WO2015077241A1 - Methods of producing d-lactic acid in cyanobacteria - Google Patents

Abstract

The present invention encompasses methods to produce optically pure D-lactate that is substantially derived from CO₂ using a recombinant phototrophic microorganism.

Description

METHODS OF PRODUCING D-LACTIC ACID IN CYANOBACTERIA

GOVERNMENTAL RIGHTS

[0001 ] This invention was made with government support under

MCB0954016 awarded by National Science Foundation. The government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the priority of US provisional application number 61/906,701 , filed November 20, 2013, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] The present invention encompasses methods to produce optically pure D-lactate that is substantially derived from CO2 using a recombinant phototrophic microorganism.

BACKGROUND OF THE INVENTION

[0004] The world faces the challenge to develop sustainable technologies to replace thousands of products that have so far been generated from fossil fuels. Due to concerns about food security, sugar-based microbial fermentation has limitations from an economical perspective. Therefore, phototrophic microbial cell factories are desired for the production of commodity chemicals and biofuels. For example, poly lactic acid (PLA) with its biodegradable properties is a sustainable, environmentally friendly alternative to polyethylene, but the present PLA production is mainly dependent on food crops such as corn and sugarcane. Moreover, optically pure lactic acid is required for the production of PLA, where D-lactate controls the thermochemical and physical properties of PLA. Thus, there remains a need in the art for methods to synthesize optically pure D-lactic acid from CO2 with solar energy. BRIEF DESCRIPTION OF THE FIGURES

[0005] The application file contains at least one photograph executed in color. Copies of this patent application publication with color photographs will be provided by the Office upon request and payment of the necessary fee.

[0006] FIG. 1 depicts an illustration of the metabolic pathway for D-lactate synthesis (A) and images and graphs showing autotrophic production of D-lactate by Synechocystis 6803 (A-D). Red arrows in (A) indicate the heterologous pathway engineered into Synechocystis 6803; (B) Colony PCR to verify the presence of the heterologous genes of the mutant glycerol dehydrogenase (Left) and transhydrogenase (Right) in the engineered strains of Synechocystis 6803; (C) Growth and (D) D-lactate production in the engineered strains (n = 3). Circles: AV08 (with gldA101). Triangles: AV10 (with gldA101-syn and sth) and Squares: AV1 1 (with gldA101-syn). Abbreviations: GlyDH^*, mutant glycerol dehydrogenase; TH, Transhydrogenase; 3PGA, 3- phosphoglycerate; CoA, Coenzyme A; G1 P, glucose 1 -phosphate; G6P, glucose 6- phosphate; PHB, ροΐν-β-hydroxybutyrate; RuBP, ribulose 1 ,5-bisphosphate.

[0007] FIG. 2 shows graphs of growth (A) and lactate production (B) in the engineered Synechocystis 6803 strain AV10 (n = 3), with the provision of additional organic carbon source, i.e., with glucose and acetate (Mixotrophic metabolism).

Squares: with acetate. Circles: with glucose.

[0008] FIG. 3 shows graphs of the isotopomer analysis showing the mass fraction of isotopomers for selected proteinogenic amino acids [TBDMS based] and D- lactate [MSTFA based] (see the materials and methods). (A) Cultures grown with 5 g/L of [1 ,2-¹³C] glucose and (B) Cultures grown with 15 mM of [1 ,2-¹³C] acetate, "white bar" m₀ - mass fraction without any labeled carbon; "grey bar" mi - mass fraction with one labeled carbon; "black bar" m₂ - mass fraction with two labeled carbon. [Note: natural ¹³C makes up about 1 .1 % of total carbon as measurement background.]

[0009] FIG. 4 depicts the nucleotide sequences and alignment of gldA101 (SEQ ID NO: 2) and the codon-optimized gldA101 (gldA-syn, synthesized by Genewiz Inc; SEQ ID NO: 3). [0010] FIG. 5 is a graph showing the autotrophic growth curve for the Synechocystis 6803 mutants. Diamond: Wild type. Square: AV08. Triangle: AV10.

Circle: AV1 1 .

[001 1 ] FIG. 6 graphically depicts extracellular D-lactate labeling. The graph demonstrates that synthesis of ¹³C-lactate with ¹³C-NaHCO3 achieved 85% fully labeled, 1 1 % partially labeled, and 4% unlabeled extracellular lactate. Overall labeling reaches a ¹³C enrichment of- 92% (percentage of carbons that are carbon-13 isotopes). Labeling enrichment may be further improved by sub-culturing ¹³C-labeled cyanobacteria in the fully labeled bicarbonate medium.

[0012] FIG. 7 graphically depicts intracellular D-lactate concentrations. There is significant increase (95% confidence) of the intracellular D-lactate

concentration when cells were placed in neutral pH (=7). This result proves that pH places important role on lactate production.

DETAILED DESCRIPTION OF THE INVENTION

[0013] A recombinant microorganism with an increased photoautotrophic D-lactate production rate, as compared to a wild type microorganism from which the recombinant microorganism was derived, has been developed. Using the

microorganism of the invention, it is now possible to produce optically pure D-lactate that is substantially derived from CO2. Advantageously, the microorganisms of the invention may be used to produce D-lactate at a price point and scale that is not only commercially feasible, but superior to existing processes. A recombinant microorganism of the invention and methods of using such a recombinant microorganism are described below.

I. Recombinant microorganism

[0014] One aspect of the present invention provides a recombinant microorganism, wherein the recombinant microorganism has an increased

photoautotrophic D-lactate production, whereas the microorganism from which it was derived lacks the ability for D-lactate production. "Photoautotrophic D-lactate

production" refers to synthesis of D-lactate from inorganic substances using light as an energy source. According to the invention, the photoautotrophic D-lactate production was achieved in the microorganism by expressing a nucleic acid encoding an

exogenous D-lactate dehydrogenase. Additional improvements in the photoautotrophic D-lactate production rate may also be achieved by A) expressing a nucleic acid encoding an exogenous soluble pyridine nucleotide dehydrogenase, B) decreasing endogenous pyruvate dehydrogenase activity, C) decreasing the amount of intracellular Coenzyme A, D) increasing pH during late phase growth, E) codon optimization of the exogenous genes, F) by utilizing combinations of the above in another fast growing photosynthetic microorganism or G) any combination of the above. As used herein, "endogenous to a microorganism" refers to a nucleic acid sequence that is typically present in the wild-type genome of the particular microorganism, while "exogenous to a microorganism" refers to a nucleic acid sequence that is not typically present in the wild- type genome of the particular microorganism. Each aspect of the invention is described in more detail below.

(a) microorganism

[0015] A microorganism of the invention may be any phototrophic or photosynthetic microorganism that may be used to produce D-lactic acid. As used herein, the terms "photosynthetic microorganism" or "phototrophic microorganism" may be used interchangeably, and refer to any microorganism capable of using photons to acquire energy. The phototrophic microorganism may be a bacterium, an alga or a phytoplankton. The phototrophic microorganism may be a thermophilic microorganism, also referred to as a thermophile. As used herein, a thermophile is a microorganism that grows at relatively high temperatures. For example, a thermophile may grow and thrive at temperatures greater than 37°C. As such, a thermophile may grow and thrive at a temperature range from about 37°C to about 45°C, about 45°C to about 55°C, about 55°C to about 65°C, about 65°C to about 75°C, about 75°C to about 85°C, about 85°C to about 95°C, or greater than 95°C. In an embodiment, a thermophile may grow and thrive at a temperature range from about 37°C to about 45°C. In another embodiment, a thermophile may grow and thrive at a temperature range from about 37°C to about 40°C. In some embodiments, a thermophile may grow and thrive at a temperature of about 37°C, about 38°C, about 39°C, about 40°C, about 41 °C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, or about 50°C.

[0016] A microorganism of the invention may be a wildtype microorganism, or may be a microorganism comprising one or mutations in its genome that decreases expression of a competing pathway. "Competing pathway", as used herein, refers to glycogen and polyhydroxybutyrate synthesizing pathways. Glycogen synthesis can be reduced by decreasing expression of glgC and polyhydroxybutyrate synthesis can be reduced by decreasing expression of both phaC and phaE together. In some

embodiments, expression may be decreased at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% compared to wildtype levels of expression. In other embodiments, expression may be decreased about 100% compared to wildtype levels of expression. Expression may be decreased by any method known in the art, including but not limited to, deletions, insertions, RNAi, siRNA, or manipulation of expression using a regulated promoter.

[0017] In some embodiments, a phototrophic microorganism of the invention is a bacterium. Non-limiting examples of phototrophic bacteria that may be used to produce a valuable product may include species in the genera Chamaesiphon, Chroococcus, Cyanobacterium, Cyanobium, Cyanothece, Dactylococcopsis,

Gloeobacter, Gloeocapsa, Gloeothece, Microcystis, Prochlorococcus, Prochloron, Synechococcus, Synechocystis, Cyanocystis, Dermocarpella, Stanieria, Xenococcus, Chroococcidiopsis, Myxosarcina, Pleurocapsa, Arthrospira, Borzia, Crinalium,

Geitlerinema, Halospirulina, Leptolyngbya, Limnothrix, Lyngbya, Microcoleus,

Oscillatoria, Planktothrix, Prochlorothrix, Pseudanabaena, Spirulina, Starria, Symploca, Trichodesmium, Tychonema, Anabaena, Anabaenopsis, Aphanizomenon, Calothrix, Cyanospira, Cylindrospermopsis, Cylindrospermum, Nodularia, Nostoc, Rivularia, Scytonema, Tolypothrix, Chlorogloeopsis, Fischerella, Geitleria, lyengariella, Nostochopsis, and Stigonema of cyanobacteha.

[0018] In other embodiments, a phototrophic microorganism of the invention may be a eukaryotic alga. Non-limiting examples of an alga that may be used for the invention may include an alga belonging to the groups archaeplastida such as rhodophyta (Red algae), chlorophyta (Green algae), or glaucophyta; rhizaria or excavata such as chlorarachniophytes and euglenids; heterokonts such as

bacillariophyceae (Diatoms), axodine, bolidomonas, eustigmatophyceae, phaeophyceae (Brown algae), chrysophyceae (Golden algae), raphidophyceae, synurophyceae and xanthophyceae (Yellow-green algae); cryptophyta; dinoflagellates; and haptophyta. In preferred embodiments, the microorganism is a phytoplankton, which includes diatoms, dinoflagellates and coccolithophores, in addition to cyanobacteria and algae.

[0019] In preferred embodiments, a microorganism of the invention may be a cyanobacterium. Non-limiting examples of cyanobacteria that may be used in the invention may include cyanobacteria belonging to the order Chroococcales,

cyanobacteria belonging to the order Nostocales, and cyanobacteria belonging to the order Stigonematales. In some embodiments, the cyanobacterium belongs to the order Nostocales. In other embodiments, the cyanobacterium belongs to the order

Stigonematales. In yet other embodiments, the cyanobacterium belongs to the order Chroococcales. In a preferred embodiment, the bacterium is derived from the genus Synechocystis. For instance, a bacterium of the invention may be derived from

Synechocystis PCC sp. 6803. As used herein, the terms "Synechocystis PCC sp. 6803" and "Synechocystis 6803" may be used interchangeably. Additionally, a bacterium of the invention may be derived from Synechocystis PCC sp. 7002.

(b) nucleic acid construct encoding an exogenous D-lactate dehydrogenase

[0020] Generally speaking, a microorganism of the invention with an increased photoautotrophic D-lactate production rate expresses a nucleic acid encoding an exogenous D-lactate dehydrogenase. As such, a microorganism of the invention may comprise a recombinant nucleic acid construct, wherein the nucleic acid construct comprises a promoter operably-linked to a nucleic acid encoding an exogenous D- lactate dehydrogenase. Such a nucleic acid construct may be chromosomally

integrated, or may be expressed on an extrachromosomal vector. Suitable

extrachromosomal vectors are known in the art. Non-limiting examples of suitable extrachromosomal vectors include pFC1 , pSL121 1 and pPMQAKL Chromosomal integration may be directed or random. Generally speaking, when using an exogenous promoter to drive expression, a suitable genomic locus for directed integration is one that is silent under most conditions. Such loci are known in the art, or may be identified with routine experimentation. A suitable Synechocystis genomic locus for chromosomal integration includes, but is not limited to, psbA1, psbA2, slr0168, and intergenic regions. In certain embodiments, a nucleic acid encoding an exogenous D-lactate

dehydrogenase is integrated at more than one chromosomal locus. For example, a nucleic acid encoding an exogenous D-lactate dehydrogenase may be integrated into at least two, at least three, at least four, or at least five chromosomal loci of a

microorganism. In some embodiments, a first nucleic acid construct is integrated into multiple loci. In other embodiments, a first nucleic acid construct is integrated into a first genomic locus, and one or more variants of the first nucleic acid construct is integrated into additional genomic loci. The variants of the first nucleic acid construct may have different promoters, ribosomal binding sites, variations in the nucleic acid sequence encoding the exogenous D-lactate dehydrogenase, or a combination thereof.

