table above), the organism or GEM is further adapted to incorporate the following additional metabolic reactions, denoted in the Applied Model Modifications list below. Applied Model Modifications: r_0226, r_1110, r_2111_constrained_4083, r_1992_constrained_4084, r_1714_constrained_4140 and pepc_4.1.1.49_atp_4139 In this embodiment, the following enzymes and reaction stoichiometries were specifically added to the Yeast 8 Saccharomyces cerevisiae GEM along with the Applied Model Modifications listed above, representing a particular embodiment of pathway 6 as “Model 11308”. Pathway EC E^{nz me Name Modeled Reaction +} >

A GEM incorporating the additional reactions and constraints set forth above was analyzed in silico using stoichiometric Flux Balance Analysis to assess uptake and secretion (boundary) fluxes as well as mass yield, molar yield, carbon molar yield, and overall specific rate of product formation. The results are set forth in the tables below: Overall Yield / Flux for 3-hydroxypropionate Mass yield 95.4%

Uptake Flux (boundary reactions and metabolites that are entering the model) Flux (uptake in millimole / Reaction Metabolite

Secreting Flux (boundary reactions and metabolites that are exiting the model) Flux (secretion in millimole / Reaction Metabolite

Model 9648 In one embodiment, the application provides a modified organism or GEM adapted to produce 3- hydroxypropionate via pathway 3. In one embodiment (and as shown in the table above), the organism or GEM is further adapted to incorporate the following additional metabolic reactions, denoted in the Applied Model Modifications list below. Applied Model Modifications: r_0226, r_1110, r_2111_constrained_4083, r_1992_constrained_4084 and r_1714_constrained_4140 In this embodiment, the following enzymes and reaction stoichiometries were specifically added to the Yeast 8 Saccharomyces cerevisiae GEM along with the Applied Model Modifications listed above, representing a particular embodiment of pathway 3 as “Model 9648”. Pathway EC E^{nz me Name Modeled Reaction} P

hydroxypropionyl-CoA 3^{h dr x r i n lC A} 3hdrx r inlCA + PPi + AMP

A GEM incorporating the additional reactions and constraints set forth above was analyzed in silico using stoichiometric Flux Balance Analysis to assess uptake and secretion (boundary) fluxes as well as mass yield, molar yield, carbon molar yield, and overall specific rate of product formation. The results are set forth in the tables below: Overall Yield / Flux for 3-hydroxypropionate Mass yield 95.4%

Uptake Flux (boundary reactions and metabolites that are entering the model) Flux (uptake in millimole / Reaction Metabolite

Secreting Flux (boundary reactions and metabolites that are exiting the model) Flux (secretion in millimole / Reaction Metabolite

The foregoing description of preferred embodiments has been presented for purposes of illustration and description only. It is not intended to be exhaustive or to limit the application to the precise form disclosed, and modifications and variations are possible and/or would be apparent in light of the above teachings or may be acquired from practice of the application. The embodiments were chosen and described in order to explain the principles of the application and its practical application to enable one skilled in the art to utilize the application in various embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the application be defined by the claims appended hereto. Additional Host Cell Modifications In all of the foregoing GEMs, pathways, and preferred embodiments, the export of 3- hydroxypropionate from the intracellular space to the extracellular matrix relies on an ATP- independent mechanism of transport. The inventors have identified that in some preferred physical embodiments of the pathways disclosed herein, additional ATP may be expended by the cell to maintain a high extracellular concentration of the protonated acid form (3-hydroxypropionic acid), particularly when the extracellular medium is maintained at a pH well below the pKa of the acid. This ATP cost may be related to 3-hydroxypropionate export, maintenance of pH homeostasis via membrane-bound proton-pumping ATP hydrolyzing enzymes (H⁺ ATPases), or increased cellular growth and maintenance energy costs. Thus, another aspect of the present application is to provide approaches for increasing ATP conservation of a fungal (yeast) cell, in particular a fungal cell harboring the biosynthetic pathways discussed herein. In some embodiments, the application provides an approach which conserves energy (ATP) by identifying an H⁺ ATPase with increased proton:ATP ratios in vivo. Thus, in some embodiments, the fungal cell contains an H⁺ ATPase with increased proton:ATP ratios, for example 2:1 or higher (2:1, 3:1, etc.). The typical P-type ATPase that fungal species use to maintain pH homeostasis pumps protons at a ratio of 1 proton per ATP hydrolyzed. The proposed invention will identify H⁺ ATPases that pump protons out of the cell at a ratio of two, three, or more protons pumped per ATP hydrolyzed. Thus, the present application also provides approaches for identifying and incorporating certain H⁺ ATPase into fungal cells. In a preferred embodiment, the approach includes identifying natural and engineered genetic sources of proton pumps having (or potentially having) an elevated proton:ATP ratio. The natural sources may include both public and proprietary metagenomic databases. P-type ATPases, which exist in the plasma membrane and maintain cellular pH homeostasis, may be preferred candidates for incorporating into fungus such as yeast based on the natural presence of P-type ATPases in these organisms. The approach also provides that putative transporters can be computationally screened and scored based on a variety of criteria, including evidence of proton:ATP ratio, predicted or actual 3D structure, and localization. Engineered sources may include mutagenesis of natural P-type ATPases either via random mutation or rational selection of residues for mutation based on predicted or actual 3D structure. The approach also provides the highest scoring natural and engineered candidates may be codon optimized, barcoded, and synthesized for expression in a fungal host. The approach also provides for concurrent development of fungal strains, for example homo-fermentative lactic and 3-hydroxypropionic acid-producing yeast strains that do not produce ethanol. Once developed, the approach provides for expression into these fungal strains of a library of potentially improved H⁺ ATPases defined through the initial computational metagenomic searches while simultaneously removing the gene(s) encoding the endogenous H⁺ ATPase(s) from the host genome (e.g., PMA1 in Saccharomyces cerevisiae) through standard genetic engineering practices. Proton pumping is an essential function for survival, thus cells that survive this transformation protocol can be sequenced to identify functional proton pumps. Finally, this library of functional H⁺ ATPases expressed in either a lactic-acid or 3-hydroxypropionic acid strain may be grown in an anaerobic chemostat at pH ~3 under carbon limited conditions with oxygen supply slowly decreased over a time course. Strains that express an H⁺ ATPase with a proton:ATP ratio of 2 or higher will have a selective advantage at lower oxygen transfer rates. At the end of the chemostat run, barcodes of surviving strains may be sequenced for identification. This approach may also be combined with a screen of potential lactate or 3-hydroxypropionate ATP-independent anion transporters. Ultimately the in vivo selection approach detailed herein can be used to find H⁺ ATPases that pump protons at a ratio of 2 or greater protons per ATP hydrolyzed. This enables fungal cells to grow and produce 3-hydroxypropionic acid in a low pH extracellular environment in the absence of oxygen using the pathways and embodiments described above.

