Microorganism modified for the assimilation of levulinic acid

Abstract

The present invention relates to a genetically modified microorganism for the fermentative conversion of levulinic acid into propionyl-CoA and acetyl-CoA, and to a fermentation process for performing said conversion.

Claims

1. A method for the fermentative conversion of levulinic acid into propionyl-CoA and acetyl-CoA, comprising the step of culturing, under fermentation conditions, a genetically modified microorganism, in a culture medium comprising as a source of carbon at least levulinic acid, wherein said genetically modified microorganism overexpresses relative to a corresponding non-genetically modified microorganism: at least one enzyme converting levulinic acid into levulinyl-CoA selected from acyl CoA: 3-ketoacid CoA/acetate CoA transferases (EC 2.8.3.5/EC 2.8.3.8) and combinations thereof, wherein said at least one enzyme comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5, and at least one enzyme converting said levulinyl-CoA into propionyl-CoA and acetyl-CoA, selected from acetyl-CoA C-acetyltransferases (EC 2.3.1.9/EC 2.3.1.16) and combinations thereof, wherein said at least one enzyme comprises the amino acid sequence of SEQ ID NO: 9.

2. A method for the fermentative production of 1,2-propanediol, said method comprising the steps of: a) culturing, under fermentation conditions, a genetically modified microorganism, in a culture medium comprising as a source of carbon at least levulinic acid, and b) recovering said 1,2-propanediol, wherein said genetically modified microorganism overexpresses relative to a corresponding non-genetically modified microorganism: at least one enzyme converting levulinic acid into levulinyl-CoA selected from acyl CoA: 3-ketoacid CoA/acetate CoA transferases (EC 2.8.3.5/EC 2.8.3.8) and combinations thereof, wherein said at least one enzyme comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5, and at least one enzyme converting said levulinyl-CoA into propionyl-CoA and acetyl-CoA, selected from acetyl-CoA C-acetyltransferases (EC 2.3.1.9/EC 2.3.1.16) and combinations thereof, wherein said at least one enzyme comprises the amino acid sequence of SEQ ID NO: 9.

3. The method according to claim 2, wherein said source of carbon is a lignocellulosic substrate.

4. The method according to claim 1, wherein said source of carbon further comprises at least one carbohydrate substrate.

5. The method according to claim 2, wherein said source of carbon further comprises at least one carbohydrate substrate.

6. The method according to claim 5, wherein said carbohydrate substrate is selected from xylose, glycerol, glucose, galactose, fructose, lactose, maltose, sucrose, and combinations thereof.

7. The method according to claim 4, wherein said carbohydrate substrate is selected from xylose, glycerol, glucose, galactose, fructose, lactose, maltose, sucrose, and combinations thereof.

8. The method according to claim 1, wherein said microorganism is selected from bacterium, yeast and fungus.

9. The method according to claim 8, wherein said microorganism belongs to the family of the bacteria Enterobacteriaceae, Clostridiaceae, Bacillaceae, Streptomycetaceae, or Corynebacteriaceae, or to the family of yeasts Saccharomycetaceae.

10. The method according to claim 9, wherein said Enterobacteriaceae bacterium is Escherichia coli, said Clostridiaceae bacterium is Clostridium acetobutylicum, said Corynebacteriaceae bacterium is Corynebacterium glutamicum, or said Saccharomycetaceae yeast is Saccharomyces cerevisiae.

11. The method according to claim 2, wherein said microorganism is selected from bacterium, yeast and fungus.

12. The method according to claim 11, wherein said microorganism belongs to the family of the bacteria Enterobacteriaceae, Clostridiaceae, Bacillaceae, Streptomycetaceae, or Corynebacteriaceae, or to the family of yeasts Saccharomycetaceae.

13. The method according to claim 12, wherein said Enterobacteriaceae bacterium is Escherichia coli, said Clostridiaceae bacterium is Clostridium acetobutylicum, said Corynebacteriaceae bacterium is Corynebacterium glutamicum, or said Saccharomycetaceae yeast is Saccharomyces cerevisiae.

