WO2014087184A1

WO2014087184A1 - Neopentyl glycol fermentative production by a recombinant microorganism

Info

Publication number: WO2014087184A1
Application number: PCT/IB2012/002833
Authority: WO
Inventors: Céline RAYNAUD; Laurence Dumon-Seignovert; Guillaume Letellier; Véronique DOUCHIN
Original assignee: Metabolic Explorer SA
Current assignee: Metabolic Explorer SA
Priority date: 2012-12-03
Filing date: 2012-12-03
Publication date: 2014-06-12
Anticipated expiration: 2015-06-03

Abstract

The invention relates to a method for the fermentative production of neopentyl glycol comprising culturing a recombinant microorganism in an appropriate culture medium comprising a source of carbon, wherein in said microorganism, the neopentyl glycol biosynthesis pathway comprises 2-dehydropantoate as intermediate product that is converted into neopentyl glycol by two or three successive steps including a decarboxylation and an aldehyde reduction.

Description

NEOPENTYL GLYCOL FERMENTATIVE PRODUCTION BY A

RECOMBINANT MICROORGANISM

FIELD OF THE INVENTION

The present invention relates to a new method for the fermentative production of neopentyl glycol by culturing a recombinant microorganism in an appropriate culture medium. The microorganism is genetically modified in a way that it comprises a neopentyl glycol biosynthesis pathway comprising 2-dehydropantoate as intermediate product. PRIOR ART

Neopentyl glycol (CAS number 126-30-7) has the following formula:

Neopentyl glycol or 2,2-dimethyl-l,3-propanediol is an organic chemical compound with two alcohol groups.

Neopentyl glycol is chemically manufactured by the aldol reaction of formaldehyde and isobutyraldehyde. This reaction creates the intermediate hydroxypivaldehyde which is converted to neopentyl glycol with excess of formaldehyde or by catalytic hydrogenation o f the al dehyde group to an al co ho l group (EP343475, WO 1999/0351 12, WO2004/078691, US20080004475, WO2008/051143, US2012/0271029).

This compound is mainly used as a building block in the synthesis of polyesters resins for coatings and enhances the stability of the products towards heat, light and water. It is also used as buildings blocks in synthesis of unsaturated polyesters, paints, lubricants and plasticizers.

Thus, there exists a need for alternative method for efficiently producing commercial quantities of neopentyl glycol. In this context, the inventors have found a biosynthesis pathway to produce neopentyl glycol by fermentation. Fermentative production of neopentyl glycol has never been described and there is no biosynthesis pathway of neopentyl glycol described in the prior art.

The biological production of neopentyl glycol requires the formation of 2- dehydropantoate (common synonym 4-hydroxy-3,3-dimethyl-2-oxobutanoate) from pyruvate. This compound is a key intermediate of the biosynthesis pathway of pantothenic acid well known in the art and disclosed in particular in patent applications WOO 1/21772, WO02/061108, WO02/057474, WO2010/018196 and WO 2012/145179. The biosynthesis pathway from pyruvate to 2-dehydropantoate disclosed in the prior art includes a biosynthesis pathway from pyruvate to 3-methyl-2-oxobutanic acid (also called a- ketoisovalerate) in three common steps with the biosynthesis pathway of isobutanol well known in the art and disclosed in particular in patent applications, WO2007/050671, WO2008/098227, WO2008/130995, US2009/0081746, WO2011/142865, US2011112334.

The problem to be solved by the present invention is the development of a biosynthesis pathway for the production of neopentyl glycol. The inventors have found a method for the fermentative production of neopentyl glycol from a source of carbon, comprising 2-dehydropantoate as intermediate product, wherein said method is performed by a recombinant microorganism on an appropriate culture medium comprising said source of carbon.

SUMMARY OF THE INVENTION

The present invention is related to a method for the fermentative production of neopentyl glycol, comprising culturing a recombinant microorganism in an appropriate culture medium comprising a source of carbon, the biosynthesis pathway comprising the following steps:

a) Production of 2-dehydropantoate from the carbon source; and b) Conversion of 2-dehydropantoate into neopentyl glycol;

wherein in said microorganism, the conversion of 2-dehydropantoate into neopentyl glycol is catalyzed by two or three successive steps including a step of decarboxylation and a step of aldehyde reduction.

In a specific embodiment of the invention the intermediate product 2- dehydropantoate is converted into neopentyl glycol by successive actions of an enzyme having alpha keto acid decarboxylase activity and then of an enzyme having aldehyde reductase activity.

In another specific embodiment of the invention the intermediate product 2- dehydropantoate is converted into neopentyl glycol by successive actions of an enzymatic complex having keto-acid dehydrogenase activity, of an enzyme having aldehyde oxidoreductase (CoA-acylating) activity and then of an enzyme having aldehyde reductase activity.

Moreover, the invention relates to a microorganism for the fermentative production of neopentyl glycol wherein said microorganism overexpresses at least one of the following genes: kivD, yqhD, adhE, bkdAl, bkdA2, bkdB and IpdV or genes originating from the evolution of these genes. BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawing which is incorporated in and constitute a part of this specification exemplifies the invention and together with the description serve to explain the principles of this invention.

Figure 1 : Metabolic pathways for the biosynthesis of neopentyl glycol from 2- dehydropantoate .

Figure 2: Metabolic pathways for the biosynthesis of pantoate from pyruvate.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified methods and may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting, which will be limited only by the appended claims.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. However, publications mentioned herein are cited for the purpose of describing and disclosing the protocols, reagents and vectors that are reported in the publications and that might be used in connection with the invention.

Furthermore, the practice of the present invention employs, unless otherwise indicated, conventional microbiological and molecular biological techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, for example, Prescott et al. (1999) and Sambrook et al. (1989) (2001).

It must be noted that as used herein and in the appended claims, the singular forms

"a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a microorganism" includes a plurality of such microorganisms, and a reference to "an exogenous gene" is a reference to one or more exogenous genes, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred materials and methods are now described.

In the claims that follow and in the consecutive description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise", "contain", "involve" or "include" or variations such as "comprises", "comprising", "containing", "involved", "includes", "including" are used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

Definitions

The term "neopentyl glycol" (IUPAC name 2,2-dimethyl-l,3-propanediol, common synonyms dimethyltrimethylene glycol, neopentylene glycol, neopentanediol) designates the organic chemical compound with the formula C5H12O2 and with the CAS number 126-30-7.

The term "biosynthesis pathway" designates the different steps catalysed by different enzymes that convert a source of carbon, such as glucose or sucrose for instance, into neopentyl glycol, with 2- dehydropantoate as intermediate product in a microorganism.

The term "convert" or "conversion" designates the chemical step that transforms a product A into the product B and catalysed by an enzyme.

The term "2-dehydropantoate" (common synonym a-ketopantoate) designates the

2-oxo monocarboxylic acid with chemical formula C₆H₉O₄, key intermediate in pantothenate biosynthesis.

The term "pyruvate" designates the 2-oxoacid or a-ketoacid with chemical formula C₃H₄O₃.

The term "microorganism", as used herein, refers to a bacterium, archae or fungus which is not modified artificially. Preferentially, the microorganism of the invention is chosen among the Enterobacteriaceae, Clostridiaceae, Bacillaceae, Streptomycetaceae, Corymb acteriaceae, and Saccharomycetaceae families. More preferentially, the microorganism is a species of Escherichia, Klebsiella, Pantoa, Salmonella, Clostridium, Bacillus, Pseudomonas, Corynebacterium, or Saccharomyces.