EXAMPLES Example 1 -- Bioproduction of 3HP in Yeast Based on embodiments described herein, a set of yeast strains were constructed to demonstrate initial 3-HP production under both aerobic and anaerobic conditions. As demonstrated below, several strains exhibited significant 3-HP production. Reagents and Media Used Standard yeast extract, peptone, dextrose growth media (YPD) was prepared as follows. 10 grams (g) yeast extract, 20 g peptone, was dissolved in 900 ml water and autoclaved to sterilize. To this 900 ml, 100 ml sterile 20% glucose was added. YPE (yeast extract, peptone, ethanol) was prepared similarly, dissolving 10 g yeast extract and 20 g peptone in 900 ml water and autoclaved for sterilization. To this solution, 100 ml sterile 20% ethanol was added. For YPDS (yeast extract, peptone, dextrose, sorbitol), 10 g yeast extract, 20 g peptone, and 182.2 g sorbitol were dissolved in 900 ml water and sterilized by autoclave. To this solution, 100 ml sterile 20% dextrose was added. Standard lithium acetate tris EDTA (Li-Acetate-TE) buffer for yeast transformation was prepared to final concentrations of 0.1 M lithium acetate, 10 mM Tris-HCl pH 7.5, 1 mM EDTA and filter sterilized through a 0.22 μ filter.50% Polyethylene glycol [PEG] 3350 (w/v) for yeast transformation was prepared by weighing the PEG and dissolving in the appropriate amount of water. The viscous solution is heated briefly to dissolve and filter sterilized while hot.50% DMSO (v/v in water) was sterilized through a 0.22 μ filter. DNA Assembly Techniques In one embodiment, DNA vectors (plasmids) for expressing the 3-HP pathway were designed to encode up to four heterologous pathway genes using yeast promoters and terminators. In a preferred embodiment, heterologous genes were codon optimized for expression in Saccharomyces cerevisiae and synthesized by a commercial DNA synthesis manufacturer. The synthesized linear DNA fragments, along with Saccharomyces cerevisiae DNA fragments amplified from yeast genomic DNA encoding promoter and terminator elements, were amplified via PCR for assembly into the final DNA vector. Plasmid backbones containing a yeast antibiotic marker (KanR, HygR, or NatR), an E. coli antibiotic marker (AmpR), a yeast 2µ origin of replication, and an E. coli pBR322 origin of replication were also PCR amplified to generate a linear fragment for assembly. All fragments were amplified with 20-50 base pair overlaps with adjacent DNA fragments on each end to provide homologous regions for recombination. Expression vectors were assembled leveraging homologous recombination by transforming a Saccharomyces cerevisiae strain with a mixture of the overlapping DNA fragments and selecting for the antibiotic resistance marker encoded on the plasmid backbone. Yeast transformation was performed using standard lithium-acetate (chemical transformation) method. Briefly, a fresh colony was inoculated into 3 mL of YPD and incubated overnight at 30 °C. The following day, 50 mL of pre-warmed YPD was inoculated to an initial optical density at 600 nm (OD600) of 0.02- 0.04 and grown at 30 °C until the OD600 reached 0.4. Cells from this culture are harvested, washed with sterile water, and then resuspended in water. The cells are then centrifuged and resuspended in Lithium-Acetate-TE Buffer, followed by another centrifugation and resuspension in a final volume of 250 µL Li-Acetate-TE Buffer for transformation. A mixture of 50 µL cells, 10 µL PCR DNA, and 5 µL boiled salmon sperm DNA is prepared, to which 120 µL fresh 50% PEG 3350 and 160 µL 10 M Lithium Acetate is added and the mixture is vortexed. After a 60-minute incubation at 30 °C, 40 µL 50% DMSO is added followed by heat shock at 42 °C for 45 minutes. The cells are then centrifuged, washed with sterile water, and resuspended in YPD medium with antibiotics. After overnight incubation at 30 °C and 250 rpm shaking speed, assembled plasmids were extracted using a commercially available yeast miniprep kit (Zymochem Yeast Miniprep Kit), and transformed into E. coli using a standard chemical transformation method for further amplification and clonal purification via standard miniprep procedures. Purified plasmids were sequenced using Illumina next generation sequencing and SeqWell multiplexing library kits and sequence-correct clones were selected after aligning the sequencing reads vs. the expected reference plasmid sequence. Strain Construction Plasmids with antibiotic resistance markers and containing the 3-HP pathway genes were transformed into Saccharomyces cerevisiae strains previously engineered to contain gal80Δ. In this embodiment, yeast galactose-inducible promoters are no longer repressible, and these promoter elements are considered constitutively expressed (e.g. GAL1, GAL10, GAL7, GAL2 promoters). In some embodiments, ethanol production is decreased by deletion of the native pyruvate PDC1 (pdc1Δ) and may also contain deletions of native alcohol dehydrogenase (e.g. adh1Δ and/or adh5Δ). These deletions were created as complete open-reading frame deletions using standard methods. Transformation of these strains, in some embodiments gal80Δ, in another embodiment gal80Δ, pdc1Δ, in a further embodiment gal80Δ, pdc1Δ adh1Δ, adh5Δ was performed using standard chemical or electroporation transformation methods. Chemical transformation was performed as described above. The yeast electroporation protocol begins by preparing a pre- growth culture, where 2 ml of YPE medium is inoculated with 100 µL of the relevant S. cerevisiae strain glycerol stock and incubated at 30 °C (250 rpm) for 20 hours. For culturing and preparing the competent cells, 100 mL YPE medium is inoculated with the pre-growth culture and incubated under similar conditions, selecting cultures with an OD600 between 5-10 for further processing. After incubation, cells are transferred, centrifuged, and washed with sterile water before combining the pellets from two centrifuge tubes into one. These cells are then resuspended in Lithium- Acetate-TE Buffer and incubated at room temperature for 60 minutes. Following this, the cells are centrifuged again, washed thrice (the second and third wash being with 1 