Description

DRAWINGS

(1) FIG. 1 represents possible enzymes involved in the assimilation of levulinic acid in Ralstonia euphora.

(2) FIG. 2 represents levulinyl-CoA chemical formula.

(3) FIG. 3 represents extracted ion chromatograms of transition 864 to 408 from a sample of Q0KBZ9_CUPNH/QOKC00_CUPNH reaction mixture (3A), and from a sample of a reaction mixture control (3B). The peaks of the chromatograms show the presence of levulinyl-CoA in the reaction mixture, which demonstrates the conversion of levulinic acid into levulinyl-CoA by the enzyme.

EXAMPLES

(4) Molecular Biology Methods

(5) Methods well known in the art were used to construct Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum or Clostridium acetobutylicum strains containing replicating vectors and/or various chromosomal deletions, and substitutions. For example, chromosomal modification in E. coli could be introduced using homologous recombination well described by Datsenko & Wanner (2000). In the same manner, the use of plasmids or vectors to express or overexpress one or several genes in a recombinant microorganisms are well known by the man skilled in the art. Examples of suitable E. coli expression vectors include pTrc, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236, etc. . . . (Studier et al. 1990, Pouwels et al. 1985). Examples of E. coli promoter leading high overexpression of the gene include Ptrc (Brosius et al., 1985), Ptac (de Boer et al., 1983), P/ac (Dickson et al., 1975) etc. . . .

(6) Examples of suitable S. cerevisiae vectors are e. g. centromeric and 2p plasmids of the pRS series (Sikorski and Hieter, 1989; Christianson et al., 1992) etc. . . . . Examples of S. cerevisiae promoters leading to high overexpression of genes include hxt7, pgk1 (Partow et al., 2010), tef1, tef2 (Nevoigt et al., 2006) etc. . . . . Examples of suitable S. cerevisiae terminators are e. g. cyc1, adh1, pgk1 etc. . . . . (Curran et al., 2013). Chromosomal modification could be introduced in S. cerevisiae using homologous recombination as described by Gldener et al. (1996).

(7) Examples of suitable C. glutamicum vectors are e. g. pClik5aMCS (WO2005059093) plasmids of pEC-X series (Kirchner et al., 2003) other can be found in Eikmanns et al. (1991) etc. . . . . Examples of C. glutamicum promoter leading high overexpression of the gene include PgapA, Ptuf, Psod, Ptrc (Eikmanns et al., 1991; Patek et al. 2013) etc. . . . . Chromosomal modifications could be introduced in C. glutamicum using homologous recombination as described by Suzuki et al. (2005).

(8) Examples of suitable C. acetobutylicum vectors are e. g. pSOS plasmids (Tummala et al. 1999), plasmids of pSYL series (Lee, 1992) others of pMTL series can be found in Chambers et al. (1988) etc. . . . . Examples of C. acetobutylicum promoter leading to high overexpression of genes include thl, adc, ptb (Tummala et al., 1999) etc. . . . . Chromosomal modifications could be introduced in C. acetobutylicum using homologous recombination as described by Croux & Soucaille in patent application WO2008/040387.

(9) Protocol 1 (chromosomal modifications by homologous recombination, selection of recombinants), Protocol 2 (transduction of phage P1) and Protocol 3 (antibiotic cassette excision, the resistance genes were removed when necessary) were used herein, and have been fully described in patent application EP2532751, incorporated herein by reference. Chromosomal modifications were verified by a PCR analysis with appropriate oligonucleotides that the person skilled in the art is able to design.

(10) Protocol 4: Construction of Recombinant Plasmids

(11) Recombinant DNA technology is described in Molecular Cloning: Sambrook and Russell, 3.sup.rd edition (2001) Cold Spring Harbor Laboratory Press, NY, Vol 1, 2, 3. Briefly, the DNA fragments were PCR amplified using oligonucleotides and appropriate genomic DNA as matrix (that the person skilled in the art will be able to define). The DNA fragments and chosen plasmid were digested with compatible restriction enzymes (that the person skilled in the art will be able to define), then ligated and transformed into competent cells. Transformants were analysed and recombinant plasmids of interest were verified by DNA sequencing.