The term "recombinant microorganism" or "genetically modified microorganism", as used herein, refers to a microorganism genetically modified or genetically engineered. It means, according to the usual meaning of these terms, that the microorganism of the invention is not found in nature and is modified either by introduction, by deletion or by modification of genetic elements. It can also be transformed by forcing the development and evolution of new metabolic pathways in combining mutagenesis and evolution under specific selection pressure (see for instance WO 2004/076659).

The term "decarboxylation" designates a chemical reaction that releases carbon dioxide (C0₂) from a carboxylic acid function.

The term "aldehyde reduction" designates a chemical reaction of hydrogenation of an aldehyde function to obtain an alcohol function. The terms "overexpression", "enhanced expression" or "increased expression" and grammatical equivalents thereof, are used interchangeably in the text and have a similar meaning. These terms mean that the expression of a gene or an enzyme is increased compared to a non modified microorganism. Increased expression of an enzyme is obtained by increasing expression of the gene encoding the said enzyme.

To increase the expression of a gene, the man skilled in the art knows different techniques:

- Increasing the number of copies of the gene in the microorganism. The gene is encoded chromosomally or extra-chromosomally. When the gene is located on the chromosome, several copies of the gene can be introduced on the chromosome by methods of recombination, known to the expert in the field, including gene replacement. When the gene is located extra-chromosomally, it may be carried by different types of plasmids that differ with respect to their origin of replication and thus their copy number in the cell. These plasmids are present in the microorganism in 1 to 5 copies, or about 20 copies, or up to 500 copies, depending on the nature of the plasmid: low copy number plasmids with tight replication (pSCl Ol , RK2), low copy number plasmids (pACYC, pRSFlOlO) or high copy number plasmids (pSK bluescript II).

- Using a promoter inducing a high level of expression of the gene. The man skilled in the art knows which promoters are the most convenient, for instance promoters Ftrc, Ftac, Viae, or the lambda promoter cl are widely used. These promoters can be "inducible" by a particular compound or by specific external condition like temperature or light. These promoters may be homologous or heterologous.

- Attenuating the activity or the expression of a transcription repressor, specific or non-specific of the gene.

- Using elements stabilizing the corresponding messenger RNA (Carrier and

Keasling, 1999) or elements stabilizing the protein (e.g., GST tags, GE Healthcare).

- Modifying Ribosome Binding Site (RBS) sequences and sequences surrounding them.

- Mutating the coding sequence leading to mutations on the amino-acid sequence.

- Mutating the coding sequence to optimize the codon usage but leading to no mutation of the amino-acid sequence.

The terms "attenuation" or "expression attenuated" mean that the expression of a gene or an enzyme is decreased or suppressed compared to a non modified microorganism. Decreasing or suppressing the expression of an enzyme is obtained by attenuating the expression of the gene encoding the said enzyme.

Attenuation of genes may be achieved by means and methods known to the man skilled in the art. Generally, attenuation of gene expression may be achieved by: - Mutating the coding region or the promoter region or,

- Deleting all or a part of the promoter region necessary for gene expression or,

- Deleting the coding region of the gene or,

- Inserting an external element into the coding region or into the promoter region or,

- Expressing the gene under the control of a weak promoter or,

- Modifying RBS sequences and sequences surrounding them.

The man skilled in the art knows a variety of promoters which exhibit different strengths and which promoter to use for a weak genetic expression.

To further modulate the activity of an enzyme by overexpression or attenuation, the man skilled in the art can use evolved enzymes.

The term "evolved enzyme" designates an enzyme that possesses at least one mutation in its sequence, in comparison with the amino-acid sequence of the wild-type enzyme from which it originates. Said mutation can lead to an enzyme which exhibits increased activity or a change of its specificity for a specific substrate and/or improved stability. These enzymes have been chosen with respect to their improved activity or specificity for the substrate and/or stability. In particular, these enzymes can be specific for a substrate in their wild-type state, and become specific for another substrate after the mutation(s) occurs. Genes encoding these selected enzymes can be heterologous or homologous.

The phrase "evolved enzyme derived from the X gene" designates an enzyme encoded by a mutated X gene, having an increased activity or a change of its specificity for a specific substrate or an improved stability.

Evolved enzymes can be obtained according to all techniques well known by the man skilled in the art, in particular such as presented in patents EP 1597364 and EP 1704230. Screening of mutated enzymes can be performed from libraries of mutated proteins generated by random mutagenesis. All evolved enzymes are tested for their activity and substrate specificity in a 'test' microorganism or in vitro; enzymes showing an improved activity and/or a change in their specificity for the substrate are identified and isolated, and preferentially are sequenced to identify the existing mutations. Genes encoding these identified evolved enzymes are cloned into a vector and introduced into the microorganism according to the invention.

All techniques for transforming the microorganism, in particular to introduce expression vectors, and regulatory elements used for enhancing the production of neopentyl glycol, are well known in the art and available in the literature, including the applicant's own patent applications on the modification of biosynthesis pathways in various microorganisms , including WO 2008/052973, WO 2008/052595, WO 2008/040387, WO 2007/144346, WO 2007/141316, WO 2007/077041, WO 2007/017710, WO 2006/082254, WO 2006/082252, WO 2005/111202, WO 2005/073364, WO 2005/047498, WO 2004/076659 , the content of which is incorporated herein by reference.

Expression vectors carrying genes encoding enzymes of the biosynthesis pathway allow the overexpression of said genes and corresponding encoded enzymes.

The terms "encoding" or "coding" refer to the process by which a polynucleotide, through the mechanisms of transcription and translation, produces an amino-acid sequence. The gene(s) encoding the enzyme(s) can be exogenous or endogenous.

The term "endogenous gene" means that the gene was present in the microorganism before any genetic modification, in the wild-type strain. Endogenous genes may be overexpressed by introducing heterologous sequences in addition to, or to replace endogenous regulatory elements, or by introducing one or more supplementary copies of the gene into the chromosome or a plasmid. Endogenous genes may also be modified to modulate their expression and/or activity. For example, mutations may be introduced into the coding sequence to modify the gene product. Modulation of an endogenous gene may result in the up-regulation and/or enhancement of the activity of the gene product, or alternatively, down regulate and/or lower the activity of the endogenous gene product.

Another way to modulate gene expression is to exchange the endogenous promoter of a gene (e.g., wild type promoter) with a stronger or weaker promoter to up or down regulate expression of the endogenous gene. These promoters may be homologous or heterologous. It is well for the person skilled in the art to select appropriate promoters.

Moreover, a microorganism may be modified to express exogenous genes if these genes are introduced into the microorganism with all the elements allowing their expression in the host microorganism.

The term "exogenous gene" or "exogenous nucleic acid sequence" or

"exogenous activity" as it is used herein is intended to mean that the gene or nucleic acid sequence or the activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of a nucleic acid sequence into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of a nucleic acid sequence refers to introduction of the nucleic acid sequence in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism. The source can be, for example, a homologous or heterologous nucleic acid sequence that expresses the activity following introduction into the host microbial organism. The term "heterologous" refers to a molecule or activity derived from a source other than the referenced species whereas "homologous" refers to a molecule or activity derived from the host microbial organism. Accordingly, exogenous expression of a nucleic acid sequence of the invention can utilize either or both a heterologous or homologous nucleic acid sequence.

In the present application, all genes are referenced with their common names and with references that give access to their nucleotidic sequences on the websites http://www.ncbi.nlm.nih.gov/gene or http ://www. ebi . ac.uk/ emb V.