M sorbitol), and finally resuspended in 1 M Sorbitol to achieve an OD600=100. Prior to electroporation, the cells may be kept at 4 °C for up to 3 hours. A high throughput electroporation cuvette with a 0.2 cm electrode gap is precooled on ice. To this, 1500-2000 ng of DNA is added, followed by 80 µL of the competent cells. The cuvette is incubated on ice for 5 minutes, then electroporation is executed on a high throughput electroporation device (in one embodiment, the BTX ECM630 device with the BTX HT100 plate handler accessory). The electroporation parameters are set to a voltage of 1.5 kV, capacitance of 25 μF, and resistance of 200 Ohms. After electroporation, the reaction volume is immediately transferred to a 96-well culture plate prepared with 150 μL of YPDS per well. The cells are then allowed to regenerate at 30 °C without shaking and are pelleted by centrifugation, washed in sterile water, and resuspended in sterile water. The cells are then spread onto selective agar plates and incubated for a period of 96 hours at 30 °C. In some preferred embodiments genes are integrated into the yeast genome by adding homology to a target integration locus by amplifying the 3-HP pathway expression cassettes using PCR with primers encoding the homology regions. In some embodiments, the integration is into a neutral locus known to enable good transcriptional expression, and in other embodiments the target integration locus may include deletion of a native gene to limit byproduct formation (e.g. ethanol via knockout of PDC1 or ADH1 or ADH5 as described above). A combination of these approaches may also be used. Transformation is performed as described above. Successful integrations may be confirmed via PCR amplification of diagnostic regions of the integration and/or native deletion, and in some embodiments may be confirmed by whole-genome sequencing. Strain Screening for 3-HP Production and By-product formation To screen strains for 3-HP production, a high-throughput, facultative anaerobic microtiter plate screen was developed. Colonies transformed with plasmid DNA expression vectors and/or expression cassettes integrated into the genome were picked into microtiter plates containing YPD medium and appropriate antibiotics. This pre-culture microtiter plate was sealed with a breathable membrane seal and grown in a shaking incubator at 30 °C for 24 hours. Once grown, 60 µL of culture from the pre-culture plate was transferred to a new 2 mL production microtiter plate containing 540 µL YPD medium with 40 g/L dextrose. The plate was sealed with an aluminum heat seal. The sealed plates were further sealed using a rubber mat and clamp system, in a preferred embodiment using the Kuhner Duetz-System plate clamps. The plates were incubated with minimal shaking at 30 °C for 72 hours. The volume of the air in the wells and amount of glucose in the medium ensure that oxygen was consumed rapidly in the first few hours of the incubation and in the remaining time the cells are under substantially anaerobic conditions. This is intended to mimic the final conditions at commercial production scale in a typical fuel ethanol facility, where yeast growth is achieved aerobically and oxygen is consumed in the early stages of production fermentation, the culture being kept in an substantially anaerobic state by low-speed mixing, lack of oxygen or air injection into the bioreactor, and the metabolism of the organism itself. For comparison, aerobic cultures were prepared by creating duplicate production microtiter plates from the same aerobic pre-culture plate, which were sealed with a breathable membrane instead of the foil seal and clamp system. Aerobic plates were placed in a shaking incubator at 30 °C for 72 hours. After cells were cultured in production plates, the culture was assayed for 3-HP, ^-alanine, aspartate, pyruvate, glucose, ethanol, glycerol, and acetate concentration. Additionally, the optical density (OD 600nm) of the culture was measured. Analytical Methods 3-HP, ^-alanine, aspartate, and pyruvate were assayed by LC-MS. Culture samples are diluted 4-fold in an extraction buffer (40% methanol, 40% acetonitrile, 20% water, with 5 mM ammonium acetate, pH=9) containing 1 mg/L of the appropriate heavy isotopic standards. Extractions were shaken at 1000 rpm at room temperature for 20 minutes, centrifuged to pellet the cell debris, and supernatant was transferred to a new plate. Analysis of the extracted samples was performed on a Waters I-class UPLC system with a Xevo quadrupole-orbitrap mass detector and a Waters BEH Amide column (2.1x100 mm, 2.5 µm packing - SKU: 186006091) using the following mobile phases: C = 5 mM ammonium acetate in water, pH=9; D = 5 mM ammonium acetate in 90% acetonitrile and 10% water, pH=9. The elution gradient was 0.5 mL/min of 100% D for three minutes, followed by a linear ramp from 100% to 65% D in C over four minutes. The column was then flushed with 35% D in C for one minute, and re-equilibrated with 100% D for three minutes. Column effluent is introduced into the mass spectrometer via a standard ESI source with negative mode ionization, vaporizer temperature of 400 °C, and ion transfer tube temperature of 375 °C. Glucose, ethanol, glycerol, and acetate were measured by HPLC with refractive index (RI) detection. Supernatant was sampled from culture broth following centrifugation of the production plates. The column used was the BioRad Aminex 87H 300x7.8 mm, 9 µm. Eluent was 5 mM H2SO4 in water and was run in isocratic mode. Flow rate was 0.6 mL/minute with a column temperature of 65 °C. Enzymes and Expression Cassette Designs Based on embodiments of the in silico designs established above, complete pathways for Pathway 1 and Pathway 2 were assembled using selected enzymes described in Table 3. Although specific enzymes were selected, based on this application, a person of ordinary skill in the art would understand that the other enzymes described herein may also be used in the pathways discussed. Note that some elements of Pathways 4, 5, and 6 were also tested in the following examples. Table 3. Enzymes Tested Gene Species Origin EC # Pathway Step(s) Sequence ID(s)

