# US 2015/0147795 A1 · Microorganism modified for the production of 1,3-propanediol

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- Number: US 2015/0147795 A1
- Document ID: US20150147795A1
- Status: Published application
- Kind: A1
- Application number: 14/410,732
- Filed: 2013-07-10
- Published: 2015-05-28
- Earliest claimed: 2012-07-11
- Assignees: Institut National Des Sciences Appliquees
- Applicants: Institut National Des Sciences AppliquéEs
- Inventors: Thomas Walther; Jean Marie Francois
- Web page: https://patentgrep.com/patent/US20150147795A1

## CPC

- C12P 7/18: polyhydric
- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12N 9/0008: {acting on the aldehyde or oxo group of donors (1.2)}
- C12N 9/1205: {Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases}
- C12N 9/88: Lyases (4.)
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12Y 101/01001: Alcohol dehydrogenase (1.1.1.1)
- C12Y 101/01027: L-Lactate dehydrogenase (1.1.1.27)
- C12Y 101/01037: Malate dehydrogenase (1.1.1.37)
- C12Y 101/01202: 1,3-Propanediol dehydrogenase (1.1.1.202)
- C12Y 401/01001: Pyruvate decarboxylase (4.1.1.1)
- C12Y 401/01072: Branched-chain-2-oxoacid decarboxylase (4.1.1.72)
- C12Y 401/01: Carboxy-lyases (4.1.1)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

The invention relates to a modified microorganism for the production of PDO from a carbon substrate wherein the microorganism includes a three-step metabolic pathway including a first step of conversion of 2,4-dihydroxybutyrate (DHB) to obtain 2-oxo-4-hydroxybutyrate (OHB) by an enzyme having 2,4-DHB dehydrogenase activity, a second step of decarboxylation of the OHB to obtain 3-hydroxypropionaldehyde by an enzyme having 2-oxo-4-hydroxybutyrate decarboxylase activity, and a third step of reduction of the obtained 3-hydroxypropionaldehyde to obtain PDO with an enzyme having 3-hydroxypropionaldehyde reductase activity and the genes enabling the microorganism for the synthesis of DHB.

## Claims

### Claim 1

A modified microorganism for the production of PDO from a carbon substrate wherein the microorganism comprises a three-step metabolic pathway comprising the following steps:
a first step of conversion of 2,4-dihydroxybutyrate (DHB) to obtain 2-oxo-4-hydroxybutyrate (OH B) by an enzyme having 2,4-DHB dehydrogenase activity, and
a second step of decarboxylation of the OHB to obtain 3-hydroxypropionaldehyde by an enzyme having 2-oxo-4-hydroxybutyrate decarboxylase activity, and
a third step of reduction of the obtained 3-hydroxypropionaldehyde to obtain PDO with an enzyme having 3-hydroxypropionaldehyde reductase activity and the genes enabling the microorganism to synthesize DHB.

### Claim 2 (depends on 1)

The modified microorganism of claim 1 comprising a pathway for the synthesis of DHB from malate.

### Claim 3 (depends on 2)

The modified microorganism according to claim 2 wherein the microorganism has been further modified by the introduction of genes encoding:
a malate kinase catalyzing the transformation of malate into 4-phospho-malate,
a malate semialdehyde dehydrogenase catalyzing the transformation of 4-phospho-malate into malate-4-semialdehyde
a malate semialdehyde reductase catalyzing the transformation of malate-4-semialdehyde into 2,4-DHB.

### Claim 4 (depends on 1)

The microorganism of claim 1 wherein the enzymes are encoded by an endogenous or a heterologous gene.

### Claim 5 (depends on 1)

The modified organism of claim 1 wherein the enzyme having enzyme having 2,4-DHB dehydrogenase activity is selected among enzymes having lactate dehydrogenase or malate dehydrogenase activity.

### Claim 6 (depends on 1)

The modified organism of claim 1 wherein the enzyme having 2,4-DHB dehydrogenase activity is obtained by at least one mutation of an enzyme, said mutation improving the activity and/or the substrate affinity of the mutated enzyme for DHB.

### Claim 7 (depends on 5)

The modified organism of claim 5 wherein the enzyme is a gene product encoded by genes selected among IdhA from Lactococcus lactis, lldD from Escherichia coli, IdD from E. coli carrying a mutation at position V108 (by reference to SEQ ID No. 122), mdh from E. coli or from Bacillus subtilis, or mdh of E. coli carrying mutations in at least one of the following positions (by reference to SEQ ID No. 124): Ile12, Lys82, Met85, Asp86, Val93, Ile117, Gly179, Thr211, or Met227 (by reference to SEQ ID No. 126).

### Claim 8 (depends on 7)

The modified organism of claim 7 wherein the enzyme is
a polynucleotide chosen among the group comprising SEQ ID No. 119, SEQ ID No. 121, SEQ ID No. 153, SEQ ID No. 155, SEQ ID No. 157, SEQ ID No. 159, SEQ ID No. 161, SEQ ID No. 163, SEQ ID No. 165, SEQ ID No. 167, SEQ ID No. 169, SEQ ID No. 171 or SEQ ID No. 173 or any sequence sharing a homology of at least 50% with said sequences,
a polypeptide chosen among the group comprising SEQ ID No. 120, SEQ ID No. 122, SEQ ID No. 154, SEQ ID No. 156, SEQ ID No. 158, SEQ ID No. 160, SEQ ID No. 162, SEQ ID No. 164, SEQ ID No. 166, SEQ ID No. 168, SEQ ID No. 170, SEQ ID No. 172 or SEQ ID No. 174 or any sequence sharing a homology of at least 50% with said sequences.