[0021 ] Methods of making a microorganism of the invention are also known in the art. Generally speaking, a microorganism is transformed with a nucleic acid construct of the invention. Methods of transformation are well known in the art, and may include electroporation, natural transformation, and calcium chloride mediated transformation. Methods of screening for and verifying chromosomal integration are also known in the art.

[0022] A nucleic acid construct of the invention may comprise a plasmid suitable for use in a bacterium. Such a vector may contain multiple cloning sites for ease in manipulating nucleic acid sequences. Numerous suitable plasmids are known in the art and include, but are not limited to, vectors comprising an origin of replication (o ) selected from the group consisting of the pSC101 o , the p15A o , the pBR o , and the pUC o .

[0023] In a preferred embodiment, the microorganism is a photosynthetic cyanobacterium. Methods of making a cyanobacterium of the invention are known in the art. For example, see Varman et al., Appl Environ Microbiol 79(3): 908-914; and Liu et al., PNAS (201 1 ) 108:6899-6904, hereby incorporated by reference in their entirety.

[0024] In other embodiments, the microorganism is a eukaryotic alga.

Nucleic acid sequences may be expressed in the nucleus or the plastid of eukaryotic algal cells. As is generally recognized in the art, chloroplasts use bacterial means for expression of nucleic acid sequences and for protein synthesis. As such, methods for regulated or constitutive expression of nucleic acid sequences in algal chloroplasts are as described for expression of nucleic acid sequences in bacteria. Methods of transforming an alga and to express nucleic acid sequences from the nucleus or the plastid of the algal cell are known in the art. For more details, see Wang et al., J. Genet. Genomics (2009) 36:387-398, Radakovits et al., Eukaryotic Cell (2010) 9(4):486-501 , Newell et al. (2003) 12:631 -634, hereby incorporated by reference in their entirety.

1 . promoter

[0025] Suitable promoters include both constitutive promoters and inducible promoters. The term "promoter", as used herein, may mean a synthetic or naturally- derived molecule that is capable of conferring, activating, or enhancing expression of a second nucleic acid sequence. A suitable promoter may or may not be a Synechocystis promoter. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or alter spatial expression and/or temporal expression of a nucleic acid. The term "operably linked", as used herein, means that expression of a nucleic acid is under the control of a promoter with which it is spatially connected. A promoter may be positioned 5' (upstream) of the nucleic acid under its control. The distance between the promoter and a nucleic acid sequence to be expressed may be approximately the same as the distance between that promoter and the native nucleic acid it controls. As is known in the art, variation in this distance may be accommodated without loss of promoter function.

[0026] In some embodiments, a nucleic acid construct of the invention encompasses a constitutive promoter. Suitable constitutive promoters are known in the art and include, but are not limited to, constitutive promoters from Gram-negative bacteria or a bacteriophage propagating in a Gram-negative bacterium. For instance, promoters for genes encoding highly expressed Gram-negative gene products may be used, such as the promoter for Lpp, OmpA, rRNA, and ribosomal proteins. Alternatively, regulatable promoters may be used in a strain that lacks the regulatory protein for that promoter. For instance Plac, Ptac, and Ptrc may be used as constitutive promoters in strains that lack Lacl. Similarly, P22 PR and PL may be used in strains that lack the P22 C2 repressor protein, and λ PR and PL may be used in strains that lack the λ C1 repressor protein. In one embodiment, the constitutive promoter is from a

bacteriophage. In another embodiment, the constitutive promoter is from a Salmonella bacteriophage. In yet another embodiment, the constitutive promoter is from a cyanophage. In some embodiments, the constitutive promoter is a Synechocystis promoter. For instance, the constitutive promoter may be the PpsbAII promoter or its variant sequences, the Prbc promoter or its variant sequences, the Pcpc promoter or its variant sequences, and the PrnpB promoter or its variant sequences. In preferred embodiments, the constitutive promoter is the Ptrc promoter or its variant sequences.

[0027] In other embodiments, a nucleic acid construct of the invention encompasses an inducible promoter. Suitable constitutive promoters are known in the art and include, but are not limited to, those induced by expression of an exogenous protein (e.g., T7 RNA polymerase, SP6 RNA polymerase), by the presence of a small molecule (e.g., IPTG, galactose, tetracycline, steroid hormone, abscisic acid), by absence of small molecules (e.g., CO2, iron, nitrogen), by metals or metal ions (e.g., copper, zinc, cadmium, nickel), and by environmental factors (e.g., heat, cold, stress, light, darkness), and by growth phase. In each of the above embodiments, the inducible promoter is preferably tightly regulated such that in the absence of induction,

substantially no transcription is initiated through the promoter. Additionally, induction of the promoter of interest should not typically alter transcription through other promoters. Also, generally speaking, the compound or condition that induces an inducible promoter should not be naturally present in the organism or environment where expression is sought. In some embodiments, a nucleic acid construct of the invention encompasses a trc promoter (P_frc; IPTG inducible), tetR promoter (P_tetR, aTc inducible), nrsB promoter (PnrsB,^' nickel inducible), and psbA2 promoter (P_pSbA2, light inducible).

2. D-lactate dehydrogenase

[0028] A suitable D-lactate dehydrogenase of the invention is an enzyme with high D-lactate dehydrogenase activity. In some embodiments, "high activity" refers to a D-lactate dehydrogenase with activity greater than the activity of endogenous D- lactate dehydrogenase of the microorganism of the invention. Methods for measuring D- lactate dehydrogenase activity are well known in the art. For example, in certain embodiments, "high activity" refers to pyruvate reduction to D-lactate activity greater than 0.01 unit per mg protein. Pyruvate reduction to D-lactate activity greater than 0.01 unit per mg protein can be at least 0.02, at least 0.03, at least 0.04, at least 0.05, at least 0.06, at least 0.07, at least 0.08, at least 0.09, at least 0.10, at least 0.1 1 , at least 0.12, at least 0.13, at least 0.14, at least 0.15, at least 0.16, at least 0.17, at least 0.18, at least 0.19, at least 0.21 , at least 0.22, at least 0.23, at least 0.24, at least 0.25, at least 0.26, at least 0.27, at least 0.28, at least 0.29, at least 0.31 , at least 0.32, at least 0.33, at least 0.34, at least 0.35, at least 0.36, at least 0.37, at least 0.38, at least 0.39, at least 0.41 , at least 0.42, at least 0.43, at least 0.44, at least 0.45, at least 0.46, at least 0.47, at least 0.48, at least 0.49, at least 0.51 , at least 0.52, at least 0.53, at least 0.54, at least 0.55, at least 0.56, at least 0.57, at least 0.58, at least 0.59, at least 0.61 , at least 0.62, at least 0.63, at least 0.64, at least 0.65, at least 0.66, at least 0.67, at least 0.68, at least 0.69, at least 0.71 , at least 0.72, at least 0.73, at least 0.74, at least 0.75, at least 0.76, at least 0.77, at least 0.78, at least 0.79, or at least 0.80 unit per mg protein. For a greater description of the methods disclosed, see J Biol Chem 235:1820- 1823 or Method Enzymol 41 :304-309, each hereby incorporated by reference in its entirety. In other embodiments, "high activity" refers to pyruvate reduction to D-lactate activity greater than 1 .0 unit per mg protein. Pyruvate reduction to D-lactate activity greater than 1 .0 unit per mg protein can be at least 1 .5, at least 2.0, at least 2.5, at least 3.0, at least 3.5, at least 4.0, at least 4.5, at least 5.0, at least 6.0, at least 6.5, at least 7.0, at least 7.5, at least 8.0, at least 8.5, at least 9.0, at least 9.5, at least 10.0, at least 1 1 .0, at least 1 1 .5, at least 12.0, at least 12.5, at least 13.0, at least 13.5, at least 14.0, at least 14.5, at least 15.0, at least 16.0, at least 16.5, at least 17.0, at least 17.5, at least 18.0, at least 18.5, at least 19.0, at least 19.5, at least 20.0, at least 20.5, at least 21 .0, at least 21 .5, at least 22.0, at least 22.5, at least 23.0, at least 23.5, at least 24.0, at least 24.5, at least 25.0, at least 26.0, at least 26.5, at least 27.0, at least 27.5, at least 28.0, at least 28.5, at least 29.0, at least 29.5, at least 30.0, at least 30.5, at least 31 .0, at least 31 .5, at least 32.0, at least 32.5, at least 33.0, at least 33.5, at least 34.0, at least 34.5, at least 35.0, at least 36.0, at least 36.5, at least 37.0, at least 37.5, at least 38.0, at least 38.5, at least 39.0, at least 39.5, at least 40.0, at least 40.5, at least 41 .0, at least 41 .5, at least 42.0, at least 42.5, at least 43.0, at least 43.5, at least 44.0, at least 44.5, at least 45.0, at least 46.0, at least 46.5, at least 47.0, at least 47.5, at least 48.0, at least 48.5, at least 49.0, at least 49.5, at least 50.0, at least 50.5, at least 51 .0, at least 51 .5, at least 52.0, at least 52.5, at least 53.0, at least 53.5, at least 54.0, at least 54.5, at least 55.0, at least 56.0, at least 56.5, at least 57.0, at least 57.5, at least 58.0, at least 58.5, at least 59.0, at least 59.5, at least 60.0, at least 60.5, at least 61 .0, at least 61 .5, at least 62.0, at least 62.5, at least 63.0, at least 63.5, at least 64.0, at least 64.5, at least 65.0, at least 66.0, at least 66.5, at least 67.0, at least 67.5, at least 68.0, at least 68.5, at least 69.0, at least 69.5, at least 70.0, at least 70.5, at least 71 .0, at least 71 .5, at least 72.0, at least 72.5, at least 73.0, at least 73.5, at least 74.0, at least 74.5, at least 75.0, at least 76.0, at least 76.5, at least 77.0, at least 77.5, at least 78.0, at least 78.5, at least 79.0, at least 79.5, at least 80.0, at least 80.5, at least 81 .0, at least 81 .5, at least 82.0, at least 82.5, at least 83.0, at least 83.5, at least 84.0, at least 84.5, at least 85.0, at least 86.0, at least 86.5, at least 87.0, at least 87.5, at least 88.0, at least 88.5, at least 89.0, at least 89.5, at least 90.0, at least 90.5, at least 91 .0, at least 91 .5, at least 92.0, at least 92.5, at least 93.0, at least 93.5, at least 94.0, at least 94.5, at least 95.0, at least 96.0, at least 96.5, at least 97.0, at least 97.5, at least 98.0, at least 98.5, at least 99.0, at least 99.5, or 100.0 unit per mg protein. In other embodiments, "high activity" refers to pyruvate reduction to D-lactate activity from about 1 .0 to about 100.0 unit per mg protein. In other embodiments, "high activity" refers to pyruvate reduction to D-lactate activity from about 1 .0 to about 10.0 unit per mg protein. In other embodiments, "high activity" refers to pyruvate reduction to D-lactate activity from about 5.0 to about 15.0 unit per mg protein. In other embodiments, "high activity" refers to pyruvate reduction to D-lactate activity from about 10.0 to about 50.0 unit per mg protein. In other embodiments, "high activity" refers to pyruvate reduction to D-lactate activity from about 1 .0 to about 50.0 unit per mg protein. In other

embodiments, "high activity" refers to pyruvate reduction to D-lactate activity from about 10.0 to about 100.0 unit per mg protein.

[0029] A suitable D-lactate dehydrogenase may be wildtype enzyme or may be an enzyme engineered to have increased activity. A wildtype enzyme may be any enzyme belonging to the enzyme class EC 1 .1 .1 .28. For example, the polypeptide encoded by SEQ ID NO: 1 (GlyDH^*) has increased activity compared to the wildtype polypeptide (Bacillus coagulans GlyDH; see Wang et al. PNAS 108(47): 18920-18925). Increased activity of the polypeptide encoded by SEQ ID NO: 1 compared to the wildtype polypeptide is the result of two amino acid mutations in the wildtype protein, D121 N and F245S (numbering based off SEQ ID NO:1 ). Similar mutations made in homologs of B. coagulans GlyDH may also increase activity compared to wildtype protein. Also contemplated within the scope of the invention are variants of SEQ ID NO:1 that have increased activity. Variants of SEQ ID NO: 1 that have increased activity may be engineered by any method known in the art. Generally speaking, variants of SEQ ID NO: 1 are generated by a mutagenesis strategy or a selective pressure, and the variants are screened for increased D-lactate dehydrogenase activity as known in the art. For example, a derivative with increased activity may be engineered by metabolic evolution, as described in Wang et al. PNAS 108(47): 18920-18925). Alternatively, a derivative with increased activity may be engineered by random mutagenesis or site- directed mutagenesis. Methods of mutagenesis are well known in the art. [0030] In some embodiments, a microorganism of the invention may comprise an exogenous nucleic acid construct, wherein the nucleic acid construct comprises a promoter operably-linked to a nucleic acid encoding a polypeptide with at least 80% identity to SEQ ID NO: 1 . For example, the polypeptide may have about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 1 . In other embodiments, a microorganism of the invention comprises an exogenous nucleic acid construct, wherein the nucleic acid construct comprises a promoter operably-linked to a nucleic acid encoding a polypeptide with (i) at least 80% identity to SEQ ID NO: 1 and (ii) an asparagine at position 121 and a serine at position 245, relative the numbering of SEQ ID NO: 1 .