Using the references given in Genbank for known genes, those skilled in the art are able to determine the equivalent genes in other organisms, bacterial strains, fungi, mammals, plants, etc. This routine work is advantageously done using consensus sequences that can be determined by carrying out sequence alignments with genes derived from other microorganisms and designing degenerated probes to clone the corresponding gene in another organism. These routine methods of molecular biology are well known to those skilled in the art, and are disclosed, for instance, in Sambrook et al, (1989) and (2001).

The term "function" or "activity" of an enzyme designates, in the context of the invention, the reaction that is catalyzed by the enzyme. The man skilled in the art knows how to measure the enzymatic activity of said enzyme.

The terms "attenuated activity" or "reduced activity" of an enzyme compared to the non modified enzyme mean either a reduced specific catalytic activity of the protein and/or a reduced specificity for its substrate obtained by mutation in the amino-acid sequence and/or a decrease of the concentration of the protein in the cell achieved by mutation of the nucleotidic sequence or by the deletion of the coding region of the gene or by the decrease of enzymatic stability of the enzyme due to its mutation(s).

The terms "enhanced activity" or "increased activity" of an enzyme compared to the non modified enzyme designate either an increased specific catalytic activity of the enzyme and/or an increased specificity for the substrate, and/or an increased concentration/availability of the enzyme in the cell, obtained for example by overexpressing the gene encoding the enzyme or by mutation(s) which enhance the stability of the enzyme in the cell.

The term "specificity" designates the affinity of an enzyme for a precise substrate. According to the invention, specificity of an enzyme means that this enzyme recognizes one substrate as preferred substrate among all other substrates. Affinity of an enzyme can be defined by the Michaelis constant value (Km). Enzymes that have a low Km value have high affinity to the substrate and act at maximal velocity at low substrate concentration whereas enzymes with a high Km value have low affinity to substrate and need high concentration of substrate to react.

The terms "fermentative production" or "culture" or "fermentation" are used to denote the growth of microorganisms. This growth is generally conducted in fermenters with an appropriate culture medium adapted to the microorganism being used and containing at least one simple carbon source, and if necessary co-substrates.

An "appropriate culture medium" or "culture medium" designates a medium (e.g., a sterile, liquid medium) comprising nutrients essential or beneficial to the maintenance and/or growth of the cell such as carbon sources or carbon substrates, nitrogen sources, phosphorus sources, for example, monopotassium phosphate or dipotassium phosphate; trace elements (e.g., metal salts), for instance magnesium salts, cobalt salts and/or manganese salts; as well as growth factors such as amino acids and vitamins. The appropriate medium for growth of a particular microorganism will be known by one skilled in the art of microbiology or fermentation science. Those skilled in the art are able to define the culture conditions for the microorganism according to the invention.

The term "source of carbon" or "carbon source" or "carbon substrate" according to the present invention denotes any source of carbon that can be used by those skilled in the art to support the normal growth of a microorganism, including monosaccharides (such as glucose, galactose, xylose, fructose or lactose), oligosaccharides, disaccharides (such as sucrose, cellobiose or maltose), molasses, starch or its derivatives, hemicelluloses and combinations thereof. An especially preferred carbon source is glucose. Another preferred carbon source is sucrose. The carbon source can be derived from renewable feed-stock. Renewable feed-stock is defined as raw material required for certain industrial processes that can be regenerated within a brief delay and in sufficient amount to permit its transformation into the desired product. Vegetal biomass treated or not, is an interesting renewable carbon source. Another interesting carbon source may be carbon monoxide (CO) containing gases, recovered from for instance industrial wastes, such as disclosed in patent applications WO2012026833 or WO2012087949. Hence it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon containing substrates and will only be limited by the choice of organism. In a specific embodiment of the invention, the substrate may be an hydrolysate of second generation carbon sources such wheat straw, miscanthus plants, sugar cane bagasses, wood and others that are known to the man skilled in the art. The carbon source can be derived from renewable microbial biomass, too. In another specific embodiment of the invention, the substrate may be a methyl group donor such as methanol, formaldehyde, formate, methylamine, methane, glycine betaine (GBT), trimethylamine (TMA), trimethylamine N- oxide (TMAO), dimethylsulfoniopropionate (DMSP), dichloromethane. These substrates may be used as co-substrates in addition to the carbon source previously described.

The term "renewable microbial biomass" is defined as raw microbial biomass issued from industrial processes that can be treated and/or refined (hydrolysis to obtain oligosaccharides and free amino -acids) within a brief delay and in a sufficient amount to permit its use in the culture medium. Fermentations may be performed under aerobic or anaerobic conditions.

The terms "anaerobic conditions" refer to conditions under which the oxygen concentration in the culture medium is too low for the microorganism to be used as terminal electron acceptor.

"Aerobic conditions" refers to concentrations of oxygen in the culture medium that are sufficient for an aerobic or facultative anaerobic microorganism to use as a terminal electron acceptor.

Those skilled in the art are able to define the culture conditions for the microorganisms according to the invention. In particular the microorganisms should be fermented at suitable temperature and pH range.

The amount of product in the fermentation medium can be determined using a number of analytic methods known in the art, for instance, high performance liquid chromatography (HPLC) or gas chromatography (GC).

The present process may employ a batch method of fermentation. A classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation the medium is inoculated with the desired organism(s), and fermentation is permitted to occur without adding anything to the system. Typically, however, a "batch" fermentation is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems the metabolite and biomass compositions of the system change constantly up to the time when the fermentation is stopped. Within batch cultures cells progress through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase generally are responsible for the bulk of production of end product or intermediate.

A Fed-Batch system may also be used in the present invention. A Fed-Batch system is similar to a typical batch system with the exception that the carbon source substrate is added in increments as the fermentation progresses. Fed-Batch systems are useful when catabolite repression (e.g. glucose repression) is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as C0₂.

Finally, continuous systems may also be used in the present invention. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology.

Fermentations are common and well known in the art and examples may be found in Weusthuis et al. (1994).

It is contemplated that the present invention may be practiced using any known mode of fermentation culture and in particular either batch, fed-batch or continuous processes and combinations thereof. Additionally, it is contemplated that cells may be immobilized on a substrate as whole cell catalysts or on inert support material generally selected among the group consisting of metal, glass, ceramic and polypropylene or on fibrous materials (porous particles, sponge or sponge-like materials, tangled fibres) and subjected to fermentation conditions for production.

Neopentyl glycol biosynthesis pathway.

a) Production of 2-dehydropantoate from the carbon source; and

b) Conversion of 2-dehydropantoate into neopentyl glycol;

In a first aspect of the invention, the 2-dehydropantoate is converted into neopentyl- glycol by successive actions of an enzyme having alpha keto acid decarboxylase activity converting 2-dehydropantoate into 3-hydroxy-2,2-dimethylpropanal and then of an enzyme having aldehyde reductase activity converting 3-hydroxy-2,2-dimethylpropanal into neopentyl glycol as it is shown in Figure 1.