Gene Species Origin EC # Pathway Step(s) Sequence ID(s) ed

on the methods described above. The complete set of plasmids used in the subsequent examples showing aerobic and facultatively anaerobic production of 3-HP are described in Table 4. These DNA constructs below represent a single codon optimization for each enzyme, in other embodiments alternative DNA codon optimizations can be used and would accomplish similar or sometimes increased levels of expression and 3-HP production performance. Table 4. DNA Vectors for Gene Overexpression DNA ID Plasmid backbone Expression cassettes 1

DNA ID Plasmid backbone Expression cassettes 2 east oriin H R east antibiotic TDH3>AtAAT2 tJEN1 tHMG1 1

DNA ID Plasmid backbone Expression cassettes E coli BR322 oriin of relication tTPI1 T, T, O O

DNA ID Plasmid backbone Expression cassettes resistance E coli antibiotic marker Am R TDH3>TcPAND* tPGK1 tHMG1, O O

Production of 3-HP using Pathway 1 and Pathway 2 Using the methods and DNA constructs described above, a set of strains were constructed to demonstrate initial 3-HP production under both aerobic and anaerobic conditions. Two plasmids containing the upstream and downstream portions of Pathway 2 were used to transform a haploid and prototrophic background of Saccharomyces cerevisiae strain isolate (CEN.PK 113-7D) that already contained four complete open reading frame deletions to enable constitutive expression of galactose-inducible promoters (gal80Δ) and to reduce the formation of ethanol and drive re- oxidation of reducing equivalents through the 3-HP pathway (pdc1Δ, adh1Δ, adh5Δ). Strain identifiers and the associated plasmids used for transformation are described in Table 5. Data from the anaerobic and aerobic production of 3-HP were collected and assayed using the plate model and analytical methods described above, and are shown in FIG.1. Table 5. Strain Genotypes for Production of 3-HP using Pathway 1 and Pathway 2 Strain ID Parent Strain Genotype Y2 CEN.PK gal80Δ Y7 Y2 gal80Δ pdc1Δ adh1Δ adh5Δ Y34 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D235, Plasmid D250 Y35 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D236, Plasmid D251 Y36 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D237, Plasmid D250 Y37 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D238, Plasmid D252 Y38 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D239, Plasmid D253 Y39 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D238, Plasmid D250 Y40 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D240, Plasmid D254 Y41 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D237, Plasmid D255 Y42 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D236, Plasmid D253 Y43 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D241, Plasmid D256 Y44 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D242, Plasmid D253 Y45 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D243, Plasmid D254 Y46 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D244, Plasmid D256 Y47 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D241, Plasmid D251 Y48 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D245, Plasmid D252 Y49 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D246, Plasmid D255 Y50 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D247, Plasmid D257

Y51 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D239, Plasmid D251 Y52 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D240, Plasmid D257 Y53 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D248, Plasmid D255 Y54 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D238, Plasmid D251 Y55 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D248, Plasmid D251 Y56 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D249, Plasmid D251 Y57 Y7 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D240, Plasmid D251 gal80Δ pdc1Δ adh1Δ adh5Δ, Plasmid D310, Plasmid D309 Y131 Y7 (control) The data in FIG.1 demonstrate that strains containing some combination of overexpression of a subset of the tested enzymes successfully produce 3-HP in an aerobic plate model for Pathways 1 and 2. Functional enzymes for each step are as follows: Step 2.1 (Cg_PYC, ScPYC1, Re_PYC), Step 1.1 (Ec_PPC), Steps 1.2 and 2.2 (AtAAT2 or ScAAT2), Steps 1.3 and 2.3 (TcPAND), Steps 1.4 and 2.4 (BcBAPAT or UmBOT), and Steps 1.5 and 2.5 (EcYDFG, MsHPDH, or CaHIBADH). The highest titers achieved include strains Y39, Y51, and Y56, which use a combination of ScPYC1 or Ec_PPC, ScAAT2 or AtAAT, TcPAND, BcBAPAT or UmBOT, EcYDFG. These data support 3-HP production from either can be produced using either Pathway 1 or Pathway 2 under aerobic conditions. The data in FIG.2 demonstrate that strains containing some combination of overexpression of a subset of the tested enzymes produce 3-HP in an anaerobic plate model also for Pathway 1 and Pathway 2. Background 3-HP production is observed in this experiment (up to 4 mg/L), which could be attributed to the fact that ^-alanine is also metabolite in the spermine pathway, and likely the native GABA aminotransferase Uga1 is deaminating ^-alanine to MSA which is converted to 3-HP by an unknown MSA reductase. Only strains Y56 shows statistically significant evidence of anaerobic 3-HP production (Y51 and Y39 trend higher than controls as well). As shown above, these are also the best-producing strains aerobically, and thus also use a combination of ScPYC1 (Pathway 2) or Ec_PPC (Pathway 1), ScAAT2 or AtAAT, TcPAND, BcBAPAT or UmBOT, EcYDFG. These data support 3-HP production from either Pathway 1 or Pathway 2 under anaerobic conditions. Table 6. Strain Genotypes for Production of 3-HP using Pathway 1 and containing PEPCK Strain ID Parent Strain Genotype Description