[0031 ] In some embodiments, a microorganism of the invention may comprise an exogenous nucleic acid construct, wherein the nucleic acid construct comprises a promoter operably-linked to a nucleic acid sequence with at least 80% identity to SEQ ID NO: 2. For example, the nucleic acid sequence may have about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 2. In certain embodiments, a microorganism of the invention comprises an exogenous nucleic acid construct, wherein the nucleic acid construct comprises a promoter operably-linked to a nucleic acid with (i) at least 80% identity to SEQ ID NO: 2 and (ii) a codon for an asparagine at positions 361 -363 and a codon for a serine at positions 733-735, relative the numbering of SEQ ID NO: 2. For example, a codon for an asparagine may be selected from the group consisting of AAT and AAC and a codon for a serine may be selected from the group consisting of TCT, TCC, TCA, TCG, AGT and AGC. 3. optional modifications

[0032] In all of the embodiments described above, the nucleic acid construct comprising a promoter operably-linked to a nucleic acid sequence encoding an exogenous D-lactate dehydrogenase may be further modified to increase expression of the nucleic acid. The optimal level of expression of the nucleic acid sequence encoding the exogenous D-lactate dehydrogenase may be estimated or may be determined by experimentation. Generally speaking, an optimal level of expression balances expression of the nucleic acid with cell growth such that D-lactate production is maximized. Non-limiting examples of methods to increase expression of a nucleic acid or a peptide encoded by a nucleic acid sequence include (1 ) use of promoters with high strength that result in high levels of expression, (2) optimizing ribosomal binding sites, (3) codon optimization, and (4) duplicating heterologous genes by integrating the nucleic acid sequence at multiple sites, as well as combinations thereof.

[0033] In some embodiments, a nucleic acid sequence encoding an exogenous D-lactate dehydrogenase may be altered to reflect the codon preference of the microorganism of the invention in order to achieve faster translation rates and higher accuracy, and thereby increased expression. Codon usage by microorganism and codon optimization tools are well known in the art. See for example, Plant Physiol 92: 1 - 1 1 or Metab Eng 12:70-79, each hereby incorporated by reference in its entirety. In an exemplary embodiment, a microorganism of the invention is Synechocystis 6803 and the microorganism comprises an exogenous nucleic acid construct, wherein the nucleic acid construct comprises a nucleic acid sequence with at least 80% identity to SEQ ID NO: 3 (gldA-syn). For example, the nucleic acid sequence may have about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 3. In certain embodiments, a microorganism of the invention comprises an exogenous nucleic acid construct, wherein the nucleic acid construct comprises a promoter operably-linked to a nucleic acid with (i) at least 80% identity to SEQ ID NO: 3 and (ii) a codon for an asparagine at positions 361 -363 and a codon for a serine at positions 733-735, relative the numbering of SEQ ID NO: 3. For example, a codon for an asparagine may be selected from the group consisting of AAT and AAC and a codon for a serine may be selected from the group consisting of TCT, TCC, TCA, TCG, AGT and AGC.

[0034] In other embodiments, a promoter operably-linked to a nucleic acid sequence encoding an exogenous D-lactate dehydrogenase may be altered to modify translation initiation, and thereby increase expression. The translation initiation rate is determined by the summary effect of multiple molecular interactions, including the hybridization of the 16S rRNA to the ribosomal binding site sequence, the binding of tRNAfMET to the start codon, the distance between the 16S rRNA binding site and the start codon, and the presence of RNA secondary structures that occlude either the 16S rRNA binding site or the standby site. In certain embodiments, variations in the consensus sequence of a ribosomal binding site may increase expression. In certain embodiments, the distance between the ribosomal binding site or other regulatory elements (e.g. RNA polymerase binding sites) and the start codon may be altered (i.e. increased or decreased) to increase expression.

[0035] In other embodiments, more than one modification or type of modification may be performed to optimize expression of an operably-linked nucleic acid sequence encoding an exogenous D-lactate dehydrogenase. Methods of modifying nucleic acid sequences are known in art. For example, a nucleic acid construct comprising a promoter operably-linked to a nucleic acid sequence encoding an exogenous D-lactate dehydrogenase may comprise at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 modifications or types of modifications that result in optimal D-lactate production.

4. additional components

[0036] In certain embodiments, nucleic acids of the invention may further comprise additional components, such as a marker, a spacer domain, and a flanking sequence. [0037] In one embodiment, a nucleic acid of the invention comprises at least one marker. Generally speaking, a marker encodes a product that the host cell cannot make, such that the cell acquires resistance to a specific compound, is able to survive under specific conditions, or is otherwise differentiate from cells that do not carry the marker. Markers may be positive or negative markers. In some embodiments, a nucleic acid of the invention may comprise both a positive marker and a negative marker. In certain embodiments, the marker may code for an antibiotic resistance factor. Suitable examples of antibiotic resistance markers may include, but are not limited to, those coding for proteins that impart resistance to kanamycin, spectinomycin,

streptomycin, neomycin, gentamicin (G418), ampicillin, tetracycline, and

chloramphenicol. Additionally, the sacB gene may be used as a negative marker. The sacB gene is lethal in many bacteria when they are grown on sucrose media.

Additionally, fluorescent proteins may be used as visually identifiable markers.

Generally speaking, markers may be present during construction of the strains, but are typically removed from the final constructs. Proteins can also be marked by adding a sequence such as FLAG, HA, His tag, that can be recognized by a monoclonal antibody using immunological methods. In some embodiments, a marker may be a unique identifier of a genetically modified cyanobacterium. In other embodiments, a marker may be a unique identifier of a genetically modified chloroplast genome in a unicellular alga.

[0038] Additionally, a nucleic acid of the invention may comprise a Shine- Dalgarno sequence, or a ribosome binding site (RBS). Generally speaking, a RBS is the nucleic acid sequence in the mRNA that binds to a 16s rRNA in the ribosome to initiate translation. For Gram-negative bacteria, the RBS is generally AGGA. The RBS may be located about 8 to about 1 1 bp 5' of the start codon of the first structural gene. One skilled in the art will realize that the RBS sequence or its distance to the start codon may be altered to increase or decrease translation efficiency.

[0039] Nucleic acid constructs of the invention may also comprise flanking sequences. The phrase "flanking sequence" as used herein, refers to a nucleic acid sequence homologous to a chromosomal sequence. A construct comprising a flanking sequence on either side of a construct (i.e., a left flanking sequence and a right flanking sequence) may homologously recombine with the homologous chromosome, thereby integrating the construct between the flanking sequences into the chromosome.

Generally speaking, flanking sequences may be of variable length. In an exemplary embodiment, the flanking sequences may be between about 300 and about 500 bp. In another exemplary embodiment, the left flanking sequence and the right flanking sequence are substantially the same length.

(c) an exogenous soluble pyridine nucleotide transhydrogenase

[0040] In another aspect, a microorganism of the invention expressing a nucleic acid encoding an exogenous D-lactate dehydrogenase may further comprise an exogenous soluble pyridine nucleotide transhydrogenase. Expression of an exogenous soluble pyridine nucleotide transhydrogenase in a microorganism of the invention will increase the ratio of NADH to NADPH, thereby increasing the photoautotrophic D- lactate production rate of the microorganism.

[0041 ] In some embodiments, a microorganism of the invention may comprise a recombinant nucleic acid construct, wherein the nucleic acid construct comprises a promoter operably-linked to a first nucleic acid encoding an exogenous D- lactate dehydrogenase and a second nucleic acid encoding an exogenous soluble pyridine nucleotide transhydrogenase. In other embodiments, a microorganism of the invention may comprise a recombinant nucleic acid construct, wherein the nucleic acid construct comprises a promoter operably-linked to a first nucleic acid encoding an exogenous soluble pyridine nucleotide transhydrogenase and a second nucleic acid encoding an exogenous D-lactate dehydrogenase. In still other embodiments, a microorganism of the invention may comprise (i) a first recombinant nucleic acid construct, wherein the first nucleic acid construct comprises a promoter operably-linked to a nucleic acid encoding an exogenous D-lactate dehydrogenase and (ii) a second recombinant nucleic acid construct, wherein the second nucleic acid construct comprises a promoter operably-linked to a nucleic acid encoding an exogenous soluble pyridine nucleotide transhydrogenase. The exogenous soluble pyridine nucleotide transhydrogenase may be expressed using the same promoter as the exogenous D- lactate dehydrogenase or may be expressed using a different promoter. Suitable promoters are described above.

[0042] Any soluble pyridine nucleotide transhydrogenase that may be expressed in a microorganism of the invention is suitable. A pyridine nucleotide transhydrogenase may be any enzyme belonging to the enzyme class EC 1 .6.1 . For example, an exogenous soluble pyridine nucleotide transhydrogenase may be encoded by the sth gene from Pseudomonas aeruginosa or a homolog thereof (Gen Bank identifier: AAG06379.1 ; NCBI Reference Sequence: NP_251681 .1 ). Methods of identifying homologs are known in the art.

[0043] In an exemplary embodiment, a nucleic acid encoding an exogenous soluble pyridine nucleotide transhydrogenase has at least 80% identity to SEQ ID NO: 4. For example, the nucleic acid may have about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 4. In other embodiments, a nucleic acid encoding an exogenous soluble pyridine nucleotide transhydrogenase encodes a polypeptide with at 80% identity to SEQ ID NO: 5. For example, the polypeptide may have about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 5.

[0044] In each of the embodiments described above, a nucleic acid encoding a pyridine nucleotide transhydrogenase may comprise one or more modifications to increase expression of the nucleic acid sequence. Suitable

modifications are described above. In a preferred embodiment, a nucleic acid encoding a pyridine nucleotide transhydrogenase may be altered to reflect the codon preference of the microorganism of the invention in order to achieve faster translation rates and higher accuracy, and thereby increased expression. (d) decreasing endogenous pyruvate dehydrogenase activity

[0045] In another aspect, a microorganism of the invention expressing a nucleic acid encoding an exogenous D-lactate dehydrogenase may further comprise decreased endogenous pyruvate dehydrogenase activity, optionally in combination with expression of an exogenous soluble pyridine nucleotide transhydrogenase. Pyruvate dehydrogenase catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA. As further detailed in the Examples, decreasing endogenous pyruvate dehydrogenase activity directs the carbon flux towards D-lactate production, thereby increasing the amount of D-lactate produced.

[0046] Activity may be decreased by any method known in the art. In some embodiments, pyruvate dehydrogenase activity is decreased by reducing expression of an endogenous pyruvate dehydrogenase. Suitable methods are known in the art and include, but are not limited to, deletions, insertions, RNAi, siRNA, or manipulation of expression using a regulated promoter. As a non-limiting example, a light or a growth responsive promoter may be used to induce D-lactate production during middle log growth phase. Without wishing to be bound by theory, this would allow the culture to maintain a good light condition and to balance carbon partition between growth and D- lactate production. In other embodiments, an inhibitor may be used to decrease pyruvate dehydrogenase activity. In still other embodiments, pyruvate dehydrogenase activity is reduced by decreasing the amount of intracellular CoA.

[0047] In a preferred embodiment, pyruvate dehydrogenase activity is decreased by culturing a microorganism of the invention in the presence of acetate. In another preferred embodiment, pyruvate decarboxylation is decreased by culturing a microorganism of the invention in the presence of acetate. In another preferred embodiment, the amount of intracellular CoA is decreased by culturing a microorganism of the invention in the presence of acetate. To obtain a substantial improvement in D- lactate titer, at least about 1 mM acetate should be used, preferably at least about 5 mM. An increase in D-lactic acid titer is expected by addition of more acetic acid, up to about 15mM. Further increases of acetic acid may be deleterious to microbial health and/or increase the operating costs without any substantial benefit. In some embodiments, cultunng a microorganism of the invention in the presence of about 1 mM to about 15 mM acetate decreases pyruvate dehydrogenase activity. In other

embodiments, culturing a microorganism of the invention in the presence of about 5 mM to about 15 mM acetate decreases pyruvate dehydrogenase activity. In other

embodiments, culturing a microorganism of the invention in the presence of about 1 mM to about 15 mM acetate decreases pyruvate decarboxylation. In other embodiments, culturing a microorganism of the invention in the presence of about 5 mM to about 15 mM acetate decreases pyruvate decarboxylation. In other embodiments, culturing a microorganism of the invention in the presence of about 1 mM to about 15 mM acetate decreases the amount of intracellular CoA. In other embodiments, culturing a

microorganism of the invention in the presence of about 5 mM to about 15 mM acetate decreases the amount of intracellular CoA. About 1 mM to about 15 mM acetate can be about 1 mM, about 1 .5 mM, about 2.0 mM, about 2.5 mM, about 3.0 mM, about 3.5 mM, about 4.0 mM, about 4.5 mM, about 5.0 mM, about 5.5 mM, about 6.0 mM, about 6.5 mM, about 7.0 mM, about 7.5 mM, about 8.0 mM, about 8.5 mM, about 9.0 mM, about 9.5 mM, about 10.0 mM, about 10.5 mM, about 1 1 .0 mM, about 1 1 .5 mM, about 12.0 mM, about 12.5 mM, about 13.0 mM, about 13.5 mM, about 14.0 mM, about 14.5 mM, or about 15.0 mM acetate, or an equivalent amount of an acetate derivative. Acetate may be provided to the culture conditions once (for example, at the start of growth or at a point during logarithmic growth), or may be added continuously to maintain a constant concentration (e.g. during long-term D-lactate production). In a specific embodiment, acetate may be provided once at the late stage of growth. The late stage of growth may be the stage where high D-lactate production is achieved. In an embodiment, the late stage of growth may be between about day 14 to about day 21 . For example, the late stage of growth may be day 14, day 15, day 16, day 17, day 18, day 19, day 20 or day 21 . In a specific embodiment, the late stage of growth may be between about day 17 to about day 19. In another specific embodiment, the late stage of growth may be day 18.