### Claim 9 (depends on 1)

The modified organism of claim 1 wherein the enzyme having 2-oxo-4-hydroxybutyrate decarboxylase activity is selected among enzyme having a 2-keto acid decarboxylase activity.

### Claim 10 (depends on 1)

The modified organism of claim 1 wherein the enzyme having 4-hydroxybutyrate decarboxylase activity is obtained by at least one mutation of an enzyme, said mutation improving the activity and/or the substrate affinity of the mutated enzyme for OHB.

### Claim 11 (depends on 9)

The modified organism of claim 9 wherein the enzyme is a gene product encoded by genes selected among PDC1, PDC5, PDC6, ARO10, THI3 genes from Saccharomyces cerevisiae, kivD, kdcA genes from Lactococcus lactis, pdc gene from Clostridium acetobutylicum, PDC2, PDC3 genes from Arabidopsis thaliana, PDC1, PDC2, AR010 genes from Pichia stipitis, the pdc gene from Zymomonas mobilis, the sucA gene from Escherichia coli, dxs gene of Escherichia coli, the pdc gene from Z. mobilis carrying a mutation in at least one of the following positions: Tyr290, Trp392, Gly413, or Ile476 ((by reference to SEQ ID No. 128), the kdcA gene from L. lactis carrying a mutation in at least one of the following positions: Gln377, Phe381, Phe382, Gly402 Val461, Ile465, or Phe542 ((by reference to SEQ ID No. 130)).

### Claim 12 (depends on 11)

The modified organism of claim 11 wherein the enzyme is
a polynucleotide chosen among the group comprising SEQ ID No. 129, SEQ ID No. 127, SEQ ID No. 207, SEQ ID No. 189, SEQ ID No. 191, SEQ ID No. 193, SEQ ID No. 195, or SEQ ID No. 197 or any sequence sharing a homology of at least 50% with said sequences,
a polypeptide chosen among the group comprising SEQ ID No. 130, SEQ ID No. 128, SEQ ID No. 208, SEQ ID No. 190, SEQ ID No. 192, SEQ ID No. 194, SEQ ID No. 196, or SEQ ID No. 198 or any sequence sharing a homology of at least 50% with said sequences.

### Claim 13 (depends on 1)

The modified organism of claim 1 wherein the enzyme having 3-hydroxypropionaldehyde reductase activity is selected among enzymes having hydroxyaldehyde reductase activity, alcohol dehydrogenase activity, lactaldehyde reductase activity, or methylglyoxal reductase activity.

### Claim 14 (depends on 13)

The modified organism of claim 13 wherein the enzyme is a gene product encoded by genes selected among yqhD, fucO, dkgA, dkgB genes from Escherichia coli, dhaT gene of K. pneumoniae, or ADH1 and ADH2 genes from Saccharomyces cerevisiae or an enzyme having 3-hydroxypropionaldehyde reductase activity obtained by at least one mutation of an enzyme, said mutation improving the activity and/or the substrate affinity of the mutated enzyme for 3-HPA.

### Claim 15 (depends on 1)

The modified organism according to claim 1 wherein the production of PDO is enhanced.

### Claim 16 (depends on 1)

The modified organism according to claim 1 wherein, the 2,4-dihydroxybutyrate dehydrogenase, 2-oxo-4-hydroxybutyrate decarboxylase, and/or 3-hydroxypropionaldehyde reductase activities and/or the enzymes activities allowing the synthesis of DHB are enhanced.

### Claim 17 (depends on 1)

The microorganism of claim 1 being a bacterium, a yeast, or a fungus.

### Claim 18 (depends on 1)

The microorganism of claim 1 wherein the expression of at least of one the enzymatic activities chosen among phosphoenolpyruvate carboxylase, phosphoenol pyruvate carboxykinase, isocitrate lyase, pyruvate carboxylase, and hexose symporter permease is increased, and/or at least one of the enzymatic activities chosen among lactate dehydrogenase, alcohol dehydrogenase, acetate kinase, phosphate acetyltransferase, pyruvate oxidase, isocitrate lyase, fumarase, 2-oxoglutarate dehydrogenase, pyruvate kinase, malic enzyme, phosphoglucose isomerase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, pyruvate-formate lyase, succinic semialdehyde dehydrogenase, sugar-transporting phosphotransferase, ketohydroxyglutarate aldolase, homoserine-O-succinyl transferase, homoserine kinase, diaminopimelate decarboxylase, and/or methylglyoxal synthase is decreased.

### Claim 19 (depends on 17)

The microorganism according to claim 17 being Escherichia coli overexpressing at least one of the genes chosen among ppc, pck, aceA, galP, asd, thrA, metL, lysC all E coli; pycA from L lactis, and/or having at least one of the genes deleted chosen among IdhA, adhE, ackA, pta, poxB, focA, pfIB, sad, gabABC, sfcA, maeB, ppc, pykA, pykF, mgsA, sucAB, ptsl, ptsG, pgi, fumABC, aldA, lldD, icIR, metA, thrB, lysA, eda.

### Claim 20

A method of production of PDO comprising the steps of
contacting the modified microorganism with a carbon substrate in an appropriate culture medium,
recovering PDO from the culture medium.

### Claim 21 (depends on 20)

The method of claim 20 wherein the PDO is further purified.

### Claim 22

(canceled)

### Claim 23

(canceled)