Strain ID Parent Strain Genotype Description Y257 Y222 Y222 + Plasmid D307 nal

heterologous gene overexpression, 3 of the upstream pathway genes (AtAAT2, Ec_PPC, and TcPAND) were integrated into Y2 (CEN.PK with gal80Δ) using methods described above. In this case, the integration simultaneously deleted the native PDC1. This strain (Y222) was then transformed with a plasmid that contained additional heterologous genes encoding the downstream portion of the pathway including ^-alanine aminotransferase activity encoded by UmBOT, malonate semialdehyde reductase activity encoded by either EcYDFG, CaHIBADH, or two different N-terminal signal sequence cleavage variants (CaHIBADH_v3 or _v4). Some plasmids also encoded additional overexpression of a PEPCK (EC # 4.1.1.49) from E. coli. Some plasmids also encoded additional overexpression of the aspartate decarboxylase, TcPAND. Strain ID’s are shown in Table 6, and plasmid details are shown in Table 4. The data in FIG.3 demonstrate the production of up to 1.6 g/L of 3-HP in an aerobic plate model. These data represent additional supporting evidence of Pathway 1 functionality. Step 1.1 (Ec_PPC), Step 1.2 (AtAAT2), and Step 1.3 (TcPAND) were all encoded in a single copy in the strains in FIG.3 in the chromosomal pdc1Δ integration as described above. An additional copy of TcPAND was overexpressed in some strains (Y244 through Y258) on the episomal vector. Steps 1.4 (encoded by UmBOT) was expressed on the episomal vector as well, and was included in all strains except the parent (Y222) and the empty vector control (Y261). The terminal malonate semialdehyde reductase was also included on the vector for all strains except the parent and control strains (encoded by EcYDFG, CaHIBADH, or the variants thereof). Three strains (Y255, Y257, Y258) also encoded overexpression of the E. coli PEPCK (phosphoenolpyruvate [PEP] carboxykinase) to increase expression and ATP availability during conversion of PEP to oxaloacetate. The highest titers achieved were with strain Y259, which did not contain additional TcPAND overexpression. In fact, comparisons of Y244 to Y259, where the only difference was additional TcPAND overexpression, suggest in this context there is no improvement from additional aspartate decarboxylase activity. Comparisons between Y245/ Y247/Y248 and Y255/Y257/Y258 clearly show the benefit of EcPEPCK overexpression. EcYDFG continues to be the most active terminal reductase, however all of the terminal reductases tested support 3-HP production. These data provide additional support for 3-HP production using Pathway 1 under aerobic conditions, and also provide support for the use of a phosphoenolpyruvate carboxykinase (EcPEPCK) to improve 3-HP production. The data in FIG.4 demonstrate the production of up to 350 mg/L of 3-HP in an anaerobic plate model, as described in the methods above. These data represent specific supporting evidence of Pathway 1 functionality under substantially anaerobic or facultative anaerobic fermentation conditions. As these are the same strains in FIG. 3, Step 1.1 (Ec_PPC), Step 1.2 (AtAAT2), and Step 1.3 (TcPAND) are all also encoded in a single copy in the chromosomal pdc1Δ integration. An additional copy of TcPAND was overexpressed in some strains (Y244 through Y258) on the episomal vector. Steps 1.4 (encoded by UmBOT) was expressed on the episomal vector as well, and was included in all strains except the parent (Y222) and the empty vector control (Y261). The terminal malonate semialdehyde reductase was also included on the vector for all strains except the parent and control strains (encoded by EcYDFG, CaHIBADH, or the variants thereof). Three strains (Y255, Y257, Y258) also encoded overexpression of the E. coli PEPCK (phosphoenolpyruvate [PEP] carboxykinase) to increase expression and ATP availability during conversion of PEP to oxaloacetate. The highest titers achieved anaerobically were also from the strain that was the highest aerobically (Y259), which did not contain additional TcPAND overexpression. Again, comparisons of Y244 to Y259, where the only difference was additional TcPAND overexpression, suggest in this context there is no improvement from additional aspartate decarboxylase activity. Comparisons between Y245/ Y247/Y248 and Y255/Y257/Y258 clearly show the benefit of EcPEPCK overexpression. EcYDFG continues to be the most active terminal reductase, however all of the terminal reductases tested support 3-HP production. These data provide additional support for 3-HP production using Pathway 1 under anaerobic conditions, and also provide support for the use of a phosphoenolpyruvate carboxykinase (EcPEPCK) to improve 3-HP production anaerobically. Table 7. Strain Genotypes for Production of 3-HP using Pathway 1 and containing PEPCK Strain ID Parent Strain Genotype Y7 CENPK gal80Δ pdc1Δ adh1Δ adh5Δ nal