[0048] In some embodiments, endogenous pyruvate dehydrogenase activity may be decreased at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to wildtype levels of activity. In other

embodiments, activity may be decreased about 100% compared to wildtype levels of activity. Methods of measuring pyruvate dehydrogenase activity are known in the art. Alternatively, a change in pyruvate dehydrogenase activity may be indirectly determined by measuring a change in the amount of d-lactate produced.

(e) increased pH during late stage growth

[0049] In another aspect, a microorganism of the invention expressing a nucleic acid encoding an exogenous D-lactate dehydrogenase may further be cultured in the presence of increased pH, optionally in combination with expression of an exogenous soluble pyridine nucleotide transhydrogenase and optionally in combination with a culture comprising acetate. To promote lactate production, a microorganism of the invention may be grown in a slightly alkaline pH and then the pH may be increased during late stage growth for enhanced extracellular production of lactate. As further detailed in the Examples, decreasing pH increased intracellular D-lactate

concentrations, thereby decreasing the amount of D-lactate secreted.

[0050] pH may be increased by any method known in the art. In some embodiments, pH is increased by addition of a base to the culture. Suitable bases to increase the pH of a culture are known in the art. For example, sodium hydroxide (NaOH) is a strong base that may be used to increase the pH of a culture. The addition of a base may increase the pH or alkalinity of a culture. A culture with a pH greater than 7 is generally a basic or alkaline culture. A slightly alkaline pH may refer to a culture with a pH of greater than 7, but less than 10. For example, a slightly alkaline pH may refer to a culture with a pH of about 7.5, about 8.0, about 8.5, about 9.0 or about 9.5. The pH may be further increased during late stage growth. For example, the pH may be increased from about 10 to about 12 such that the pH may be about 10.0, about 10.5, about 1 1 .0, about 1 1 .5 or about 12.0 during late stage growth. Late stage growth may be as defined above. [0051 ] In a preferred embodiment, lactate secretion is increased by culturing a microorganism of the invention in the presence of alkaline pH. In another preferred embodiment, lactate secretion is increased by culturing a microorganism of the invention in the presence of increased pH. In another preferred embodiment, extracellular lactate is increased by culturing a microorganism of the invention in the presence of alkaline pH. In each of the foregoing embodiments, the pH may initially be slightly alkaline and then the alkalinity of the culture may be further increased during late stage growth. To obtain a substantial improvement in D-lactate titer, the pH may initially be greater than 7 but less than 10. In an embodiment, the pH may initially be about 7 to about 9, or about 7 to about 8. For example, to obtain a substantial improvement in D-lactate titer, the pH may initially be about 7.1 , about 7.5, about 8.0, about 8.5, about 9.0, about 9.5 or about 9.9. In another embodiment, to obtain a substantial improvement in D-lactate titer, the pH may initially be greater than 7 but less than 10 and then the alkalinity of the culture may be increased to a pH from about 10 to about 12 during late stage growth. In an embodiment, the alkalinity of the culture may be increased to a pH from about 10 to about 1 1 . For example, the pH of the culture may be increased to about 10.0, about 10.5, about 1 1 .0, about 1 1 .5 or about 12.0 during late stage growth. A base may be added to the culture conditions once (for example, at the start of growth or at a point during logarithmic growth), or may be added continuously to maintain a constant pH (e.g. during long-term D-lactate production). In a specific embodiment, pH may be adjusted once at the late stage of growth. The late stage of growth may be the stage where high D-lactate production is achieved. In an

embodiment, the late stage of growth may be between about day 14 to about day 21 . For example, the late stage of growth may be day 14, day 15, day 16, day 17, day 18, day 19, day 20 or day 21 . In a specific embodiment, the late stage of growth may be between about day 17 to about day 19. In another specific embodiment, the late stage of growth may be day 18. (f) increased photoautotrophic D-lactate production rate

[0052] In another aspect, a microorganism of the invention has an increased photoautotrophic D-lactate production rate. As stated above,

"photoautotrophic D-lactate production" refers to synthesis of D-lactate from inorganic substances using light as an energy source. Without wishing to be bound by theory, the rate of D-lactate production may be measured as g/L/day, though other measurements known in the art are suitable. For example, D-lactate production may also be reported as mmol/g dry weight/hr.

[0053] Without wishing to be bound by theory, the maximum D-lactate production rate can be equivalent to the maximum biomass production rate. In some embodiments, a maximum D-lactate production rate is about 2 g/L/day. In other embodiments, a maximum D-lactate production rate is about 0.5 g/L/day. In still other embodiment, a maximum D-lactate production rate is about 1 g/L/day. In still yet other embodiments, autotrophic peak production rate is about 0.05 g/L day to about 2.00 g/L/day. For example, autotrophic peak production rate may be about 0.05, about 0.06, about 0 .07, about 0. .08, about 0. .09, about 0. .10, about 0. .1 1 , about 0 .12, about 0. .13 about 0 .14, about 0. .15, about 0. .16, about 0. .17, about 0. .18, about 0 .19, about 0. .20 about 0 .21 , about 0. 22, about 0. .23, about 0. ■24, about 0. .25, about 0 .26, about 0. .27 about 0 .28, about 0. .29, about 0. .30, about 0. .31 , about 0. .32, about 0 .33, about 0. .34 about 0 .35, about 0. .36, about 0. .37, about 0. .38, about 0. .39, about 0 .40, about 0. .41 about 0 .42, about 0. .43, about 0. ■44, about 0. .45, about 0. .46, about 0 .47, about 0. .48 about 0 .49, about 0. .50, about 0. .51 , about 0. .52, about 0. .53, about 0 .54, about 0. .55 about 0 .56, about 0. .57, about 0. .58, about 0. .59, about 0. .60, about 0 .61 , about 0. .62 about 0 .63, about 0. .64, about 0. .65, about 0. .66, about 0. .67, about 0 .68, about 0. .69 about 0 .70, about 0. ■71 , about 0. 72, about 0. .73, about 0. ■74, about 0 .75, about 0. .76 about 0 .77, about 0. .78, about 0. .79, about 0. .80, about 0. .81 , about 0 .82, about 0. .83 about 0 .84, about 0. .85, about 0. .86, about 0. .87, about 0. .88, about 0 .89, about 0. .90 about 0 .91 , about 0. .92, about 0. .93, about 0. .94, about 0. .95, about 0 .96, about 0. .97 about 0 .98, about 0. .99, about 1 . .00, about 1 . .01 , about 1 . .02, about 1 .03, about 1 . .04 about 1 .05, about 1 .06, about 1 .07, about 1 .08, about 1 .09, about 1 .10, about 1 .1 1 about 1 .12, about 1 .13, about 1 .14, about 1 .15, about 1 .16, about 1 .17, about 1 .18 about 1 .19, about 1 .20, about 1 .21 , about 1 .22, about 1 .23, about 1 .24, about 1 .25 about 1 .26, about 1 .27, about 1 .28, about 1 .29, about 1 .30, about 1 .31 , about 1 .32 about 1 .33, about 1 .34, about 1 .35, about 1 .36, about 1 .37, about 1 .38, about 1 .39 about 1 .40, about 1 .41 , about 1 .42, about 1 .43, about 1 .44, about 1 .45, about 1 .46 about 1 .47, about 1 .48, about 1 .49, about 1 .50, about 1 .51 , about 1 .52, about 1 .53 about 1 .54, about 1 .55, about 1 .56, about 1 .57, about 1 .58, about 1 .59, about 1 .60 about 1 .61 , about 1 .62, about 1 .63, about 1 .64, about 1 .65, about 1 .66, about 1 .67 about 1 .68, about 1 .69, about 1 .70, about 1 .71 , about 1 .72, about 1 .73, about 1 .74 about 1 .75, about 1 .76, about 1 .77, about 1 .78, about 1 .79, about 1 .80, about 1 .81 about 1 .82, about 1 .83, about 1 .84, about 1 .85, about 1 .86, about 1 .87, about 1 .88 about 1 .89, about 1 .90, about 1 .91 , about 1 .92, about 1 .93, about 1 .94, about 1 .95 about 1 .96, about 1 .97, about 1 .98, about 1 .99, or about 2.0 g/L day.

(g) preferred embodiments

[0054] In a preferred embodiment, a phototrophic microorganism of the invention comprises an exogenous D-lactate dehydrogenase; wherein the D-lactate dehydrogenase is (i) encoded by a nucleic acid that is codon-optimized for expression in the microorganism, and (ii) comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 . In an exemplary embodiment, the microorganism is

Synechosystis 6803 and the nucleic acid has at least 80% identity to SEQ ID NO: 3.

[0055] In another preferred embodiment, a phototrophic microorganism of the invention comprises an exogenous D-lactate dehydrogenase and an exogenous soluble pyridine nucleotide transhydrogenase; wherein the D-lactate dehydrogenase is (i) encoded by a nucleic acid that is codon-optimized for expression in the

microorganism, and (ii) comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 . In an exemplary embodiment, the microorganism is Synechosystis 6803, the exogenous soluble pyridine nucleotide transhydrogenase has at least 80% identity to SEQ ID NO: 5, and the nucleic acid has at least 80% identity to SEQ ID NO: 4. [0056] In another preferred embodiment, a phototrophic microorganism of the invention comprises an exogenous nucleic acid construct, wherein the nucleic acid construct comprises a promoter operably-linked to (a) a nucleic acid sequence with at least 80% identity to SEQ ID NO: 3, or (b) a nucleic acid sequence encoding a polypeptide with at least 80% identity to SEQ ID NO: 1 .

[0057] In another preferred embodiment, a phototrophic microorganism of the invention comprises an exogenous nucleic acid construct, wherein the nucleic acid construct comprises a promoter operably-linked to (i) (a) a nucleic acid sequence with at least 80% identity to SEQ ID NO: 3, or (b) a nucleic acid sequence encoding a polypeptide with at least 80% identity to SEQ ID NO: 1 ; and (ii) a nucleic acid sequence with at least 80% identity to SEQ ID NO: 4, or (b) a nucleic acid sequence encoding a polypeptide with at least 80% identity to SEQ ID NO: 5.

II. Method for producing D-lactate using a photosynthetic microorganism

[0058] Another aspect of the present invention encompasses a method for producing D-lactate using a photosynthetic microorganism. Typically, the method comprises culturing a photosynthetic microorganism comprising an exogenous D-lactate dehydrogenase in the presence of light and inorganic carbon sources, such that the D- lactate is substantially derived from the inorganic carbon source. In a specific

embodiment, the inorganic carbon source may be CO₂. Accordingly, increasing the inorganic carbon source may increase the production of D-lactate. In a specific embodiment, increasing the CO₂ may increase the production of D-lactate. In certain embodiments, the method of the invention comprises a photosynthetic microorganism further comprising an exogenous soluble pyridine nucleotide transhydrogenase and/or decreased endogenous pyruvate dehydrogenase activity. Suitable microorganisms, D- lactate dehydrogenases, and soluble pyridine nucleotide transhydrogenases are described above.

[0059] Preferably, a method of the invention produces optically pure D- lactate substantially derived from CO₂. The phrase "optically pure", as used herein, refers to the presence of only a single enantiomer. Thus, stated another way, a method of the invention favors synthesis of D-lactate over synthesis of L-lactate, such that about 100% of the lactate produced is the D-enantiomer. D-lactate dehydrogenase is a highly specific enzyme, allowing production of optically pure lactic acid. The phrase

"substantially derived from CO2" means that greater than 50% of the carbon atoms of the D-lactate molecules produced by the method of the invention are derived from CO2 as opposed to another carbon source. For example, about 51 %, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61 %, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71 %, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the carbon atoms of the D-lactate molecules produced by the method of the invention are derived from CO2. Methods for determining the percentage of the carbon atoms derived from CO2 are known in the art and detailed in the Examples. For example, a microorganism of the invention may be cultured in the presence of labeled organic carbon and the abundance of the label in the D-lactate can be measured after a given period of time. Generally speaking, if D-lactate produced by the microorganism is substantially derived from the labeled carbon source, then the carbon atoms in D-lactate will be labeled. Alternatively, if D-lactate produced by the microorganism is substantially derived from a non-labeled carbon source (i.e. CO2), then the carbon atoms in D-lactate will not be labeled.