(12) Strain Cultivation Methods

(13) Wild-type E. coli, S. cerevisiae, C. glutamicum and C. actetobutylicum strains and their derivatives overexpressing the levulinic acid pathway were cultivated in shake flasks as described in Sambrook and Russel as described above, van Dijken et al. (2000), Keilhauer et al. (1993) and Holt et al. (1984), respectively. Industrial strains producing 1,2-propanediol, glycolic acid, 1,4-butanediol, ethanol, succinic acid, lysine or butanol were cultivated as described in patents WO 2008/116852, WO 2012/055798, WO 2011/157728, WO 2010/141920, US 2015/104822, WO 2010/003728, U.S. Pat. No. 9,109,242 or WO 2008/052973, respectively. When needed, the appropriate plasmid antibiotics were included in the culture medium.

(14) For each wild-type and industrial strain not expressing the levulinic acid pathway (parent strains), levulinic acid was added to the culture medium at different concentrations ranging from 0.01 g/L (0.1% w/v) to 10 g/L (1% w/v). The half maximal inhibitory concentration (IC50) of levulinic acid was determined as the concentration that led to a 50% decrease of final OD (optical density, reflecting biomass concentration) by comparison to the control culture without levulinic acid.

(15) The strain derivatives overexpressing the levulinic acid pathway were then cultivated with levulinic acid at the IC50 determined for their respective parent strains. Growth was evaluated by measuring the final OD. For industrial strains, production was evaluated by measuring the final concentration of 1,2-propanediol, glycolic acid, 1,4-butanediol, ethanol, succinic acid, lysine or butanol as described in patents WO 2008/116852, WO 2012/055798, WO 2011/157728, WO 2010/141920, US 2015/104822, WO 2010/003728, U.S. Pat. No. 9,109,242 or WO 2008/052973, respectively.

Example 1: Identification of Ralstonia eutropha Putative Enzymes and Corresponding Genes Involved in the Assimilation of Levulinic Acid by a Bioinformatics Approach

(16) Ralstonia eutropha is known to assimilate levulinic acid. The products of this assimilation have however not been identified to this day. The Inventors hypothesized that assimilation of levulinic acid by Ralstonia eutropha proceeds through 2 enzymatic steps as shown in FIG. 1: a first step leading to levulinyl-CoA (A); and a second step leading to acetyl-CoA and propionyl-CoA (B); acetyl-CoA and propionyl-CoA being then integrated into the central carbon metabolism, respectively through the citric acid and the 2-methylcitric acid cycles, or used for the production of polyhydroxyalkanoates (Steinbchel and Schlegel 1991, Bramer and Steinbuchel 2001, Yu and Si 2004).

(17) Based on this hypothesis, it was considered that the following enzymes might be involved in such conversion: for step A, in addition to acyl-CoA synthetases (A1: EC 6.2.1.-), coA transferases (A2: EC 2.8.3.-) or a combination (A3) of carboxy-phosphotransferases (A3a: EC 2.7.2.-) and phosphate transacylases (A3b: EC 2.3.1.8/2.3.1.19/2.3.1.222); for step B, in addition to beta-ketothiolases (EC 2.3.1.16), other thiolases (EC 2.3.1.9/2.3.1.174/2.3.1.194).

(18) Enzymes displaying the above activities were then searched in Ralstonia eutropha annotated genome: 37 candidates were found for reaction A1 (as described in FIG. 1 and Table 2), 5 for reaction A2 (as described in FIG. 1 and Table 3), 5 for reaction A3a (as described in FIG. 1 and Table 4), none for reaction A3b, and 24 for reaction B (as described in FIG. 1 and Table 5).