heterologous gene overexpression in an alternative genetic context, 3 of the upstream pathway genes (AtAAT2, Ec_PPC, and TcPAND) were integrated into Y7 (CEN.PK with gal80Δ pdc1Δ adh1Δ adh5Δ) using methods described above. In this case, the DNA construct encoding the upstream pathway was integrated into a “neutral” or non-coding locus (denoted 1309a in Table 7). This strain (Y227) was then transformed with a similar set of plasmids used to create the strains in Table 6. These contained additional heterologous genes encoding the downstream portion of the pathway including UmBOT ( ^-alanine aminotransferase), malonate semialdehyde reductase activity encoded by either EcYDFG, CaHIBADH, or two different N-terminal signal sequence cleavage variants (CaHIBADH_v3 or _v4). Again, some plasmids also encoded additional overexpression of a PEPCK (EC # 4.1.1.49) from E. coli. Some plasmids also encoded additional overexpression of the aspartate decarboxylase, TcPAND. Strain ID’s are shown in Table 7, and plasmid details are shown in Table 4. The data in FIG.5 demonstrate production of up to 1.5 g/L 3-HP in an aerobic plate model in an alternative genetic context. These data further support evidence of Pathway 1 functionality and use of the E. coli PEPCK to improve 3-HP titer. Step 1.1 (Ec_PPC), Step 1.2 (AtAAT2), and Step 1.3 (TcPAND) were all encoded in a single copy in the strains in FIG.5 in the chromosomal non-coding 1309a locus integration as described above. An additional copy of TcPAND was overexpressed in some strains (Y262 through Y276) on the episomal vector. Step 1.4 (encoded by UmBOT) was expressed on the episomal vector as well, and was included in all strains except the parent (Y227) and the empty vector control (Y279). The terminal malonate semialdehyde reductase was also included on the vector for all strains except the parent and control strains (encoded by EcYDFG, CaHIBADH, or the variants thereof). Three strains (Y273, Y275, Y276) also encoded overexpression of the E. coli PEPCK (phosphoenolpyruvate [PEP] carboxykinase) to increase expression and ATP availability during conversion of PEP to oxaloacetate. The highest titer was achieved in Y277. Comparisons between Y263/Y273 and Y266/Y276 clearly show the benefit of EcPEPCK overexpression. The data in FIG. 6 provide further evidence for anaerobic production of 3-HP from Pathway 1. Strain expressing the terminal reductase enzymes EcYDFG, CaHIBADH, CaHIBADH_v4 all demonstrated production in this experiment. Strains expressing the extra TcPAND produced less 3-HP. Comparison of strains Y263/Y273 and Y266/Y276 further demonstrate the benefit of expressing EcPEPCK to increase 3-HP titer. The highest producing strain is Y277, which produces 399 g/L anaerobically. Together, these data further support anaerobic production of 3-HP from Pathway 1 and the benefit of expressing EcPEPCK for increasing 3-HP titer. These data also demonstrate that beginning to remove the ability of yeast to reoxidize NADH via the ethanol pathway through deletion of PDC1 (native pyruvate decarboxylase) and ADH1 and ADH5 (native alcohol dehydrogenase) starts to improve oxidation of redox cofactors through the 3-HP pathway. Compared to a strain that just contains pdc1Δ (Y259 and Y260 in FIG.4) with strain that contains pdc1Δ and adh1Δ, adh5Δ deletions (e.g., strains Y277 and Y278 in FIG. 6) we can see an improvement in anaerobic 3-HP titer, and less difference between the aerobic and anaerobic titers. This forcing of redox cofactor re-oxidation through the terminal reduction of MSA to 3-HP could be further driven by complete pyruvate decarboxylase gene knockouts (pdc1Δ, pdc5Δ, pdc6Δ in Saccharomyces) and/or deletion of all native alcohol dehydrogenase genes (e.g., adh1Δ, adh2Δ, adh3Δ, adh4Δ, adh5Δ, adh6Δ). The replacement of NADH oxidation via ethanol with the 3-HP product pathway is ultimately important for anaerobic production, and it follows that the 3-HP pathway should ultimately use NADH rather than NADPH for its electron carrier.

Claims

CLAIMS 1. A method for bioproducing 3-hydroxypropionate comprising: a) providing a recombinant microorganism; and b) providing feedstock-derived substrate to the recombinant microorganism wherein the recombinant microorganism bioproduces 3-hydroxypropionate.

2. The method of claim 1, wherein the recombinant organism is a yeast.

3. The method of claims 1-2, wherein the recombinant organism is derived from a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Pichia, Hansenula, Issatchenkia, Candida, Zygosaccharomyces, Debaryomyces, Dekkera (Brettanomyces), or Schizosaccharomyces. 4. The method of claims 1-3, wherein the recombinant microorganism bioproduces 3- hydroxypropionic acid under facultatively or substantially anaerobic conditions via at least one of the following metabolic pathways: a) Reacting the metabolite phosphoenolpyruvate using an enzyme from the class 4.1.1.X to obtain oxaloacetate, then subsequently reacting, in order, oxaloacetate using an enzyme from the class 2.6.1.X to L-aspartate, L-aspartate using an enzyme from the class 4.1.1.X to ^-alanine, ^-alanine using an enzyme from the class 2.6.1.X to malonate semialdehyde, malonate semialdehyde using an enzyme from the class 1.1.1.X to 3-hydroxypropionate; and b) Reacting the metabolite pyruvate using an enzyme from the class 6.

4.1.X to obtain oxaloacetate, then subsequently reacting, in order, oxaloacetate using an enzyme from the class 2.6.1.X to L-aspartate, L-aspartate using an enzyme from the class 4.1.1.X to ^-alanine, ^-alanine using an enzyme from the class 2.6.1.X to malonate semialdehyde, malonate semialdehyde using an enzyme from the class 1.1.1.X to 3-hydroxypropionate

5. The method of claim 4, wherein the enzyme classes are encoded by one of the following sequence ID NOS.: wherein class 4.1.1.X is encoded by SEQ ID NOS.1-2, 8-9, 18-56, 159-187, 189- 193, 384-391, or 393-400, wherein class 2.6.1.X is encoded by SEQ ID NOS. 6-7, 10-11, 57-123, 142-158, 194-213, 228-238, or 383, wherein class 1.1.1.X is encoded by SEQ ID NOS.12-17, 214, 216-227, 243, 248, 265, 269-276, 278-279, 284-310, or 320-358, and wherein class 6.4.1.X is encoded by SEQ ID NOS.3-5, 188, 215, 392, or 401-404. 6. The method of claims 3-5, wherein the recombinant microorganism comprises at least one of the following additional metabolic modifications for bioproduction under facultatively or substantially anaerobic conditions: an addition of heterologous genes encoding one or more enzymes that catalyze a reaction selected from the group consisting of: i) diphosphate + D-fructose 6-phosphate <=> phosphate + D-fructose 1,