[0060] In another embodiment, a method of the invention produces labeled optically pure D-lactate substantially derived from a labeled carbon source. Labeled optically pure D-lactate may be useful in various aspects of medical research, including metabolism studies. Metabolism studies are often used in cancer research, diagnosis and/or treatment. A labeled carbon source may be any molecule comprising labeled carbon useful in the production of D-lactate. In some embodiments, a carbon may be labeled with a carbon isotope. Non-limiting examples of carbon isotopes include ¹¹C, ¹³C or ¹⁴C. In a specific embodiment, a carbon may be labeled with a carbon isotope such as ¹³C. The phrase "substantially derived from a labeled carbon source" means that greater than 50% of the carbon atoms of the D-lactate molecules produced by the method of the invention are labeled. For example, about 51 %, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61 %, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71 %, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the carbon atoms of the D-lactate molecules produced by the method of the invention are labeled. For example, a microorganism of the invention may be cultured in the presence of labeled organic carbon and the abundance of the label in the D-lactate can be measured after a given period of time. Generally speaking, if D-lactate produced by the microorganism is substantially derived from the labeled carbon source, then the carbon atoms in D-lactate will be labeled. By limiting the exposure to CO₂, the supplied labeled carbon source may be the only utilized carbon source by the microorganism such that the produced D- lactate is substantially labeled. In an embodiment, the labeled carbon source is ¹⁴C bicarbonate or ¹⁴C-labeled NaHCO3.ln a specific embodiment, the labeled carbon source is ¹³C bicarbonate or ¹³C-labeled NaHCO3.

[0061 ] Methods of the invention can produce D-lactate at a final titer of at least about 0.4 g/L. In some embodiments, the amount of D-lactate produced by a method of the invention is about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1 .0, about 1 .1 , about 1 .2, about 1 .3, about 1 .4, about 1 .5, about 1 .6, about 1 .7, about 1 .8, about 1 .9, about 1 .2, about 1 .3, about 1 .4, about 1 .5, about 1 .6, about 1 .7, about 1 .8, about 1 .9, about 2.0, about 2.1 , about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1 , about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1 , about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, or about 5.0 g/L. In a preferred embodiment, a method of the invention can produce D-lactate at a final titer of at least about 0.5 g/L. In another preferred embodiment, a method of the invention can produce D-lactate at a final titer of at least about 1 .0 g/L. In another preferred embodiment, a method of the invention can produce D-lactate at a final titer of at least about 2.0 g/L. In another preferred embodiment, a method of the invention can produce D-lactate at a final titer of about 2.0 g/L to about 5 g/L. The final titer may be measured at any suitable time, including, but not limited to, at the end of stationary phase or at the onset of death phase.

[0062] In other embodiments, the amount of D-lactate produced by a method of the invention is about 1 .0 g/L to about 20 g/L, for example, about 1 .0, about 2.0, about 3.0, about 4.0, about 5.0, about 6.0, about 7.0, about 8.0, about 9.0, about 10.0, about 1 1 .0, about 12.0, about 13.0, about 14.0, about 15.0, about 16.0, about 17.0, about 18.0, about 19.0, or about 20.0. In still other embodiments, the amount of D- lactate produced by a method of the invention is about 1 .0 g/L to about 5 g/L, about 5.0 g/L to about 10.0 g/L, about 10.0 g/L to about 15.0 g/L, about 15.0 g/L to about 20.0 g/L, about 1 .0 g/L to about 10.0 g/L, about 5.0 g/L to about 15.0 g/L, about 10.0 g/L to about 20.0 g/L, about 1 .0 g/L to about 15.0 g/L, or about 5.0 g/L to about 20.0 g/L.

[0063] In a preferred embodiment, a method of the invention comprises culturing a phototrophic microorganism comprising an exogenous D-lactate

dehydrogenase in the presence of light and CO₂, such that the D-lactate is substantially derived from CO₂; wherein the D-lactate dehydrogenase is (i) encoded by a nucleic acid that is codon-optimized for expression in the microorganism, and (ii) comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 . In an exemplary embodiment, the microorganism is Synechosystis 6803, the nucleic acid has at least 80% identity to SEQ ID NO: 3, and the amount of D-lactate produced is at least about 0.5 g/L.

[0064] In another preferred embodiment, a method of the invention comprises culturing a phototrophic microorganism comprising an exogenous D-lactate dehydrogenase and an exogenous soluble pyridine nucleotide transhydrogenase in the presence of light and CO₂, such that the D-lactate is substantially derived from CO₂; wherein the D-lactate dehydrogenase is (i) encoded by a nucleic acid that is codon- optimized for expression in the microorganism, and (ii) comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 . In an exemplary embodiment, the microorganism is Synechosystis 6803, the exogenous soluble pyridine nucleotide transhydrogenase has at least 80% identity to SEQ ID NO: 5, the nucleic acid has at least 80% identity to SEQ ID NO: 3, and the amount of D-lactate produced is at least about 1 .0 g/L.

[0065] In another preferred embodiment, a method of the invention comprises culturing a phototrophic microorganism comprising an D-lactate

dehydrogenase and an exogenous soluble pyridine nucleotide transhydrogenase in the presence of acetate, light and CO2, such that the D-lactate is substantially derived from CO2; wherein the D-lactate dehydrogenase is (i) encoded by a nucleic acid that is codon-optimized for expression in the microorganism, and (ii) comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 . In an exemplary embodiment, the microorganism is Synechosystis 6803, the exogenous soluble pyridine nucleotide transhydrogenase has at least 80% identity to SEQ ID NO: 5, the nucleic acid has at least 80% identity to SEQ ID NO: 3, the amount of acetate is about 5 mM to about 15 mM, and the amount of D-lactate produced is at least about 2.0 g/L.

[0066] In a different preferred embodiment, a method of the invention comprises culturing a phototrophic microorganism comprising an exogenous D-lactate dehydrogenase in the presence of light, CO2, and increased pH, such that the D-lactate is substantially derived from CO2; wherein the D-lactate dehydrogenase is (i) encoded by a nucleic acid that is codon-optimized for expression in the microorganism, and (ii) comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 . In an exemplary embodiment, the microorganism is Synechosystis 6803, the nucleic acid has at least 80% identity to SEQ ID NO: 3, and the amount of D-lactate produced is at least about 0.5 g/L.

[0067] In another preferred embodiment, a method of the invention comprises culturing a phototrophic microorganism comprising an exogenous D-lactate dehydrogenase and an exogenous soluble pyridine nucleotide transhydrogenase in the presence of light, CO2, and increased pH, such that the D-lactate is substantially derived from CO2; wherein the D-lactate dehydrogenase is (i) encoded by a nucleic acid that is codon-optimized for expression in the microorganism, and (ii) comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 . In an exemplary embodiment, the microorganism is Synechosystis 6803, the exogenous soluble pyridine nucleotide transhydrogenase has at least 80% identity to SEQ ID NO: 5, the nucleic acid has at least 80% identity to SEQ ID NO: 3, and the amount of D-lactate produced is at least about 1 .0 g/L.

[0068] In still another preferred embodiment, a method of the invention comprises culturing a phototrophic microorganism comprising an D-lactate

dehydrogenase and an exogenous soluble pyridine nucleotide transhydrogenase in the presence of acetate, light, CO2, and increased pH, such that the D-lactate is

substantially derived from CO2; wherein the D-lactate dehydrogenase is (i) encoded by a nucleic acid that is codon-optimized for expression in the microorganism, and (ii) comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 . In an exemplary embodiment, the microorganism is Synechosystis 6803, the exogenous soluble pyridine nucleotide transhydrogenase has at least 80% identity to SEQ ID NO: 5, the nucleic acid has at least 80% identity to SEQ ID NO: 3, the amount of acetate is about 5 mM to about 15 mM, and the amount of D-lactate produced is at least about 2.0 g/L.

[0069] In a different preferred embodiment, a method of the invention comprises culturing a phototrophic microorganism comprising an exogenous D-lactate dehydrogenase in the presence of light and a labeled carbon source, such that the D- lactate is substantially labeled; wherein the D-lactate dehydrogenase is (i) encoded by a nucleic acid that is codon-optimized for expression in the microorganism, and (ii) comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 . In an exemplary embodiment, the microorganism is Synechosystis 6803, the nucleic acid has at least 80% identity to SEQ ID NO: 3, and the amount of labeled D-lactate produced is at least about 0.5 g/L.

[0070] In another different preferred embodiment, a method of the invention comprises culturing a phototrophic microorganism comprising an exogenous D-lactate dehydrogenase and an exogenous soluble pyridine nucleotide transhydrogenase in the presence of light and a labeled carbon source, such that the D-lactate is substantially labeled; wherein the D-lactate dehydrogenase is (i) encoded by a nucleic acid that is codon-optimized for expression in the microorganism, and (ii) comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 . In an exemplary embodiment, the microorganism is Synechosystis 6803, the exogenous soluble pyridine nucleotide transhydrogenase has at least 80% identity to SEQ ID NO: 5, the nucleic acid has at least 80% identity to SEQ ID NO: 3, and the amount of labeled D-lactate produced is at least about 1 .0 g/L.

[0071 ] In still another different preferred embodiment, a method of the invention comprises culturing a phototrophic microorganism comprising an D-lactate dehydrogenase and an exogenous soluble pyridine nucleotide transhydrogenase in the presence of acetate, light and a labeled carbon source, such that the D-lactate is substantially labeled; wherein the D-lactate dehydrogenase is (i) encoded by a nucleic acid that is codon-optimized for expression in the microorganism, and (ii) comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 . In an exemplary embodiment, the microorganism is Synechosystis 6803, the exogenous soluble pyridine nucleotide transhydrogenase has at least 80% identity to SEQ ID NO: 5, the nucleic acid has at least 80% identity to SEQ ID NO: 3, the amount of acetate is about 5 mM to about 15 mM, and the amount of labeled D-lactate produced is at least about 2.0 g/L.