(19) TABLE-US-00002 TABLE 2 Putative candidates for reaction A1 Uniprot entry names Protein names Gene names ACSA_CUPNH Acetyl-coenzyme A synthetase (AcCoA synthetase) (Acs) acsA acoE H16_A2525 (EC 6.2.1.1) (Acetate--CoA ligase) (Acyl-activating enzyme) Q0K466_CUPNH H16_B0409 protein (EC 6.2.1.) H16_B0409 SUCC_CUPNH + Succinyl-CoA ligase [ADP-forming] (EC 6.2.1.5) sucC H16_A0547 + Q0KE74_CUPNH sucD H16_A0548 Q0K2Z8_CUPNH Acetyl-coenzyme A synthetase (AcCoA synthetase) (Acs) acsA H16_B0834 (EC 6.2.1.1) (Acetate--CoA ligase) (Acyl-activating enzyme) Q0K489_CUPNH Acetyl-CoA synthetase (NDP-forming) (EC 6.2.1.1) H16_B0386 Q0K471_CUPNH Acyl-CoA synthetase (EC 6.2.1.) H16_B0404 Q0K8X0_CUPNH Propionate-CoA ligase (EC 6.2.1.17) prpE H16_A2462 Q0KB73_CUPNH Acetyl-coenzyme A synthetase (EC 6.2.1.1) H16_A1616 Q0KDD5_CUPNH Acyl-CoA synthetase (AMP-forming)/AMP-acid ligase II H16_A0839 (EC 6.2.1.) Q0KA99_CUPNH Acyl-CoA synthetase (AMP-forming)/AMP-acid ligase II H16_A1971 (EC 6.2.1.) Q0K3I3_CUPNH Acyl-CoA synthetase (AMP-forming)/AMP-acid ligase II H16_B0644 (EC 6.2.1.) Q0K4B6_CUPNH Acyl-CoA synthetase (EC 6.2.1.) H16_B0358 Q0KAZ6_CUPNH Acyl-CoA synthetase (AMP-forming)/AMP-acid ligase II H16_A1700 (EC 6.2.1.) Q0K0I0_CUPNH Acyl-CoA synthetase (AMP-forming)/AMP-acid ligase II H16_B1709 (EC 6.2.1.) Q0KCD3_CUPNH Acyl-CoA synthetase (EC 6.2.1.) H16_A1197 Q0K281_CUPNH Acetyl-CoA synthetase (EC 6.2.1.1) H16_B1102 Q0K1J9_CUPNH Acyl-CoA synthetase (AMP-forming)/AMP-acid ligase II H16_B1335 (EC 6.2.1.) Q0K7Y6_CUPNH Acyl-CoA synthetase (AMP-forming)/AMP-acid ligase II H16_A2807 (EC 6.2.1.) Q0K3F0_CUPNH Acyl-CoA synthetase (AMP-forming)/AMP-acid ligase II H16_B0677 (EC 6.2.1.) Q0KDA8_CUPNH Acyl-CoA synthetase (AMP-forming)/AMP-acid ligase II H16_A0866 (EC 6.2.1.) Q0K3D1_CUPNH Acyl-CoA synthetase (AMP-forming) (EC 6.2.1.1) H16_B0696 Q0K4U9_CUPNH Acyl-CoA synthetase (AMP-forming)/AMP-acid ligase II H16_B0174 (EC 6.2.1.) Q0K2S2_CUPNH Acyl-CoA synthetase (AMP-forming)/AMP-acid ligase II H16_B0910 (EC 6.2.1.) Q0K1G2_CUPNH Acyl-CoA synthetase (AMP-forming)/AMP-acid ligase II H16_B1373 (EC 6.2.1.) Q0K7G7_CUPNH Acyl-CoA synthetase (EC 6.2.1.) H16_A2978 Q0K7Z8_CUPNH Acyl-CoA synthetase (AMP-forming)/AMP-acid ligase II H16_A2794 (EC 6.2.1.) Q0K9H2_CUPNH Acyl-CoA synthetase (EC 6.2.1.) H16_A2252 Q0KAX9_CUPNH Acyl-CoA synthetase (AMP-forming)/AMP-acid ligase II H16_A1718 (EC 6.2.1.) Q0KCA1_CUPNH Acyl-CoA synthetase (AMP-forming)/AMP-acid ligase II H16_A1230 (EC 6.2.1.) Q0KC36_CUPNH Acyl-CoA synthetase (AMP-forming)/AMP-acid ligase II H16_A1295 (EC 6.2.1.) Q0KDA3_CUPNH Acyl-CoA synthetase (AMP-forming)/AMP-acid ligase II H16_A0871 (EC 6.2.1.) Q0K3N6_CUPNH Acetyl-CoA synthetase (EC 6.2.1.1) H16_B0591 Q0KBH0_CUPNH Acyl-CoA synthetase (AMP-forming)/AMP-acid ligase II H16_A1519 (EC 6.2.1.) Q0K235_CUPNH Acyl-CoA synthetase (EC 6.2.1.) H16_B1148 Q0K7T4_CUPNH Acetoacetyl-CoA synthetase (EC 6.2.1.16) H16_A2860 Q0K1S0_CUPNH Acyl-CoA synthetase (AMP-forming)/AMP-acid ligase II H16_B1264 (EC 6.2.1.) Q0K1D7_CUPNH Acyl-CoA synthetase (AMP-forming)/AMP-acid ligase II H16_B1398 (EC 6.2.1.)