The term "alpha keto-acid decarboxylase enzyme" designates an enzyme catalyzing the following reaction: R-CO-COOH (alpha keto-acid) R-CHO (aldehyde) + C0₂ This enzyme belongs to the enzyme class EC 4.1.1. The alpha keto-acid decarboxylase enzyme of the invention has specific activity for 2-dehydropantoate and converts it into 3- hydroxy-2,2-dimethylpropanal. This enzyme is also called 2-dehydropantoate decarboxylase in this invention. In one embodiment of the invention, the enzymatic activity of 2-dehydropantoate decarboxylase may be catalysed by an enzyme encoded by a gene chosen among a list of genes well known in the art (de la Plaza et ah, 2004; Li et ah, 2004; Alexander et ah, 1978; Dickinson et ah, 2000 and 2003) including but not limited to the genes listed below:

- kivD from Lactococcus lactis

kdcA from Lactococcus lactis

ubiD and ubiX from Escherichia coli.

kdc from Mycobacterium tuberculosis

- ARO10 from Saccharomyces cerevisiae.

Any polypeptide having at least 90% sequence identity to any of the polypeptides encoded by those genes may be used.

In a preferred embodiment of the invention, the 2-dehydropantoate decarboxylase is encoded by kivD from Lactococcus lactis.

In a more preferred embodiment of the invention, the enzyme having keto acid decarboxylase activity is an evolved enzyme. In particular, the evolution of the enzyme is obtained by mutating an encoding gene in order to improve:

- the enzymatic catalytic efficiency of conversion of 2-dehydropantoate into 3- hydroxy-2,2-dimethylpropanal and/or

- the enzyme specificity for the 2-dehydropantoate as substrate and/or

- the enzyme stability.

Evolution of the enzyme is carried out by means and methods well known by the man skilled in the art in order to obtain enzymes having specificity for the substrate 2- dehydropantoate and able to convert it into 3-hydroxy-2,2-dimethylpropanal. The selection of the evolved enzyme is performed either by expressing the evolved enzyme in the microorganism of the invention or in vitro with 2-dehydropantoate as substrate and by quantifying the product 3-hydroxy-2,2-dimethylpropanal or with 3-hydroxy-2,2- dimethylpropanal as substrate and by quantifying 2-dehydropantoate.

In a preferred embodiment of the invention, the enzyme having keto acid decarboxylase activity is an evolved enzyme derived from the kivD gene.

In another embodiment of the invention the enzyme having 2-dehydropantoate decarboxylase activity may be obtained by screening metagenomic libraries or libraries of evolved enzymes.

The term "aldehyde reductase enzyme" designates an enzyme catalyzing the following reaction:

R-CHO (aldehyde) + 2e-⁺ R-CH₂OH (alcool)

This enzyme always functions with a carrier of electrons, such as NADH, or other specific carriers. The activity of this enzyme is the conversion of an aldehyde into an alcohol. The other common name is alcohol dehydrogenase and this enzyme belongs to the enzyme class EC 1.1.1.

According to the present invention, the aldehyde reductase enzyme has specific activity for 3-hydroxy-2,2-dimethylpropanal and converts it into neopentyl glycol. This enzyme is also called 3-hydroxy-2,2-dimethylpropanal reductase in the invention.

In one embodiment of the invention, the enzymatic activity of 3-hydroxy-2,2- dimethylpropanal reductase may be catalyzed by an enzyme encoded by a gene chosen among a list of genes well known in the art (Perez et al, 2008; Habrych et al, 2002; Autieri et al, 2007; Rath et al, 2009; Xu et al, 2006; Ehrensberger et al, 2004; Lei et al, 2009; Rodriguez et al, 2012) including but not limited to the genes listed below:

yqhD from Escherichia coli

yqhE from Escherichia coli

yafB from Escherichia coli

fucO from Escherichia coli

air from Leishmania donovani

sakRl from Synechococcus sp.

yhdN from Bacillus subtilis

ytbE from Bacillus subtilis

yiaY from Escherichia coli.

In a preferred embodiment of the invention, the 3-hydroxy-2,2-dimethylpropanal reductase is encoded by yqhD from Escherichia coli.

More preferably, this enzyme has been evolved by mutating the encoding gene in order to improve:

the enzymatic catalytic efficiency of conversion of 3-hydroxy-2,2- dimethylpropanal into neopentyl glycol and/or

the enzyme specificity for the 3-hydroxy-2,2-dimethylpropanal as substrate and/or

- the enzyme stability.

Evolution of the enzyme is carried out by means and methods well known by the man skilled in the art in order to obtain enzymes having specificity for the substrate 3-hydroxy- 2,2-dimethylpropanal and enabling to convert it into neopentyl glycol. The selection of the evolved enzyme is performed by expressing the evolved enzyme in the microorganism of the invention or in vitro with 3-hydroxy-2,2-dimethylpropanal as substrate and by quantifying the product neopentyl glycol or with neopentyl glycol as substrate and by quantifying 3-hydroxy-2,2-dimethylpropanal. In a preferred embodiment of the invention, the enzyme having aldehyde reductase activity is an evolved enzyme derived from yqhD gene.

In another embodiment of the invention the enzyme having 3-hydroxy-2,2- dimethylpropanal reductase activity may be obtained by screening metagenomic libraries or libraries of evolved enzymes.

In a second aspect of the invention, the 2-dehydropantoate is converted into neopentyl-glycol by successive actions of:

an enzyme having keto-acid dehydrogenase activity converting 2-dehydropantoate into 3-hydroxy-2,2-dimethylpropanoyl-CoA,

- an enzyme having aldehyde oxidoreductase (CoA-acylating) converting 3-hydroxy-

2,2-dimethylpropanoyl-CoA into 3-hydroxy-2,2-dimethylpropanal, and then of an enzyme having aldehyde reductase activity converting 3-hydroxy-2,2- dimethylpropanal into neopentyl glycol

as it is shown in Figure 1.

In this second aspect of the invention, the decarboxylation step is catalyzed by the successive actions of an enzyme having keto-acid dehydrogenase activity and then of an enzyme having aldehyde oxidoreductase (CoA-acylating) activity.

The term "keto-acid dehydrogenase enzyme" designates an enzyme catalyzing the following reaction:

R-CO-COOH (alpha keto-acid) + CoA-SH R-CO-S-CoA (Acyl-CoA) + C0₂ + 2e-. This enzyme belongs to the EC 1.2.1. enzyme class.

This enzyme always functions with a carrier of electrons, such as NADH, or other specific carriers. The activity of this enzyme is the conversion of an alpha keto-acid into an acyl-CoA. Preferably the enzyme having keto-acid dehydrogenase activity is a branched- chain keto-acid dehydrogenase complex which is an enzymatic complex composed of four subunits El α, Ε ΐβ, E2 and E3. This complex contains three enzymatic functions: a keto- acid decarboxylase activity permitting the removal of one molecule of carbon dioxide, a keto-acid dehydrogenase activity and a keto-acid CoA acylase activity allowing both the conversion of a keto-acid into an acyl-CoA. Common examples of branched-chain keto- acid dehydrogenase complex are: pyruvate dehydrogenase complex and a-ketoglutarate dehydrogenase complex.

According to the present invention, the branched-chain keto-acid dehydrogenase complex has specific activity for 2-dehydropantoate and converts it into 3-hydroxy-2,2- dimethylpropanoyl-CoA. This enzyme is also named 2-dehydropantoate dehydrogenase complex in the invention.

In a specific embodiment of the invention, this enzyme is encoded by one gene chosen among a list of genes well known in the art (Sokatch et al, 1981; McCully et al, 1986; Lowe et al, 1983) including but not limited to the genes listed below: Genes bkdAl, bkdA2, bkdB and IpdV from Pseudomonas putida

Genes bkdAl, bkdA2, bkdB and IpdV from Pseudomonas aeruginosa

Genes bkdAA, bkdAB, bkdB and IpdV from Bacillus subtilis

Genes bfinBAA, bfinBAB, bfinBB and pdhD from Bacillus subtilis strain 168. - Operon bkdABCD from Enterococcus faecalis.