sequence) AAGGCTTGGCAAAAAAGCATTTATTATTGCGGATGATTT

TGTCACCGGCCTTGTCGGCAAAACGGTTGAAGAAAGCT

ATGCCGGCAAAGAAACGGGGTATCAAATGGCATTATTC

GGTGGTGAGTGTTCTAAACCGGAAATCGAACGGC I M G

TGAAATGAGCAAATCCGAGGAAGCCGATGTCGTTGTCG

GAATCGGCGGCGGAAAAACATTGGATACCGCAAAAGCA

GTCGGGTATTACAATAACATTCCGGTGATTGTCGCGCC

GACCATCGCTTCCACCAATGCCCCGACAAGCGCCCTGT

CTGTTATTTACAAAGAGAACGGCGAGTTTGAAGAATACT

TGATGCTGCCGCTGAACCCGAC I I I I GTCATTATG G ATA

CGAAAGTGATTGCCTCTGCCCCTGCCCGCCTGCTCGTT

TCCGGCATGGGAGATGCGCTTGCAACGTA I I I I GAAGC

GCGCGCCACTAAGCGGGCAAATAAAACGACGATGGCA

GGCGGGCGTGTTACGGAAGCGGCGATCGCGCTTGCAA

AACTTTGTTATGACACGCAAATTTCGGAAGGTTTAAAAG

CAAAACTGGCAGCGGAAAAACATCTTGTTACGGAAGCA

GTGGAAAAAATCATTGAAGCGAATACGTATCTGAGCGG

AATCGGTTCTGAAAGCGGCGGCCTTGCTGCGGCACATG

CGATCCATAATGGGCTTACCGTGCTCGAAGAAACCCAT

CATATGTACCACGGCGAAAAAGTGGCATTCGGTACCCT

CGCCCAGCTGA I I I I GGAAGATGCGCCGAAAGCGGAAA

TTGAAGAGGTGGTCTCCTTCTGCCTGAGTGTCGGACTT

CCCGTCACGCTCGGGGA I I I GGGCGTGAAAGAACTGAA

TGAGGAAAAGCTCCGAAAAGTGGCTGAAC I I I CCTGTG

CGGAAGGCGAAACGATTTATAACATGCCGTTTGAAGTC

ACGCCTGACCTTGTGTACGCAGCAATCGTTACCGCTGA

TTCCGTCGGGCGGTATTATAAGGAAAAATGGGCATGA

ATGACCAAAATCATCACCTCCCCCTCCAAATTCATCCAG

GGCCCCGACGAGTTGTCCCGGTTGAGTGCCTATACCGA

ACGGTTGGGCAAGAAGGCC I I I ATCATCGCTGACGACT

TCGTGACCGGCTTGGTGGGTAAGACTGTGGAGGAGTC

CTATGCCGGCAAAGAAACCGGTTACCAGATGGCCTTGT

TTGGCGGCGAATGCAGTAAGCCTGAGATCGAGCGCTCT

TGCGAGATGTCCAAATCCGAAGAAGCCGACGTTGTGGT

gldA101-syn GGGCATCGGCGGTGGCAAAACCTTAGACACTGCCAAG

(nucleotide GCCGTGGGCTACTACAATAACATTCCCGTGATCGTGGC sequence) CCCCACTATTGCTTCCACCAATGCCCCCACCTCCGCCT

TGAGTGTGATCTATAAGGAGAACGGCGAATTCGAGGAA

TACTTGATGTTGCCTTTGAACCCTACCTTTGTGATCATG

GACACTAAGGTGATCGCCTCCGCTCCTGCCCGCTTATT

GGTTTCCGGCATGGGCGACGCCTTGGCCACTTACTTTG

AAGCCCGCGCCACTAAGCGGGCCAATAAGACCACCAT

GGCTGGTGGCCGCGTTACTGAGGCCGCCATCGCCTTG

GCCAAGTTGTGCTACGACACCCAGATCAGTGAGGGCTT GAAGGCCAAGTTAGCCGCCGAGAAACACTTGGTGACC

GAGGCCGTTGAGAAGATCATCGAGGCCAACACTTACTT

GTCCGGTATTGGCTCCGAAAGTGGCGGCTTAGCCGCC

GCTCACGCCATTCACAACGGCTTGACCGTGTTGGAGGA

GACCCACCACATGTACCACGGCGAGAAGGTGGC 1 1 1 CG

GCAC 1 1 1 GGCCCAGTTGATCTTGGAGGATGCCCCCAAG

GCCGAGATTGAGGAGGTGGTGTCCTTCTG 1 1 1 GTCCGT

GGGCTTGCCTGTTACCTTGGGCGACTTGGGCGTGAAG

GAATTGAACGAGGAAAAATTGCGGAAGGTGGCCGAATT

GTCCTGCGCCGAGGGCGAAACCATCTACAACATGCCCT

TTGAGGTGACCCCCGA 1 1 1 GGTGTACGCCGCCATCGTT

ACCGCTGACTCCGTGGGCCGCTATTACAAGGAAAAGTG

GGCCTAA

ATGGCTGTCTACAACTACGACGTGGTGATTTTGGGCAC

GGGTCCGGCGGGCGAAGGGGCGGCGATGAACGCCTC

CAAGTACGGACGCAAGCTGGCGGTAGTCGACAGCCGT

CGCGTGGTCGGAGGCAACTGCACGCACCTCGGCACCA

TTCCTTCCAAGGCCCTGCGCCACTCGGTGAAGCAGATC

ATCGAGTTCAACACCAACCCGATGTTCCGCCAGATCGG

CGAGCCGCGCTGGTTCTCCTTCCCCGACGTGCTGAAGA

GCGCCGACCGGGTGATCTCCAAGCAGGTCGCTTCGCG

TACCGGCTACTACGCGCGCAATCGCATCGACATGTTCA

CCGGCACCGCCAGCTTCGTCGACGAACGCACCGTCGA

AGTGGTGACGCCGAGCGGCGCGGTGGAGCGCCTGGT

CGCCGACCAGTTCGTGATCGCCACCGGCTCGCGCCCG

TATCGTCCTTCGGACATCAA 1 1 1 CAACCACCCGCGGGT

CTACGACAGCGACACCATCCTGTCCCTGAGCCACACCC

P. aeruginosa CGCGCCGGCTGATCATCTACGGAGCCGGGGTGATCGG sth (nucleotide CTGCGAATACGCGTCGATCTTCAGCGGCCTGGGCGTG sequence) CTGGTGGACCTGATCGATACGCGCGACCAGTTGCTCAG

CTTCCTCGATGACGAGA 1 1 1 CCGATGCGCTGAGCTACC

ACCTGCGCAACAACAACGTGCTGATCCGCCACAACGAG

GAGTACGAGCGCGTCGAGGGGCTGGACAACGGTGTCA

TCCTGCACCTGAAGTCGGGCAAGAAGATCAAGGCGGA

CGCGCTGCTCTGGTGCAACGGCCGGACCGGCAACACC

GACAAGCTCGGCCTGGAGAACGTCGGCATCAAGGTCA

ACAGCCGTGGCCAGATCGAGGTGGACGAGAACTACCG

CACTTCGGTGAGCAACATCTTCGCCGCTGGCGACGTGA

TCGGCTGGCCGAGCCTGGCCAGCGCTGCCTACGACCA

GGGCCGCTCGGCCGCCGGCAACATCGTCGAGAGCGAC

AGCTGGCGCTTCGTCAACGATGTGCCGACCGGCATCTA

CACCATTCCGGAGATCAGCTCGATCGGCAAGAACGAAA

GCGAACTGACCGCGGCGAAGATTCCCTACGAAGTGGG

CAAGGCGTTCTTCAAGGGCATGGCCCGGGCGCAGA I 1 1 CCAACGAGCCGGTGGGCATGCTGAAGATCCTGTTCCAT

CGCGAGACCCTGGAGATCCTCGGCGTGCACTGCTTCG

GCGACCAGGCTTCGGAAATCGTCCACATCGGCCAGGC

GATCATGAACCAGCCGGGCGAGCTGAATACCCTGAAGT

ACTTCGTCAACACCACCTTCAACTACCCGACCATGGCG

GAAGCCTATCGGGTAGCGGCGTTCGACGGCCTCAACC

GGCTTTTTTGA

MAVYNYDWILGTGPAGEGAAMNASKYGRKLAWDSRRV

VGGNCTHLGTIPSKALRHSVKQIIEFNTNPMFRQIGEPRWF

SFPDVLKSADRVISKQVASRTGYYARNRIDMFTGTASFVD

ERTVEWTPSGAVERLVADQFVIATGSRPYRPSDINFNHP

RVYDSDTILSLSHTPRRLIIYGAGVIGCEYASIFSGLGVLVDL

P. aeruginosa

IDTRDQLLSFLDDEISDALSYHLRNNNVLIRHNEEYERVEG

5 STH (amino

LDNGVILHLKSGKKIKADALLWCNGRTGNTDKLGLENVGIK

acid sequence)

VNSRGQIEVDENYRTSVSNIFAAGDVIGWPSLASAAYDQG

RSAAGNIVESDSWRFVNDVPTGIYTIPEISSIGKNESELTAA

KIPYEVGKAFFKGMARAQISNEPVGMLKILFHRETLEILGV

HCFGDQASEIVHIGQAIMNQPGELNTLKYFVNTTFNYPTM

AEAYRVAAFDGLNRLF

EXAMPLES

[0072] The following examples illustrate various iterations of the invention.

Example 1. Production of a D-lactate producing Synechocystis strain

[0073] Cyanobacteria need a lactate dehydrogenase to synthesize lactate from pyruvate (FIG. 1 A). As a first step, the activity of GlyDH^* for D-lactate production¹⁴ in Synechocystis was tested by transferring the gene from Bacillus coagulans to

Synechocystis 6803. A plasmid pYY1 was constructed that contained the gene gldA101 under the control of an Isopropyl β-D-l -thiogalactopyranoside (IPTG) inducible promoter, P_frc. The gldA101 gene was then subsequently transferred to the glucose tolerant wild type Synechocystis 6803 through natural transformation, generating the strain AV08. The optical density and the D-lactate concentration of the AV08 cultures were monitored in shake flasks. As can be verified from FIG. 1 D, AV08 did not show any significant levels of D-lactate in the initial 12 days. The D-lactate levels started increasing steadily at late autotrophic growth phase and reached a final titer of 0.4 g/L, whereas a wild type strain of Synechococcus 7002 was able to produce only ~ 7 mg/L of D-lactate through glucose fermentation¹⁵. Example 2. Optimization to increase D-lactate production by Synechocystis 6803

[0074] Cyanobacteria are known to have their own preference in the use of codons for synthesizing amino acids¹⁶. The codon optimized gene gldA101-syn

(synthesized by Genewiz Inc, South Plainfield, NJ) was integrated into the genome of the WT Synechocystis 6803 using the plasmid pDY3 to obtain the strain AV1 1 .

[0075] Further improvements in product synthesis can be achieved by rectification of bottlenecks in the metabolic pathway. The lactate dehydrogenase enzyme utilizes NADH as its cofactor. The ratio of NADH to NADPH is much lower in cyanobacteria. In Synechococcus 7942 under light conditions, the ratio of NADH to NADPH was estimated to be 0.15 ^{19, 2}°, and in Synechocystis 6903 under

photoautotrophic conditions the intracellular NADH concentration was only 20 nmol/g fresh weight, whereas the NADPH intracellular concentration was about 140 nmol/g fresh weight. To determine whether NADH is a major limiting factor for synthesizing D- lactate in cyanobacteria, a soluble transhydrogenase, sth from Pseudomonas aeruginosa² was introduced downstream of the gene gldA101-syn. This engineered strain was called AV10. The heterologous genes in AV10 and AV1 1 are under the control of the same, single promoter, P_frc, located upstream of gldA101-syn and sth in AV10 and located upstream of gldA101-syn in AV1 1 .

[0076] The three strains (AV08, AV10 and AV1 1 ) had similar growth rates (FIG. 1 C), and the production of D-lactate did not introduce any growth defects in the engineered strains under photoautotrophic conditions (FIG. 5). However, the three strains differed completely in the production rate of D-lactic acid. The strain AV1 1 with codon optimization (gldA101-syn) had an improved productivity for D-lactate compared to the AV08 strain (FIG. 1 D). Both strains produced D-lactate mainly during the later growth state. Introduction of the transhydrogenase improved the D-lactate synthesis further in AV10, and this strain produced D-lactate in both growth phase and non-growth phase. The rate of photoautotrophic D-lactate production by AV10 increased

significantly (achieving a maximum productivity of 0.19 g/L/day; Table 1) during the late phase of the culture and the final titer of D-lactate reached 1 .14 g/L. Table 1. Autotrophic peak production rate

Strain Autotrophic peak production rate + 15 mM acetate

Wildtype - -

AV08 0.05 g/L/day N/A

AV10 0.08 g/L/day 0.19/g/L/day

AV1 1 0.05 g/L/day N/A

Example 3. Supplementation with acetate

[0077] It was observed that the D-lactate production rate reaches its peak in the later stages of cultivation, suggesting that more carbon flux has been directed to lactate production during the non-growth phase. This is because the lactate precursor (pyruvate) is a key metabolic node occupying a central position in the synthesis of biomass components and more pyruvate becomes available for lactate synthesis when biomass growth becomes slow.

[0078] In order to enhance lactate production, supplementation with pyruvate was first considered²². However, our experiments found that addition of pyruvate did not yield apparent improvements in D-lactate synthesis. An alternate option was to grow AV10 with glucose and increase the glycolysis flux towards pyruvate synthesis (note: glucose significantly increased Synechocystis 6803 isobutanol production²³). The growth rate of the AV10 cells in the mixotrophic cultures

(supplemented with 5 g/L glucose), was initially higher than the autotrophic cultures, but the rate started decreasing after 9 days (FIG. 2A). As a result, the final mixotrophic D- lactate productivity of the AV10 strain was only 1 g/L (similar to autotrophic condition).

[0079] A second approach to increase the pyruvate pool for lactate production was tested, by adding exogenous acetate. Supplementing cultures with acetate can redirect more carbon from pyruvate to lactate by three possible means²⁴: (1 ) acetate is used as a building block for lactate production; (2) acetate provides additional carbon source for biomass synthesis and reduce pyruvate consumption; (3) acetate conversion by acetyl-CoA synthetase consumes Coenzyme-A (CoA), decreasing the CoA pool available for pyruvate decarboxylation. To test this hypothesis, the AV10 cultures were supplemented with 15mM acetate. The growth rate of the AV10 cultures with acetate (FIG. 2A) remained comparable to their completely autotrophic condition, but there was substantial improvement in D-lactate productivity (the final titer reached 2.17 g/L, FIG. 2B).