(20) TABLE-US-00003 TABLE 3 Putative candidates for reaction A2 Uniprot entry names Protein names Gene names Q0KC00_CUPNH + Succinyl-CoA:3-ketoacid-coenzyme A transferase (EC H16_A1331 + Q0KBZ9_CUPNH 2.8.3.5) H16_A1332 Q0K874_CUPNH Acetate CoA-transferase YdiF (EC 2.8.3.8) pct H16_A2718 Q0K4S5_CUPNH + 3-Oxoadipate CoA-transferase subunit A (EC 2.8.3.6) pcal H16_B0198 + Q0K4S4_CUPNH pcaJ H16_B0199 Q0K3Y9_CUPNH Predicted acyl-CoA transferase (EC 2.8.3.) H16_B0488 Q0K3H2_CUPNH + Acyl CoA:acetate/3-ketoacid CoA transferase (EC 2.8.3.) H16_B0655 + Q0K3H1_CUPNH H16_B0656

(21) TABLE-US-00004 TABLE 4 Putative candidates for reaction A3a Uniprot entry names Protein names Gene names PGKC_CUPNH Phosphoglycerate kinase, chromosomal (EC 2.7.2.3) cbbKC cbbK2 H16_B1385 Q0K0Q9_CUPNH Acetate kinase (EC 2.7.2.1) (Acetokinase) ackA H16_B1630 Q0KE56_CUPNH Phosphoglycerate kinase (EC 2.7.2.3) pgk H16_A0566 Q0KDV3_CUPNH Acetate kinase (EC 2.7.2.1) (Acetokinase) ackA2 ackA H16_A0670 PGKP_CUPNH Phosphoglycerate kinase, plasmid (EC 2.7.2.3) cbbKP PHG417