In a preferred embodiment of the invention, the 2-dehydropantoate dehydrogenase complex is encoded by bkdAl, bkdA2, bkdB and IpdV genes from Pseudomonas putida. More preferably, the enzyme has been evolved by mutating the encoding gene in order to improve the enzymatic catalytic efficiency of conversion of 2-dehydropantoate into 3- hydroxy-2,2-dimethylpropanoyl-CoA and/or the enzyme specificity fo r th e 2- dehydropantoate as substrate and/or the enzyme stability.

Evolution of these enzymes is carried out by means and methods well known by the man skilled in the art in order to obtain enzymes having specificity for the substrate 2- dehydropantoate and enabling to convert it into 3-hydroxy-2,2-dimethylpropanoyl-CoA.

The selection of the evolved enzyme is performed by expressing the evolved enzyme in the microorganism of the invention or in vitro with 2-dehydropantoate as substrate and by quantifying the product 3-hydroxy-2,2-dimethylpropanoyl-CoA or with 3-hydroxy-2,2- dimethylpropanoyl-CoA and by quantifying 2-dehydropantoate.

In a preferred embodiment of the invention, the enzyme having keto-acid dehydrogenase is an evolved enzyme derived from the bkdAl, bkdA2, bkdB and IpdV genes.

In another embodiment of the invention the enzyme having 2-dehydropantoate dehydrogenase complex activity may be obtained by screening metagenomic libraries or libraries of evolved enzymes.

The term "aldehyde oxidoreductase (CoA-acylating) enzyme" designates an enzyme catalyzing the conversion of an acyl-CoA into an aldehyde among the following reaction :

R-CO-S-CoA (Acyl-CoA) + 2e- R-COH (aldehyde) + CoA-SH.

This enzyme belongs to the EC 1.2.1. enzyme class. This enzyme always functions with a carrier of electrons, such as NADH, or other specific carriers.

According to the present invention, the aldehyde oxidoreductase (CoA-acylating) enzyme has specific activity for 3-hydroxy-2,2-dimethylpropanoyl-CoA and converts it into 3-hydroxy-2,2-dimethylpropanal. This enzyme is also named 3-hydroxy-2,2- dimethylpropanal oxidoreductase (CoA-acylating) in the invention. This enzyme may be encoded by one gene chosen among a list of genes well known in the art (Goodlove et al, 1989; Fontaine et al, 2002; Manjasetty et al, 2003; Shone et al, 1981) including but not limited to the genes listed below:

adhE from Escherichia coli

- adhE2 from Clostridium acetobutylicum

dmpF from Pseudomonas sp. CF600

todl from Pseudomonas putida Fl

cmtH from Pseudomonas putida Fl

mhpF from Escherichia coli.

In a preferred embodiment of the invention, the 3-hydroxy-2,2-dimethylpropanal oxidoreductase (CoA-acylating) is encoded by the adhE gene of Escherichia coli.

More preferably, the enzyme has been evolved by mutating the encoding gene in order to improve the enzymatic catalytic efficiency of conversion of 3-hydroxy-2,2- dimethylpropanoyl-CoA into 3-hydroxy-2,2-dimethylpropanal and/or the enzyme specificity for the 3-hydroxy-2,2-dimethylpropanoyl-CoA as substrate and/or the enzyme stability.

Evolution of these enzymes is carried out by means and methods well known by the man skilled in the art in order to obtain enzymes having specificity for the substrate 3-hydroxy- 2,2-dimethylpropanoyl-CoA an d enab ling to c o nv ert it into 3-hydroxy-2,2- dimethylpropanal. The selection of the evolved enzyme is performed by expressing the evolved enzyme in the microorganism of the invention or in vitro with 3-hydroxy-2,2- dimethylpropanoyl-CoA as substrate and by quantifying the product 3-hydroxy-2,2- dimethylpropanal or with 3-hydroxy-2,2-dimethylpropanal as substrate and by quantifying 3-hydroxy-2,2-dimethylpropanoyl-CoA.

In a preferred embodiment of the invention, the enzyme having 3-hydroxy-2,2- dimethylpropanal oxidoreductase (CoA-acylating) activity is an evolved enzyme derived from the adhE gene.

In another embodiment of the invention the enzyme having 3-hydroxy-2,2- dimethylpropanal oxidoreductase (CoA-acylating) activity may be obtained by screening metagenomic libraries or libraries of evolved enzymes.

The term "aldehyde reductase enzyme" designates an enzyme belonging to the enzyme class EC 1.1.1.- and catalyzing the following reaction, the conversion of an aldehyde into an alcohol:

R-CHO (aldehyde) + 2e- R-CH₂OH (alcool) According to the present invention, the aldehyde reductase enzyme has specific activity for 3-hydroxy-2,2-dimethylpropanal and converts it into neopentyl glycol. This enzyme is also named 3-hydroxy-2,2-dimethylpropanal reductase in the invention.

This enzyme may be encoded by a gene chosen among a list of genes well known in the art (Perez et al, 2008; Habrych et al, 2002; Autieri et al, 2007; Rath et al, 2009; Xu et al, 2006; Ehrensberger et al, 2004; Lei et al, 2009; Rodriguez et al, 2012) including but not limited to the genes listed below:

- yqhD from Escherichia coli

- yqhE from Escherichia coli

- yafB from Escherichia coli

fucO from Escherichia coli

air from Leishmania donovani

sakRl from Synechococcus sp.

- yhdN from Bacillus subtilis

- ytbE from Bacillus subtilis

- yiaY from Escherichia coli.

Preferentially, the enzyme has been evolved by mutating the encoding gene in order to improve the enzymatic catalytic efficiency of conversion of 3-hydroxy-2,2- dimethylpropanal into neopentyl glycol and/or the enzyme specificity for the 3-hydroxy- 2,2-dimethylpropanal as substrate and/or the enzyme stability.

Evolution of the enzyme is carried out by means and methods well known by the man skilled in the art in order to obtain enzymes having specificity for the substrate 3- hydroxy-2,2-dimethylpropanal and enabling them to convert it into neopentyl glycol. The selection of the evolved enzyme is performed by expressing the evolved enzyme in the microorganism of the invention or in vitro with 3-hydroxy-2,2-dimethylpropanal as substrate and by quantifying the product neopentyl glycol or with neopentyl glycol as substrate and by quantifying 3-hydroxy-2,2-dimethylpropanal.

Preferentially, 3-hydroxy-2,2-dimethylpropanal oxidoreductase (CoA-acylating) activity and 3-hydroxy-2,2-dimethylpropanal reductase activity are catalyzed by the same enzyme capable of performing both functions: aldehyde oxidoreduction (CoA-acylating) and aldehyde reduction converting 3-hydroxy-2,2-dimethylpropanoyl-CoA into neopentyl glycol, as it is shown in Figure 1. Enzymes possessing both activities are enzymes called 'Afunctional aldehyde-CoA/aldehyde reductase enzyme" and generally belong to the enzyme class EC 1.2.1.

According to the present invention, the bifunctional aldehyde-CoA/aldehyde reductase enzyme has specific activity for 3-hydroxy-2,2-dimethylpropanoyl-CoA and converts it into neopentyl glycol . This enzyme is also called 3-hydroxy-2,2- dimethylpropanoyl-CoA/3-hydroxy-2,2-dimethylpropanal reductase in the invention.