[0080] To further understand the role played by glucose and acetate in D- lactate synthesis, AV10 cultures were grown with [1 ,2-¹³C] glucose and [1 ,2-¹³C] acetate (Sigma, St. Louis). Cultures were collected from the mid-log phase and were used for amino acid and D-lactate analysis. GC-MS analysis was used to evaluate culture supernatants for D-lactate. AV10 cultures grown with ¹³C glucose and acetate were compared to a standard. The mass spectrum of D-lactate from a cyanobacterial culture was compared to the mass spectrum of standard. A suitable short chain fatty acid standard is propionic acid (or propanoic acid). The ¹³C abundance in the amino acids and lactate were obtained as mass fraction m,, where T indicates the number of ¹³C in the molecule. As can be seen from FIG. 3A, glucose-fed cells have significant ¹³C- carbon distributed in amino acids (indicated by an increase in mi and m₂). Also, D- lactate from glucose-fed cultures was partially ¹³C-labeled (m2 -0.22). The isotopomer data in FIG. 3A, proves that ¹³C-glucose provided the carbon source for both biomass and lactate production. However, glucose-based mixotrophic fermentation is not beneficial to D-lactate production compared to autotrophic cultures, possibly because carbon flux from glycolysis may cause some carbon and energy imbalance²³. For the acetate cultures (FIG. 3B), leucine and glutamate (both use acetyl-CoA as their precursor) were labeled (an m₂ of 0.31 and 0.32 respectively), while other amino acids (e.g., aspartate and alanine) were nonlabeled (note: natural ¹³C makes up about 1 .1 % of total carbon as measurement background). Interestingly, D-lactate from acetate-fed culture was almost nonlabeled, indicating that the carbons of lactate molecules were mainly derived from CO2. Our measurements also showed that the acetate in the culture medium was not used significantly during the cell growth. Therefore, the observation of acetate enhancement for lactate production can be explained by two complementary mechanisms. First, acetate is an additional carbon source for synthesizing biomass building blocks, such as fatty acids and some amino acids, thus redirecting the extra carbon flux from CO2 to lactate. Secondly, acetate may limit the pyruvate decarboxylation reaction by reducing the CoA pool by the formation of acetyl- CoA and thus improve D-lactate production.

Example 4. Further modifications

[0081 ] The results reported here are for the autotrophic production of D- lactate in cyanobacteria via rational metabolic engineering approaches. Other molecular strategies may also be applied to further improve the D-lactate production: (1 ) by seeking stronger promoters; (2) optimizing ribosomal binding sites; (3) improving activity of GlyDH^* via protein engineering; (4) introducing powerful lactate transporter; (5) knocking out competing pathways (such as the glycogen and polyhydroxybutyrate synthesizing pathways); (6) duplicating the heterologous genes by integrating at multiple sites; (7) switching to a photosynthetic microorganism that has a higher growth rate; and (8) limiting biomass production by nutrient control or knocking down pyruvate decarboxylation reaction. Also, considering the future outdoor algal processes for scaling up D-lactate production, removal of polyhydroxybutyrate (PHB) and glycogen pathway may be deleterious to the algal growth under day-night cultivation modes²⁵. On the other hand, process optimization by employing better light conditions to increase the cell density, along with proper CO2, pH and temperature control, may also be employed to increase the D-lactate productivity in a scaled-up system.

[0082] Acetate for the algal culture can be supplemented from various cheap sources. For example, acetate can be accumulated in a modified waste/sludge anaerobic digestion process. [Xiao Y, Ruan Z, Liu Z, Wu SG, Varman AM, Liu Y, Tang YJ. Engineering Escherichia coli to convert acetic acid to free fatty acids, Biochemical Engineering Journal. 2013. 76: 60-69.] This will reduce the production cost of the D- lactate synthesis by providing cheaper acetate and also by acting as a cheap source for macronutrients.

References for Examples 1 -4

1 . A partial list of products made from Petroleum, www.ranken- energy.com/Products%20from%20Petroleum.htm. 2. O. G. Piringer and A. L. Baner, Plastic packaging : interactions with food and pharmaceuticals, Wiley-VCH, Weinheim, 2008.

3. J. Vijayakumar, R. Aravindan and T. Viruthagiri, Recent trends in the production, purification and application of lactic acid, Chem Biochem Eng Q, 2008, 22, 245-264.

4. D. Garlotta, A literature review of poly(lactic acid), Journal of Polymers and the Environment, 2001 , 9, 63-84.

5. S. Taskila and H. Ojamo, The Current Status and Future Expectations in Industrial Production of Lactic Acid by Lactic Acid Bacteria, 2013.

6. B. Wang, J. Wang, W. Zhang and D. R. Meldrum, Application of synthetic biology in cyanobacteria and algae, Front Microbiol, 2012, 3, 344.

7. Y. Yu, L. You, D. Liu, W. Hollinshead, Y. Tang and F. Zhang, Development of Synechocystis sp. PCC 6803 as a Phototrophic Cell Factory, Marine Drugs, 2013, 1 1 , 2894-2916.

8. G. F. Wu, Z. Y. Shen and Q. Y. Wu, Modification of carbon partitioning to enhance PHB production in Synechocystis sp PCC6803, Enzyme and Microbial Technology, 2002, 30, 710-715.

9. Y. Xiao, Z. H. Ruan, Z. G. Liu, S. G. Wu, A. M. Varman, Y. Liu and Y. J. J. Tang, Engineering Escherichia coli to convert acetic acid to free fatty acids, Biochemical Engineering Journal, 2013, 76, 60-69.

10. H.-H. Huang, D. Camsund, P. Lindblad and T. Heidorn, Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology, Nucleic acids research, 2010, 38, 2577-2593.

1 1 . H. H. Huang and P. Lindblad, Wide-dynamic-range promoters engineered for cyanobacteria, Journal of biological engineering, 2013, 7, 10.

12. A. Joseph, S. Aikawa, K. Sasaki, Y. Tsuge, F. Matsuda, T. Tanaka and A. Kondo, Utilization of Lactic Acid Bacterial Genes in Synechocystis sp. PCC 6803 in the

Production of Lactic Acid, Bioscience, Biotechnology, and Biochemistry, 2013, 77, 966- 970. 13. S. A. Angermayr, M. Paszota and K. J. Hellingwerf, Engineering a Cyanobacterial Cell Factory for Production of Lactic Acid, Applied and Environmental Microbiology, 2012, 78, 7098-7106.

14. Q. Wang, L. O. Ingram and K. T. Shanmugam, Evolution of D-lactate

dehydrogenase activity from glycerol dehydrogenase and its utility for D-lactate production from lignocellulose, Proceedings of the National Academy of Sciences, 201 1 , 108, 18920-18925.

15. K. McNeely, Y. Xu, N. Bennette, D. A. Bryant and G. C. Dismukes, Redirecting Reductant Flux into Hydrogen Production via Metabolic Engineering of Fermentative Carbon Metabolism in a Cyanobacterium, Applied and Environmental Microbiology, 2010, 76, 5032-5038.

16. W. H. Campbell and G. Gowri, Codon Usage in Higher-Plants, Green-Algae, and Cyanobacteria, Plant Physiology, 1990, 92, 1 -1 1 .

17. P. Lindberg, S. Park and A. Melis, Engineering a platform for photosynthetic isoprene production in cyanobacteria, using Synechocystis as the model organism, Metabolic Engineering, 2010, 12, 70-79.

18. J. Ungerer, L. Tao, M. Davis, M. Ghirardi, P.-C. Maness and J. Yu, Sustained photosynthetic conversion of CO2 to ethylene in recombinant cyanobacterium

Synechocystis 6803, Energy & Environmental Science, 2012, 5, 8998-9006.

19. W. F. J. Vermaas, in el_S, John Wiley & Sons, Ltd, 2001 .

20. M. Tamoi, T. Miyazaki, T. Fukamizo and S. Shigeoka, The Calvin cycle in

cyanobacteria is regulated by CP12 via the NAD(H)/NADP(H) ratio under light/dark conditions, The Plant Journal, 2005, 42, 504-513.

21 . B. Wermuth and N. O. Kaplan, Pyridine nucleotide transhydrogenase from

Pseudomonas aeruginosa: Purification by affinity chromatography and physicochemical properties, Archives of Biochemistry and Biophysics, 1976, 176, 136-143.

22. T. M. Bricker, S. Zhang, S. M. Laborde, P. R. Mayer, L. K. Frankel and J. V.

Moroney, The Malic Enzyme Is Required for Optimal Photoautotrophic Growth of Synechocystis sp. Strain PCC 6803 under Continuous Light but Not under a Diurnal Light Regimen, Journal of Bacteriology, 2004, 186, 8144-8148. 23. A. M. Varman, Y. Xiao, H. B. Pakrasi and Y. J. Tang, Metabolic engineering of Synechocystis 6803 for isobutanol production, Applied and Environmental Microbiology, 2012, 79, 908-914.

24. V. F. Wendisch, A. A. de Graaf, H. Sahm and B. J. Eikmanns, Quantitative

Determination of Metabolic Fluxes during Coutilization of Two Carbon Sources:

Comparative Analyses With Corynebacterium glutamicum during Growth on Acetate and/or Glucose, Journal of Bacteriology, 2000, 182, 3088-3096.

25. M. Griindel, R. Scheunemann, W. Lockau and Y. Zilliges, Impaired glycogen synthesis causes metabolic overflow reactions and affects stress responses in the cyanobacterium Synechocysti sp. PCC 6803, Microbiology, 2012, 158, 3032-3043.

Methods for Examples 1 -4

[0083] Chemicals and reagents. Restriction enzymes, Phusion DNA polymerase, T4 DNA ligase and 10-Beta electro-competent E. coli kit were purchased from Fermentas or New England BioLabs. Oligonucleotides were purchased from Integrated DNA Technologies (IDT). All organic solvents, chemicals, ¹³C-labeled acetate, and glucose used in this study were purchased from Sigma-Aldrich (St. Louis, MO).

[0084] Medium and growth conditions. E. coli strain 10-Beta was used as the host for all plasmids constructed in this study. E. coli cells were grown in liquid Luria-Bertani (LB) medium at 37°C in a shaker at 200 rpm or on solidified LB plates. Ampicillin (100 pg/ml) or kanamycin (50 pg/ml) was added to the LB medium when required for propagation of the plasmids in E. coli. The wild-type (glucose-tolerant) and the recombinant strain of Synechocystis 6803 were grown at 30°C in a liquid blue-green medium (BG-1 1 medium) or on solid BG-1 1 plates at a light intensity of 100 μιτιοΙ of photons m-2s-1 in ambient air. Kanamycin (20 pg/ml) was added to the BG-1 1 growth medium as required. Growth of the cells was monitored by measuring their optical density at 730 nm (OD730) with an Agilent Cary 60 UV-vis spectrophotometer. Cultures for the synthesis of D-lactate were grown (initial OD730, 0.4) in 50-ml shake flasks without any antibiotic and 1 mM Isopropyl β-D-l -thiogalactopyranoside (IPTG) was added for induction. Mixotrophic cultures of Synechocystis 6803 were started in BG-1 1 medium containing a known amount of glucose (0.5%) or acetate (15mM) as an organic carbon source.

[0085] Plasmid construction and transformation. The vector pTKA3 served as the backbone for all the plasmids constructed in this study. The gene gldA101 encoding GlyDH^* 2, was amplified from the plasmid pQZ1 15 with the primers gldA-o-F2 and gldA-o-R (Table 2 and 3). The obtained 1 .2 kb fragment was digested with

BamHI/Nhel and cloned into the same restriction sites of pTKA3, yielding the vector pYY1 . A gene cassette, which consists of the codon optimized gldA101 (gldA-syn) with the promoter Ptrc in the upstream and the transhydrogenase (sth) gene from

Pseudomonas aeruginosas in the downstream, was chemically synthesized by Genewiz Inc (South Plainfield, NJ). The whole gene cassette was digested with BamHI/Nhel, and the yielding 2.6 kb fragment was cloned into the corresponding restriction sites of pTKA3, resulting in the vector pDY2. The vector pDY3 was constructed by self-ligation of the 8.2 kb fragment obtained through the digestion of pDY2 with Kpnl.

[0086] Natural transformation of Synechocystis 6803 was performed by using a double homologous-recombination procedure as described previously4.

Recombinant colonies appeared between 7 and 10 days post inoculation. For segregation, the positive colonies were propagated continuously onto BG-1 1 plates containing kanamycin. The genes of interest were finally integrated into the psbA1 gene loci into the genome of Synechocystis 6803. For segreagation, the positive colonies were propagated continuously onto BG-1 1 plates containing kanamycin and

segregation of colonies was verified through a colony PCR with primers AMV17R and ps1_up_fwda (Table 2). The promoter and the heterologous genes in the engineered strains were PCR amplified with respective primers (ptka3-F, CO-F, O-F, sth-F) and sent for sequencing to Genewiz to verify the cloning accuracy.

[0087] D (-) lactate analysis. D(-)/L(+) lactic acid detection kit (R-biopharm) was used to measure the D-lactate concentration. Samples of the cyanobacterial culture (50 μΙ) were collected every 3 days and centrifuged at 12,000 rpm for 5 min. The supernatant was collected and the D-lactate concentration assay was performed following the manufacturer's instruction. All the reactions were performed in a 96-well plate reader at room temperature (Infinite 200 PRO microplate photometer, TECAN).