6- bisphosphate + H+, ii) acetoacetyl-CoA + NADPH + H+ <=> (S)-3-hydroxybutanoyl-CoA + NADP(+), iii) acetoacetyl-CoA + NADH + H+ <=> 3-hydroxybutanoyl-CoA + NAD, iv) 3-hydroxybutanoyl-CoA <=> crotonoyl-CoA + H2O, v) crotonoyl-CoA + NADH + H+ <=> NAD+ + butyryl-CoA, vi) crotonoyl-CoA + NADPH + H+ <=> NADP(+) + butyryl-CoA, vii) butyryl-CoA + NADH + H+ <=> NAD+ + butyraldehyde + CoA, viii) butyraldehyde + NADH + H+ <=> NAD+ + n-butanol, ix) ADP + phosphoenolpyruvate + carbon dioxide <=> ATP + oxaloacetate, x) D-fructose 6-phosphate + phosphate <=> acetyl-phosphate + D-erythrose 4-phosphate + H2O, xi) D-xylulose 5-phosphate + phosphate <=> acetyl-phosphate + D- glyceraldehyde 3-phosphate + H2O, xii) coenzyme A + acetyl-phosphate <=> acetyl-CoA + phosphate, and xiii) acetaldehyde + coenzyme A + NAD+ <=> acetyl-CoA + NADH + H+ b) a knockout of genes encoding enzymes catalyzing a reaction selected from the group consisting of: i) ATP + formate + THF <=> 10-formyl-THF + ADP + phosphate ii) acetaldehyde + NADH + H+ <=> ethanol + NAD+ iii) pyruvate <=> acetaldehyde + CO2

7. The method of claims 3-6, wherein the recombinant organism bioproduces 3- hydroxypropionic acid under facultatively or substantially anaerobic conditions via the heterologous expression of an enzyme selected from the group consisting of: a) a phosphoenolpyruvate carboxykinase (EC 4.1.1.49) encoded by SEQ ID NO.2 or a phosphoenolpyruvate carboxylase (EC 4.1.1.31) encoded by SEQ ID NO.1 or a pyruvate carboxylase (EC 6.4.1.1) encoded by SEQ ID NOS.3, 4, or 5; b) an aspartate aminotransferase (EC 2.6.1.1) encoded by SEQ ID NOS.6 or 7; c) an aspartate decarboxylase (EC 4.1.1.11) encoded by SEQ ID NOS.8 or 9; d) a ^-alanine aminotransferase (EC 2.6.1.18 or EC 2.6.1.19) encoded by SEQ ID NOS.10 or 11; and e) a malonate semialdehyde reductase (EC 1.1.1.298 or 1.1.1.59) encoded by one of SEQ ID NOS.12-17.

8. The method of claim 7, wherein the recombinant organism bioproduces 3- hydroxypropionic acid under facultatively or substantially anaerobic condition using a malonate semialdehyde reductase that utilizes NADH as the electron donor (redox cofactor).

9. The method of claim 7, wherein the recombinant organism bioproduces 3- hydroxypropionic acid under facultatively or substantially anaerobic condition using a malonate semialdehyde reductase that utilizes NADPH as the electron donor (redox cofactor).

10. The method of claims 7-9, wherein the recombinant organism bioproduces 3- hydroxypropionic acid under facultatively or substantially anaerobic condition using a ^- alanine aminotransferase of EC 2.6.1.19 ( ^-alanine-oxoglutarate transaminase) that uses 2- oxoglutarate as the amino acceptor, producing malonate semialdehyde and L-glutamate. 11. The method of claim 10, where the ^-alanine aminotransferase is encoded by SEQ ID NO.

11.

12. A recombinant organism comprising a pathway for bioproducing 3-hydroxypropionic acid via anaerobic, substantially anaerobic, or facultatively anaerobic fermentation.

13. The recombinant organism of claim 12, wherein the organism is a yeast.

14. The recombinant organism of claims 12-13, wherein the organism is derived from a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Pichia, Hansenula, Issatchenkia, Candida, Zygosaccharomyces, Debaryomyces, Dekkera (Brettanomyces), or Schizosaccharomyces.

15. The recombinant organism of claims 12-14, wherein the organism comprises at least one of the following metabolic pathways: a) reacting the metabolite phosphoenolpyruvate using an enzyme from the class 4.1.1.X to obtain oxaloacetate, then subsequently reacting, in order, oxaloacetate using an enzyme from the class 2.6.1.X to L-aspartate, L-aspartate using an enzyme from the class 4.1.1.X to ^-alanine, ^-alanine using an enzyme from the class 2.6.1.X to malonate semialdehyde, malonate semialdehyde using an enzyme from the class 1.1.1.X to 3-hydroxypropionate; and b) reacting the metabolite pyruvate using an enzyme from the class 6.4.1.X to obtain oxaloacetate, then subsequently reacting, in order, oxaloacetate using an enzyme from the class 2.6.1.X to L-aspartate, L-aspartate using an enzyme from the class 4.1.1.X to ^-alanine, ^-alanine using an enzyme from the class 2.6.1.X to malonate semialdehyde, malonate semialdehyde using an enzyme from the class 1.1.1.X to 3-hydroxypropionate.

16. The recombinant organism of claim 15, wherein the enzyme classes of claim 15 are encoded by at least one of the following sequence ID’s: wherein class 4.1.1.X is encoded by SEQ ID NOS.1-2, 8-9, 18-56, 159-187, 189- 193, 384-391, or 393-400, wherein class 2.6.1.X is encoded by SEQ ID NOS. 6-7, 10-11, 57-123, 142-158, 194-213, 228-238, or 383, wherein class 1.1.1.X is encoded by SEQ ID NOS.12-17, 214, 216-227, 243, 248, 265, 269-276, 278-279, 284-310, or 320-358, and wherein class 6.4.1.X is encoded by SEQ ID NOS.3-5, 188, 215, 392, or 401-404.

17. The recombinant organism of claims 12-16, wherein the recombinant organism comprises a nucleic acid sequence for the heterologous expression of an enzyme selected from the group consisting of the following enzymes: a) a phosphoenolpyruvate carboxykinase (EC 4.1.1.49) encoded by SEQ ID NO.2 or a phosphoenolpyruvate carboxylase (EC 4.1.1.31) encoded by SEQ ID NO.1 or a pyruvate carboxylase (EC 6.4.1.1) encoded by SEQ ID NOS.3, 4, or 5; and b) an aspartate aminotransferase (EC 2.6.1.1) encoded by SEQ ID NOS.6 or 7; and c) an aspartate decarboxylase (EC 4.1.1.11) encoded by SEQ ID NOS.8 or 9; and d) a ^-alanine aminotransferase (EC 2.6.1.18 or EC 2.6.1.19) encoded by SEQ ID NOS.10 or 11; and e) a malonate semialdehyde reductase (EC 1.1.1.298 or 1.1.1.59) encoded by one of SEQ ID NOS.12-17.