In one embodiment of the invention, this enzyme is encoded by one gene chosen among a list of genes well known in the art (Goodlove et ah, 1989; Fontaine et ah, 2002) including but not limited to the genes listed below:

adhE from Escherichia coli

adhE2 from Clostridium acetobutylicum

In a preferred embodiment of the invention, the 3-hydroxy-2,2-dimethylpropanoyl-

CoA/3-hydroxy-2,2-dimethylpropanal reductase is encoded by adhE from Escherichia coli.

More preferably, the enzyme has been evolved by mutating the encoding genes in order to improve the enzymatic catalytic efficiency of conversion of 3-hydroxy-2,2- dimethylpropanoyl-CoA into neopentyl glycol and/or the enzyme specificity for the 3- hydroxy-2,2-dimethylpropanoyl-CoA as substrate and/or the enzyme stability.

Evolution of these enzymes is carried out by means and methods well known by the man skilled in the art in order to obtain enzymes having specificity for the substrate 3-hydroxy- 2,2-dimethylpropanoyl-CoA and enabling them to convert it into neopentyl glycol. The selection of the evolved enzyme is performed by expressing the evolved enzyme in the microorganism of the invention or in vitro with 3-hydroxy-2,2-dimethylpropanoyl-CoA as substrate and by quantifying the product neopentyl glycol or with neopentyl glycol as substrate and by quantifying 3-hydroxy-2,2-dimethylpropanoyl-CoA.

In a preferred embodiment of the invention, the enzyme having both activities is an evolved enzyme derived from the adhE gene.

In another embodiment of the invention the enzyme having 3-hydroxy-2,2- dimethylpropanoyl-CoA/3-hydroxy-2,2-dimethylpropanal reductase activity may b e obtained by screening metagenomic libraries or libraries of evolved enzymes

The biosynthesis pathway of 2-dehydropantoate from the source of carbon includes the following intermediate products: pyruvate, acetolactate, 2,3-dihydroxy-3- methylbutanoate and 3-methyl-2-oxobutanoate, as it is shown in Figure 2. In a specific aspect of the invention, the microorganism is further modified to improve the availability of 2-dehydropantoate, by improving availability of at least one of these biosynthesis intermediates.

In order to optimize pyruvate availability, the microorganism is modified to overexpress at least one gene involved in the pyruvate biosynthesis pathway, chosen among genes coding for phosphoglycerate mutase (gpmA and pgml genes from Escherichia coli or homologous gene), enolase (eno from Escherichia coli or homologous gene) or pyruvate kinase (pykA and pykF genes from Escherichia coli or homologous gene). Alternatively or in combination, at least one gene involved in the pyruvate degradation pathway is attenuated. This gene is chosen among pyruvate oxidase (poxB from Escherichia coli or homologous gene), phosphate acetyltransferase (pta from Escherichia coli or homologous gene), acetate kinase (ackA from Escherichia coli or homologous gene) or lactate dehydrogenase (UdD, IdhA or did from Escherichia coli or homologous gene).

In order to optimize acetolactate and 2,3-dihydroxy-3-methylbutanoate availabilities, the microorganism is further modified to overexpress at least one gene chosen among genes coding for acetolactate synthase (ilvl, ilvH, ilvN and ilvB from Escherichia coli or alsS from Klebsiella pneumoniae or homologous gene) or ketol-acid reductoisomerase (ilvC from Escherichia coli or homologous gene). In another embodiment of the invention, the ilvN gene is modified so as to produce an IlvN protein which is feedback deregulated. Such mutations of ilvN are disclosed in Park et al. , 2011.

In order to optimize the pool of 3-methyl-2-oxobutanoate, the microorganism is modified to overexpress at least one gene chosen among genes coding for dihydroxy-acid dehydratase (ilvD, yagF and yjhG from Escherichia coli or homologous gene). Alternatively or in combination, at least expression of one gene involved in the 3-methyl- 2-oxobutanoate degradation pathway is attenuated. This gene is chosen among genes coding for 2-isopropylmalate synthase (leuA from Escherichia coli or homologous gene), glutamate-pyruvate aminotransferase (alaA, alaC from Escherichia coli or homologous gene), valine-pyruvate aminotransferase (avtA from Escherichia coli or homologous gene), branched-chain amino-acid transferase (ilvE from Escherichia coli or homologous gene). Preferably this gene is expressed by use of a promoter with a weak strength and/or by use of an inducible promoter.

In a more preferred aspect of the invention, the microorganism is further modified to improve the availability of 2-dehydropantoate by overexpressing one gene coding for a 3-methyl-2-oxobutanoate hydroxymethyltransferase enzyme. Preferably, this gene is chosen among the panB gene from Escherichia coli, Mycobacterium tuberculosis, Cory neb acterium glutamicum, or Bacillus subtilis (or homologous gene). Alternatively or in combination, one gene coding for a 2-dehydropantoate 2-reductase enzyme is attenuated (panE from Escherichia coli or homologous gene). In another embodiment of the invention, the panB gene is modified so as to produce a PanB protein which is feedback deregulated for Coenzyme A (US7220561).

In an embodiment of the invention, at least one of the following enzymes is overexpressed: acetolactate synthase, ketol-acid reductosiomerase, dihydroxy-acid dehydratase and 3- methyl-2-oxobutanoate hydro xymethyltransferase. Preferentially, at least one of these enzymes is encoded by an exogenous gene.

In a further embo diment , the activity o f the 3-methyl-2-oxobutanoate hydroxymethyltransferase enzyme is improved by optimizing the pool of CI units in form of methylene-THF (WO2009/043803) acting as co-substrate for this enzyme. In order to improve the pool of methylene-THF, the recombinant microorganism is further modified as described below:

- The expression of at least one of the following genes is increased: glyA, gcvTHP, Ipd, serA, serB, serC, folA, folM and pabABC.

- The expression of at least one of the following genes is attenuated: thy A, metF, purU, purR, ItaE, tdh and kbl.

In a specific aspect of the invention the recombinant microorganism overexpresses at least one gene chosen among: ilvl, ilvH, ilvN, ilvB, alsS, ilvC, yagF, ilvD, yjhG and panB.

The invention is also related to a recombinant microorganism for the the fermentative production of neopentyl glycol. In particular, said microorganism overexpresses at least one of the following genes: kivD, yqhD, adhE, bkdAl, bkdA2, bkdB and IpdVox genes derived from the evolution of these genes.

In an aspect of the invention, said microorganism further overexpresses at least one gene chosen among: ilvl, ilvH, ilvN, ilvB, alsS, ilvC, yagF, ilvD, yjhG and panB.

In another aspect of the invention, said microorganism further comprises at least one of the following modifications:

- Attenuation of at least one gene selected among: leuA, alaA, avtA, alaC, ilvE, panE, thy A, metF, purU, purR, poxB, pta, ackA, lldD, IdhA, did, ItaE, tdh and/or kbl;

- Overexpression of at least one gene selected among: glyA, gcvTHP, Ipd, serA, serB, serC, folA, folM, pabABC gpmA, pgml, eno, pykA and/or pykF. Preferably the recombinant microorganism is modified by overexpressing:

an evolved kivD gene and

an evolved yqhD gene.

More preferably the recombinant microorganism is modified by overexpressing:

an evolved kivD gene

an evolved yqhD gene

- serB, serA and serC genes ilvB, ilvN, ilvC and ilvD genes

- the panB gene

and gcvTHP and Ipd genes.