[0088] ¹³C isotopomer experiment. To estimate the carbon contributions of glucose and acetate for biomass and D-lactic acid synthesis a ¹³C labeling experiment was performed. The mutant AV10 was grown in a BG-1 1 medium with 0.5% glucose (1 ,2-¹³C₂ glucose) or 15mM acetate (U-¹³C₂ acetate) (Sigma, St. Louis). Cultures were started at an OD730 of 0.4 and were grown with labeled glucose or acetate for over 48 hours. The biomass samples and supernatant were collected for measurement of lactate and amino acid labeling.

[0089] The proteinogenic amino acids from biomass were hydrolyzed and then derivatized with TBDMS [N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide], as described previously⁵. The derivatized amino acids were analyzed for their ¹³C mass fraction by GC-MS (Hewlett Packard 7890A and 5975C, Agilent Technologies, USA) equipped with a DB5-MS column (J&W Scientific) 5. The fragment [M-57]⁺ containing information of the entire amino acid was used for calculating the ¹³C mass fractions. The m/z of [M-15]⁺ was used only for leucine, since its [M-57]⁺ overlaps with other mass peak 6. For isotopomer analysis of extracellular D-lactic acid, the supernatant was first freeze-dried at -50°C. The dried samples were then pre-derivatized with 200 μΙ_ of 2% methoxyamine hydrochloride in pyridine for 60 minutes at 37 °C and then derivatized with 300 μΙ_ N-Methyl-N-(trimethylsilyl) trifluroacetamide (TMS) for 30 minutes at room temperature. The derivatized D-lactic acid was analyzed for their ¹³C mass fractions by GC-MS. The detailed measurement protocol can be found in a previous paper⁷.

References for Methods

1 . A. M. Varman, Y. Xiao, H. B. Pakrasi and Y. J. Tang, Metabolic engineering of Synechocystis 6803 for isobutanol production, Applied and Environmental

Microbiology, 2012, 79, 908-914.

2. Q. Wang, L. O. Ingram and K. T. Shanmugam, Evolution of D-lactate

dehydrogenase activity from glycerol dehydrogenase and its utility for D-lactate production from lignocellulose, Proceedings of the National Academy of Sciences, 201 1 , 108, 18920-18925.

3. B. Wermuth and N. O. Kaplan, Pyridine nucleotide transhydrogenase from

Pseudomonas aeruginosa: purification by affinity chromatography and

physicochemical properties, Archives of biochemistry and biophysics, 1976, 176, 136- 143.

4. X. N. Zang, B. Liu, S. M. Liu, K. Arunakumara and X. C. Zhang, Optimum conditions for transformation of Synechocystis sp. PCC 6803, Journal of Microbiology, 2007, 45, 241 -245.

5. L. You, L. Page, X. Feng, B. Berla, H. B. Pakrasi and Y. J. Tang, Metabolic pathway confirmation and discovery through 13C-labeling of proteinogenic amino acids, J Vis Exp, 2012, e3583.

6. M. R. Antoniewicz, J. K. Kelleher and G. Stephanopoulos, Accurate assessment of amino acid mass isotopomer distributions for metabolic flux analysis, Analytical chemistry, 2007, 79, 75547559.

7. Y. Tang, W. Shui, S. Myers, X. Feng, C. Bertozzi and J. Keasling, Central metabolism in Mycobacterium smegmatis during the transition from O2-rich to O2-poor conditions as studied by isotopomer-assisted metabolite analysis, Biotechnology Letters, 2009, 31 , 1233-1240.

8. J. Brosius, M. Erfle and J. Storella, Spacing of the -10 and -35 regions in the tac promoter. Effect on its in vivo activity, J Biol Chem, 1985, 260, 3539-3541 .

Table 2. Primer sequences

10 gldA-o-R2 TTAGGCCCACTTTTCCTTGTAATAGC

1 1 tranNADH-F CCTAAGCTAGCGGAGGACTAGCATGG

12 tranNADH-R GCTAGCGGTACCTCAAAAAAGCCGG

13 ptka3-F CCCGAAGTGGCGAGCCCGAT

14 CO-F TTGATGTTGCCTTTGAACCC

15 O-F ATGGATACGAAAGTGATTGC

16 sth-F GAGCTACCACCTGCGCAACA

17 GCGCGACTCCCCGTCTTTGACTATCCTTTTTAGG

AMV17R

ATGGGGCA

18 ps1_up_fwda TACCGGAACAGGACCAAGCCTT

Table 3. Plasmids and strains

Example 5. Synthesis of C-labeled D-lactate from engineered Synechocystis 6803 strain.

[0090] Method: The D-lactate strain uses CO₂ or NaHCO₃ for lactate production. Our hypothesis was that if we feed cell with only ¹³C-labeled NaHCO3, lactate will be fully labeled. The fully labeled lactate product is useful for medical research (such as cancer metabolism studies) and has much higher commercial price (~1000$/g). Thereby, we can use cheap ¹³C-NaHCO₃ (50$/g) to synthesize ¹³C-lactate. In our study, we grew the engineered cyanobacteria strain in a closed bottle with ¹³C bicarbonate (-98% labeled NaHCOs) as the only carbon source (no atmospheric ¹²CO₂) to middle log growth phase.

[0091 ] Results: FIG. 6 displays, we have successfully achieved 85% fully labeled, 1 1 % partially labeled, and 4% unlabeled of extracellular lactate. Overall labeling reaches a ¹³C enrichment of- 92% (percentage of carbons that are carbon-13 isotopes). Thereby, we can use cheap ¹³C bicarbonate to synthesize expensive labeled D-lactate as reagents for research use.

Example 6. Determine the influence of acetate and pH on D-Lactate

synthesis/accumulation inside the cell.

[0092] Method: To delineate the lactate secretion from its

production/accumulation inside the cell, we measured the intracellular concentration of D-Lactate. To quantify the intracellular D-lactate, we grew the cells under normal conditions in open shaking flasks. For time series data, we extracted on day 3 and day 12 (for the early/exponential phase of AV10 growth) and day 18 and 24 (for the late/stationary growth). To measure the influence of acetate and neutral pH on the accumulation of D-Lactate intracellularly, the cells were grown under normal conditions until day 18 (late stage/high D-lactate performance). Then placed under the influence of 1 g/L of sodium acetate or reduction of pH to pH 7 (adjusted using 6M HCI and determined by pH strips), respectively. Quantification of D-lactate was done by using a 99% 3-¹³C L-Lactate. [0093] Statistical analysis. Simple t-test proved that there is significant increase (95% confidence) of the intracellular D-lactate concentration when cells were placed in neutral pH (=7) (FIG. 7). This result proves that pH places important role on lactate production. If cells were under neutral pH, cells cannot secrete D-lactate outside and thus causing inhibition to cell physiology. To promote lactate production, we should grow cells in slight alkaline first, then increase pH to 10-1 1 during late growth phase for milking lactate out.

[0094] Future studies. We hope to expand our analysis of acetate and neutral culture conditions on the intracellular D-lactate accumulation by increasing our sample sizes. In addition, we are currently investigating the influence of glucose mixotrophic growth on lactate intracellular accumulation and strain growth. We will also engineer lactate transporter to improve the lactate secretion.

Claims

CLAIMS What is claimed is:

1 . A method for producing D-lactate using a phototrophic microorganism, the

method comprising culturing a photosynthetic microorganism comprising an exogenous D-lactate dehydrogenase in the presence of light and CO2, such that the D-lactate is substantially derived from CO2; wherein the D-lactate

dehydrogenase (a) comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 , or (b) is encoded by a nucleic acid sequence with at least 80% identity to SEQ ID NO: 2.

2. The method of claim 1 , wherein the D-lactate is optically pure.

3. The method of claim 1 , wherein the phototrophic microorganism further

comprises an exogenous soluble pyridine nucleotide transhydrogenase.

4. The method of claim 3, wherein the exogenous soluble pyridine nucleotide

transhydrogenase (a) is encoded by a nucleic acid sequence with at least 80% identity to SEQ ID NO: 4, or (b) comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 5.

5. The method of claim 1 , wherein the method further comprises decreasing the amount of endogenous Coenzyme A in the phototrophic microorganism.

6. The method of claim 1 , wherein the method further comprises decreasing

pyruvate decarboxylation activity in the phototrophic microorganism.

7. The method of claim 5 or 6, wherein the method comprises culturing the

microorganism in the presence of exogenous acetate.

8. The method of claim 7, wherein the amount of acetate is at least about 15 mM and the final titer of D-lactate is at least about 2 g/L.

9. The method of claim 1 , wherein the phototrophic microorganism is a

Synechocystis PCC sp. 6803 cyanobacterium.

10. The method of claim 1 , wherein the nucleic acid encoding the D-lactate

dehydrogenase is codon-optimized for expression in the microorganism.

1 1 . The method of claim 10, wherein the microorganism is a Synechocystis PCC sp.

6803 cyanobacterium and the nucleic acid has at 80% identity to SEQ ID NO: 3.

12. The method of claim 1 , further comprising culturing the photosynthetic

microorganism in the presence of elevated pH.

13. The method of claim 12, wherein the pH is initially slightly alkaline and then the alkalinity of the culture is further increased during late stage growth.

14. The method of claim 13, wherein the pH is initially greater than 7 but less than 10.

15. The method of claim 13, wherein the pH is increased to about 10 to about 12 during late stage growth.

16. A phototrophic microorganism comprising an exogenous nucleic acid construct, wherein the nucleic acid construct comprises a promoter operably-linked to (a) a nucleic acid sequence with at least 80% identity to SEQ ID NO: 2, or (b) a nucleic acid sequence encoding a polypeptide with at least 80% identity to SEQ ID NO: 1 .

17. The phototrophic microorganism of claim 16, wherein the promoter is an inducible promoter or a constitutive promoter.

18. The phototrophic microorganism of claim 16, wherein the photosynthetic

microorganism further comprises an exogenous soluble pyridine nucleotide transhydrogenase.

19. The phototrophic microorganism of claim 16, wherein the exogenous soluble pyridine nucleotide transhydrogenase (a) is encoded by a nucleic acid sequence with at least 80% identity to SEQ ID NO: 4, or (b) comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 5.

20. The phototrophic microorganism of claim 16, wherein the photosynthetic

microorganism is a Synechocystis PCC sp. 6803 cyanobacterium.

21 . The phototrophic microorganism of claim 16, wherein the microorganism has a photoautotrophic D-lactate production rate of at least about 0.15 g/L/day.

22. The phototrophic microorganism of claim 16, wherein the microorganism has a photoautotrophic D-lactate production rate of at least about 0.19 g/L/day.

23. The phototrophic microorganism of claim 16, wherein the nucleic acid encoding the D-lactate dehydrogenase is codon-optimized for expression in the microorganism.

24. The phototrophic microorganism of claim 16, wherein the microorganism is a Synechocystis PCC sp. 6803 cyanobacterium and the nucleic acid has at 80% identity to SEQ ID NO: 3.

25. A method for producing labeled D-lactate using a phototrophic microorganism, the method comprising culturing a photosynthetic microorganism comprising an exogenous D-lactate dehydrogenase in the presence of light and a labeled carbon source, such that the D-lactate is substantially labeled; wherein the D- lactate dehydrogenase (a) comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 , or (b) is encoded by a nucleic acid sequence with at least 80% identity to SEQ ID NO: 2.

26. The method of claim 25, wherein the D-lactate is greater than 80% labeled.

27. The method of claim 25, wherein the phototrophic microorganism further

comprises an exogenous soluble pyridine nucleotide transhydrogenase.

28. The method of claim 27, wherein the exogenous soluble pyridine nucleotide transhydrogenase (a) is encoded by a nucleic acid sequence with at least 80% identity to SEQ ID NO: 4, or (b) comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 5.

29. The method of claim 25, wherein the method further comprises decreasing the amount of endogenous Coenzyme A in the phototrophic microorganism.

30. The method of claim 25, wherein the method further comprises decreasing

pyruvate decarboxylation activity in the phototrophic microorganism.

31 . The method of claim 29 or 30, wherein the method comprises culturing the

microorganism in the presence of exogenous acetate.

32. The method of claim 31 , wherein the amount of acetate is at least about 15 mM and the final titer of D-lactate is at least about 2 g/L.

33. The method of claim 25, wherein the phototrophic microorganism is a Synechocystis PCC sp. 6803 cyanobacterium.

34. The method of claim 25, wherein the nucleic acid encoding the D-lactate

dehydrogenase is codon-optimized for expression in the microorganism.

35. The method of claim 34, wherein the microorganism is a Synechocystis PCC sp.

6803 cyanobacterium and the nucleic acid has at 80% identity to SEQ ID NO: 3.

36. The method of claim 25, wherein the labeled carbon source is ¹³C labeled.

37. The method of claim 25, wherein the labeled carbon source is ¹³C labeled

NaHCO₃.

38. The method of claim 25, further comprising culturing the photosynthetic

microorganism in the presence of elevated pH.

39. The method of claim 38, wherein the pH is initially slightly alkaline and then the alkalinity of the culture is further increased during late stage growth.

40. The method of claim 39, wherein the pH is initially greater than 7 but less than 10.

41 . The method of claim 39, wherein the pH is increased to about 10 to about 12 during late stage growth.