18. The recombinant organism of claims 12-17, wherein the recombinant organism comprises a plurality of nucleic acid sequences for the heterologous expression of each of the following enzymes: a) a phosphoenolpyruvate carboxykinase (EC 4.1.1.49) encoded by SEQ ID NO.2 or a phosphoenolpyruvate carboxylase (EC 4.1.1.31) encoded by SEQ ID NO.1; and b) an aspartate aminotransferase (EC 2.6.1.1) encoded by SEQ ID NO.6; and c) an aspartate decarboxylase (EC 4.1.1.11) encoded by SEQ ID NO.9; and d) a ^-alanine aminotransferase (EC 2.6.1.19) encoded by SEQ ID NO.11; and e) a malonate semialdehyde reductase (EC 1.1.1.298 or 1.1.1.59) encoded by SEQ ID NOS.12 or 15.

19. The recombinant organism of claims 12-18, wherein the recombinant organism comprises a nucleic acid sequence for the heterologous expression of a malonate semialdehyde reductase that utilizes NADH as the electron donor (redox cofactor).

20. The recombinant organism of claims 12-18, wherein the recombinant organism comprises a nucleic acid sequence for the heterologous expression of a malonate semialdehyde reductase that utilizes NADPH as the electron donor (redox cofactor).

21. The recombinant organism of claims 12-20, wherein the recombinant organism comprises a nucleic acid sequence for the heterologous expression of a ^-alanine aminotransferase of EC 2.6.1.19 ( ^-alanine-oxoglutarate transaminase) that uses 2-oxoglutarate as the amino acceptor, producing malonate semialdehyde and L-glutamate.

22. The recombinant organism of claim 21, wherein the ^-alanine aminotransferase is encoded by SEQ ID NO.11.

23. A protein comprising at least 80% sequence identity to an enzyme selected from the group consisting of an enzyme of class 4.1.1.X encoded by SEQ ID NOS. 1-2, 8-9, 18-56, 159-187, 189-193, 384-391, or 393-400, an enzyme of class 2.6.1.X encoded by SEQ ID NOS.6-7, 10-11, 57-123, 142-158, 194-213, 228-238, or 383, an enzyme of class 1.1.1.X encoded by SEQ ID NOS. 12-17, 214, 216-227, 243, 248, 265, 269-276, 278-279, 284-310, or 320-358, and an enzyme of class 6.4.1.X encoded by SEQ ID NOS.3-5, 188, 215, 392, or 401- 404,

24. The protein of claim 23, wherein the protein comprises at least 90% sequence identity to the selected enzyme.

25. The protein of claim 23, wherein the protein comprises at least 95% sequence identity to the selected enzyme.

26. A protein comprising at least 80% sequence identity to an enzyme selected from the group consisting of a phosphoenolpyruvate carboxykinase (EC 4.1.1.49) encoded by SEQ ID NO.2 or a phosphoenolpyruvate carboxylase (EC 4.1.1.31) encoded by SEQ ID NO.1 or a pyruvate carboxylase (EC 6.4.1.1) encoded by SEQ ID NOS.3, 4, or 5; an aspartate aminotransferase (EC 2.6.1.1) encoded by SEQ ID NOS.6 or 7; an aspartate decarboxylase (EC 4.1.1.11) encoded by SEQ ID NOS.8 or 9; a ^-alanine aminotransferase (EC 2.6.1.18 or EC 2.6.1.19) encoded by SEQ ID NOS.10 or 11; and a malonate semialdehyde reductase (EC 1.1.1.298 or 1.1.1.59) encoded by one of SEQ ID NOS.12-17.

27. The protein of claim 26, wherein the protein comprises at least 90% sequence identity to the selected enzyme.

28. The protein of claim 26, wherein the protein comprises at least 95% sequence identity to the selected enzyme.

29. A protein comprising at least 80% sequence identity to an enzyme selected from the group consisting of a phosphoenolpyruvate carboxykinase (EC 4.1.1.49) encoded by SEQ ID NO.2 or a phosphoenolpyruvate carboxylase (EC 4.1.1.31) encoded by SEQ ID NO.1; an aspartate aminotransferase (EC 2.6.1.1) encoded by SEQ ID NO.6; an aspartate decarboxylase (EC 4.1.1.11) encoded by SEQ ID NO.9; a ^-alanine aminotransferase (EC 2.6.1.19) encoded by SEQ ID NO.11; and a malonate semialdehyde reductase (EC 1.1.1.298 or 1.1.1.59) encoded by SEQ ID NOS.12 or 15.

30. The protein of claim 29, wherein the protein comprises at least 90% sequence identity to the selected enzyme.

31. The protein of claim 29, wherein the protein comprises at least 95% sequence identity to the selected enzyme.

32. A nucleic acid sequence encoding a protein of claims 23-31.

33. A vector comprising a nucleic acid sequence of claim 32.

34. A cell transformed with a vector of claim 33.

35. The cell of claim 34, wherein the cell is a eukaryotic cell.

36. The cell of claims 34-35, wherein the cell is a yeast.

37. The cell of claims 34-36, wherein the yeast is from a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Pichia, Hansenula, Issatchenkia, Candida, Zygosaccharomyces, Debaryomyces, Dekkera (Brettanomyces), or Schizosaccharomyces.

38. A method of bioproducing 3-hydroxypropionic acid, comprising: providing a plurality of cells according to claims 34-37; providing feedstock-derived substrate to the cells; establishing an environment substantially free of oxygen such that the cells produce the 3-hydroxypropionic acid under anaerobic, substantially anaerobic, or facultatively anaerobic conditions.