In another preferred embodiment the recombinant microorganism is modified by overexpressing:

evolved genes bkdAl, bkdA2, bkdB and IpdV genes and

an evolved adhE gene.

More preferably the recombinant microorganism is modified by overexpressing:

evolved genes bkdAl, bkdA2, bkdB and IpdV genes

an evolved adhE gene

- serB, serA and serC genes

ilvB, ilvN, ilvC and ilvD genes

- the panB gene

and gcvTHP and Ipd genes.

In a particular aspect of the invention the recombinant microorganism comprises the following modifications:

- overexpression of evolved kivD and yqhD genes, and of genes ilvl, ilvH, ilvN, ilvB, alsS, ilvC, yagF, ilvD, yjhG and panB, glyA, gcvTHP, Ipd, serA, serB, serC, folA, folM, pabABC gpmA, pgml, eno, pykA and pykF genes ; attenuation of genes leuA, alaA, avtA, alaC, ilvE, panE, thy A, metF, purU, purR, poxB, pta, ackA, lldD, IdhA, did, ItaE, tdh and kbl genes.

In another particular aspect of the invention the recombinant microorganism comprises the following modifications:

- overexpression of evolved genes bkdAl, bkdA2, bkdB, IpdV and adhE and genes, ilvl, ilvH, ilvN, ilvB, alsS, ilvC, yagF, ilvD, yjhG and panB, glyA, gcvTHP, Ipd, serA, serB, serC, folA, folM, pabABC gpmA, pgml, eno, pykA and pykF genes ;

attenuation of genes leuA, alaA, avtA, alaC, ilvE, panE, thy A, metF, purU, purR, poxB, pta, ackA, lldD, IdhA, did, ItaE, tdh and kbl genes. According to a specific aspect of the invention the fermentative production of neopentyl glycol comprises steps of recovery and purification of the neopentyl glycol from the culture medium. It may be achieved by a number of techniques well known in the art including but not limited to, distillation, liquid extraction, gas-stripping, pervaporation or cation exchange. Preferably neopentyl glycol is isolated from the culture medium by liquid extraction using solvents such as 1-pentanol, 1-butanol, hexanol, heptanol and mixtures thereof (WO2012/130316). EXAMPLES

The present invention is further defined in the following examples. It should be understood that these examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From above disclosure and these examples, the man skilled in the art can make various changes of the invention to adapt it to various uses and conditions without modifying the essentials means of the invention.

Exemplary genes and enzymes required for constructing microorganisms with these capabilities are described as well as methods for cloning and transformation, monitoring product formation and using the engineered microorganisms for production.

In particular, examples show modified Escherichia coli (E. coli) strains, but these modifications can easily be performed in other microorganisms of the same family or other microorganisms .

Escherichia coli belongs to the Enterobacteriaceae family, which comprises members that are Gram-negative, rod-shaped, non-spore forming and are typically 1-5 μιη in length. Most members have flagella used to move about, but a few genera are non- motile. Many members of this family are a normal part of the gut flora found in the intestines of humans and other animals, while others are found in water or soil, or are parasites on a variety of different animals and plants. E. coli is one of the most important model organism, but other important members of the Enterobacteriaceae family include Klebsiella, in particular Klebsiella terrigena, Klebsiella planticola or Klebsiella oxytoca, Pantoea and Salmonella.

EXAMPLE 1:

Calculation of maximum yields for neopentyl glycol production on glucose.

Simulations were performed with the METEX proprietary software METOPT™ based on flux balance analysis methods (Varma and Palsson, 1994). A simplified metabolic network of E. coli was used, including a core network, metabolic pathways for all biomass precursors and specific production pathways as described above and presented in Figures 1 and 2. A classical biomass composition for E. coli was used (Pramanik and Keasling, 1997). Simulations were performed using glucose as carbon source, imported via the phosphotransferase system (Escalante et al, 2012).

Calculation of a theoretical maximum yield was performed, taking into account neither cell growth nor maintenance energy requirements. Results of the simulations:

The biochemical balance equation is identical for the two pathways described above. Based on aerobic glucose oxidation for ATP production, the theoretical yield of these pathways is 0.462 g of neopentyl glycol per g of glucose.

Considering an ATP maintenance of 5 mmol/g/h, a substrate uptake rate of 3 mmol/g/h, and a growth rate of 0.1 h^"1 the production yield is estimated at 0.306 gram of neopentyl glycol per gram of glucose.

EXAMPLE 2:

Construction of a neopentyl glycol producer strain

MG1655 PtrcOl/RBSOl-gcvTHP pCL1920-Ptrc01/RBS01*2-i7vfi V* G 20- 22DDF)CD-Vtrc01/KBS01*2-panB-TT07 pBBRlMCS5-Ptrc01/RBS01*2-ji//iD- kivDll-ΎΎΟΙ pCClBAC-serB-serA-serC

The E. coli strain engineered to produce neopentyl glycol is generated using procedures described in patent application WO2010/076324.

In order to increase the methylene-tetrahydrofolate pool into the cell, the glycine cleavage complex encoded by gcvTHP operon is overproduced by adding a constitutive Vtrc promoter and an optimized ribosome binding site sequence upstream of the gcvTHP operon. The PtrcOl/RBSOl -gcvTHP chromosomal modification has been described in patent application WO2009/043803 which is incorporated by reference in this application. The resulting strain is called MG1655 PtrcOl/RBSOl -gcvTHP.

To increase the flux into the serine pathway, the serB, serA and serC genes are over-expressed under the control of their own promoter on the bacterial artificial chromosome pCClBAC plasmid (Epicentre). Plasmid pCClBAC-serB-serA-serC plasmid has been described in patent application WO2009/043803 which is incorporated by reference in this application.

The last two steps of the neopentyl glycol production pathway are catalysed by the alpha-keto-acid decarboxylase KivD and an alcohol dehydrogenase YqhD. To increase the conversion of 2-dehydropantoate into 3-hydroxy-2,2-dimethylpropanal and then into neopentyl glycol, kivD and yqhD genes are overexpressed in an operon from plasmid pBBRlMCS5 under the control of a constitutive Vtrc promoter and optimized ribosome binding site. Plasmid pBBRl MCS5-Ptrc01/RBS01 ^!i:2-3 AZ⁾-^VZ⁾//-TT 07 has b een described in patent application US2011/0294178 which is incorporated by reference in this application. To increase the 3-methyl-2-oxobutanoate and the 2-dehydropantoate pool into the cell, the ilvBN*(GMV20-22DDF)CD operon and panB gene are overexpressed on the plasmid pCL1920 (Lerner and Inouye, 1990) under the control of a constitutive Vtrc promoter and an optimized ribosome binding site.

Construction of pCL1920-Ptrc01/RBS01 *2-ilvBN*fGMV20-22DDF)CD-Ptrc01/KBS01 *2- ραηΒ-ΎΎΟΊ plasmid

pCL\920-Ptrc01/RBS01 *2-ilvBN*(GVM20-22DDF)CD-Ptrc0\/KBS0\ *2-panB- TT07 plasmid is derived from pCL1920-Ptrc01/RBS01 *2-ilvBN*(GVM20-22DDF)CD described in patent application PCT/EP2012/070160 which is incorporated by reference in this application and the panB gene from Escherichia coli coding for the 3-methyl-2- oxobutanoate hydroxymethyltransferase.

The panB gene is PCR amplified with primers Ptrc01/RBS01 *2-panB F (SEQ ID

N°01

CCGTACGTAGAGCTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAAT AAGGAGGTTATAAATGAAACCGACCACCATCTCC) and panB-TT07 R (SEQ ID N°02

GCGGGATCCGCAGAAAGGCCCACCCGAAGGTGAGCCAGGTATACTTAATGGA AACTGTGTTCTTCGC) using E. coli MG1655 genomic DNA as template. The PCR product is digested and cloned between the SnaBl and BamHl sites of the pCL1920- Ptrc01/RBS01 *2-itvBN*(GVM20-22DDF)CD-TT07. The resulting plasmid is called pCLl 920-PtrcO 1/RBSO 1 *2-ilvBN*(GVM20-22DDF)CD-Ptrc01/KBS01 *2-panB-TT07.

Construction of a strain with increased neopentyl glycol pathway flux is done by transforming pCL1920-Ptrc01/RBS01 *2-ilvBN*(GMV20-22DDF)CD-Ptrc01/KBS01 *2- ραηΒ-ΎΎΟΊ, pBBRlMCS5-Ptrc01/RBS01 *2-.y¾rA -fcvD//-TT07 and pCClBAC-serB- serA-serC plasmids into MG1655 PtrcOl/RBSOl-gcvTHP. The resulting strain is called MG1655 PtrcO 1/RBSO 1 -gcvTHP pCL 1920-PtrcO 1/RBSO 1 *2-ilvBN*(GMV20-22DDF) CD- PtrcO 1/RBSO 1 *2-panB-T T 0 7 p B B R l M C S 5-PtrcO 1/RBSO l *2-yqhD-kivDll-TT07 pCClBAC-serB-serA-serC.

This strain grown in appropriate conditions produces neopentyl glycol in amounts higher than 0,1 mg/L. EXAMPLE 3:

Construction of a neopentyl glygol producer strain via 3-hydroxy-2.2- dimethylpropanoyl-CoA

MG1655 PtrcOl/RBSOl-gcvTHP pCL1920-Ptrc01/RBS01*2-i7vfi V* G 20- 22DDF)CD-Vtrc01/KBS01*2-panB-T T 07 p BBRl M C S 5-Vtrc0l-bkdA12B+lpdV^- TT07 pUCl^PtrcOl/OPOl/RBSOl-fli Zi^ca-T^

Construction of this strain with an increased neopentyl glycol pathway flux via 3 -hydro xy- 2.2-dimethylpropanoyl-CoA is done by transforming into MG1655 Ptrc01/RBS01-gcv7HP plasmids pCL 1920-PtrcO 1/RBSO 1 *2-ilvBN*(GMV20-22DDF)CD-Ptrc01/KBS01 *2-panB- TT07 described in example 2, pBBRlMCS5-Ptrc01-6M4i25+/prf pp-TT07 described in patent application PCT/EP2012/070160 and pUC19-Ptrc01/OP01/RBSO l-a AE2ca-TT02 described in patent application WO2010/076324. The resulting strain is called MG1655 PtrcO 1/RBSO 1 -gcvTHP pCL1920-Ptrc01/KBS01 *2-ilvBN*(GMV20-22DDF)CD- PtrcO 1/RBSO 1 *2-ραηΒ-ΊΊ0Ί pBBRlMCS5-PtrcO 1 -bkdA12B+lpdVpp-TT01 pUC 19- Ptrc01/OP01/RBSO \-adhE 2 ca-ΎΎ 02 .

This strain grown in appropriate conditions produces neopentyl glycol in amounts higher than 0,1 mg/L.

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Claims

1. A method for the fermentative production of neopentyl glycol, comprising culturing a recombinant microorganism in an appropriate culture medium comprising a source of carbon, the biosynthesis pathway comprising the following steps:

a) Production of 2-dehydropantoate from the carbon source; and b) Conversion of 2-dehydropantoate into neopentyl glycol; wherein in said microorganism, the conversion of 2-dehydropantoate into neopentyl glycol is catalyzed by two or three successive steps including a step of decarboxylation and a step of aldehyde reduction.

2. The method according to claim 1 wherein 2-dehydropantoate is converted into neopentyl glycol by successive actions of :

An enzyme having alpha keto acid decarboxylase activity converting 2- dehydropantoate into 3-hydroxy-2,2-dimethylpropanal, and - An enzyme having aldehyde reductase activity converting 3-hydroxy-

2,2-dimethylpropanal into neopentyl glycol.

3. The method according to claim 2 wherein the enzyme having keto acid decarboxylase activity is an evolved enzyme derived from the kivD gene.

4. The method according to claim 2 wherein the enzyme having aldehyde reductase activity is an evolved enzyme derived from yq hD gene.

5. The method according to claim 1 wherein 2-dehydropantoate is converted into neopentyl glycol by successive actions of

An enzyme having keto-acid dehydrogenase activity converting 2- dehydropantoate into 3-hydroxy-2,2-dimethylpropanoyl-CoA, and - An enzyme having aldehyde oxidoreductase (CoA-acylating) activity converting 3-hydroxy-2,2-dimethylpropanoyl-CoA into 3-hydroxy-2,2- dimethylpropanal, and

An enzyme having aldehyde reductase activity converting 3-hydroxy- 2,2-dimethylpropanal into neopentyl glycol.

6. The method according to claim 5 wherein the enzyme having keto-acid dehydrogenase is an evolved enzyme derived from the bkdAl, bkdA2, bkdB and IpdV genes.

7. The method according to claim 5 wherein the aldehyde oxidoreductase (CoA- acylating) activity and the aldehyde reductase activity are catalyzed by the same enzyme capable of performing both functions: aldehyde oxidoreduction (CoA- acylating) and aldehyde reduction converting 3-hydroxy-2,2-dimethylpropanoyl- CoA into neopentyl glycol.

8. The method according to claim 7 wherein the enzyme is an evolved enzyme derived from the adhE gene.

9. The method according to claim 1 wherein the biosynthesis pathway of 2- dehydropantoate from the source of carbon includes the following intermediate products: pyruvate, acetolactate, 2,3-dihydroxy-3-methylbutanoate and 3-methyl-2- oxobutanoate.

10. The method according to claim 9 wherein at least one of the following enzymes is overexpressed: acetolactate synthase, ketol-acid reductosiomerase, dihydroxy-acid dehydratase and 3-methyl-2-oxobutanoate hydroxymethyltransferase.

11. The method according to claim 10 wherein at least one of the enzymes is encoded by an exogenous gene.

12. The method according to claim 11 wherein at least one of the following genes is overexpressed: ilvl, ilvH, ilvN, ilvB, alsS, ilvC, yagF, ilvD, yjhG and panB.

13. A recombinant microorganism for the fermentative production of neopentyl glycol wherein said microorganism overexpresses at least one of the following genes: kivD, yqhD, adhE, bkdAl, bkdA2, bkdB and IpdV or genes derived from the evolution of these genes.

14. The microorganism according to claim 13 wherein said microorganism further overexpresses at least one gene chosen among: ilvl, ilvH, ilvN, ilvB, alsS, ilvC, yagF, ilvD, yjhG and panB.

15. The microorganism according to claims 13 to 14 wherein said microorganism further comprises at least one of the following modifications:

Overexpression of at least one gene selected among: glyA, gcvTHP, Ipd, serA, serB, serC, folA, folM, pabABC gpmA, pgml, eno, pykA and/or pykF.