(22) TABLE-US-00005 TABLE 5 Putative candidates for reaction B Uniprot entry names Protein names Gene names BKTB_CUPNH Beta-ketothiolase (EC 2.3.1.16/EC 2.3.1.9) bktB H16_A1445 Q0K4S3_CUPNH Beta-ketoadipyl CoA thiolase (EC 2.3.1.16) pcaF H16_B0200 Q0KEF9_CUPNH Acetyl-CoA C-acyltransferase (EC 2.3.1.16) H16_A0462 Q0KAX7_CUPNH Acetyl-CoA acetyltransferase (EC 2.3.1.9) H16_A1720 Q0K0C1_CUPNH Acetyl-CoA acetyltransferase (EC 2.3.1.9) H16_A1713 H16_B1771 Q0K3F9_CUPNH Acetyl-CoA acetyltransferase (EC 2.3.1.9) H16_B0668 Q0K494_CUPNH Acetyl-CoA acetyltransferase (EC 2.3.1.9) H16_B0381 Q0KBG1_CUPNH Acetyl-CoA acetyltransferase (EC 2.3.1.9) H16_A1528 Q0KC41_CUPNH Acetyl-CoA C-acyltransferase (EC 2.3.1.16) H16_A1290 Q0KDD4_CUPNH 3-Ketoacyl-CoA-thiolase P-44/SCP2 (EC 2.3.1.16) paaJ H16_A0840 Q0KDA2_CUPNH Acetyl-CoA acetyltransferase (EC 2.3.1.9) H16_A0872 Q0K1G6_CUPNH Acetyl-CoA acetyltransferase (EC 2.3.1.9) H16_B1369 Q0K3G5_CUPNH Acetyl-CoA acetyltransferase (EC 2.3.1.9) H16_B0662 Q0KF99_CUPNH Acetyl-CoA acetyltransferase (EC 2.3.1.9) H16_A0170 Q0KAI3_CUPNH Acetyl-CoA acetyltransferase (EC 2.3.1.9) H16_A1887 Q0K368_CUPNH Acetyl-CoA acetyltransferase (EC 2.3.1.9) H16_B0759 Q0K9S6_CUPNH Acetyl-CoA acetyltransferase (EC 2.3.1.9) H16_A2148 Q0K495_CUPNH Acetyl-CoA acetyltransferase (EC 2.3.1.9) H16_B0380 Q0K485_CUPNH Acyl-CoA transferase (EC 2.3.1.16) H16_B0390 Q0K497_CUPNH Acetyl-CoA C-acyltransferase (EC 2.3.1.16) H16_B0378 Q0KDA7_CUPNH Acetyl-CoA acetyltransferase (EC 2.3.1.9) H16_A0867 Q0K469_CUPNH Acetyl-CoA acetyltransferase (EC 2.3.1.9) H16_B0406 Q0KDA6_CUPNH Acetyl-CoA acetyltransferase (EC 2.3.1.9) H16_A0868 Q0KC34_CUPNH Acetyl-CoA acetyltransferase (EC 2.3.1.9) H16_A1297

(23) In order to identify the enzymes of Tables 2 to 5 that are involved in the assimilation of levulinic acid, a quantitative proteomics approach was carried out, which allowed the discrimination of the enzymes undergoing a variation in their expression level when Ralstonia eutropha is cultured in presence of levulinic acid, from the enzymes that are not affected by the presence of levulinic acid. The enzymes that were overexpressed (i.e. upregulated) were assumed to be involved in the conversion of levulinic acid.

Example 2: Identification of Ralstonia eutropha Enzymes and Corresponding Genes Involved in the Assimilation of Levulinic Acid by a Quantitative Proteomics Approach

(24) Ralstonia eutropha DSM428 strain was cultivated in shake flasks with LB rich medium with 0 or 5 g/L levulinic acid. Cells were collected by centrifugation and resuspended in potassium phosphate buffer 100 mM pH 7.6. Proteins were extracted by sonication and crude extracts were clarified by centrifugation. The supernatant fractions were then digested with trypsin and analyzed by nanoLC-MS/MS on a Synapt G2 QTOF mass spectrometer. Protein abundancies were calculated as % of total proteins using Waters Identity bioinformatics pipeline.

(25) TABLE-US-00006 TABLE 6 Quantitative proteomics of Ralstonia eutropha DSM428 cultivated with or without levulinic acid (in bold characters: proteins up-regulated in response to levulinic acid, eg for which the quantity is at least multiplied by 2). % of total proteins Uniprot entry Gene Without With names Protein names names LA LA ACOA_CUPNH Acetoin:2,6-dichlorophenolindophenol oxidoreductase acoA 0.23 <0.06 subunit alpha (Acetoin:DCPIP oxidoreductase-alpha) H16_B0144 (Ao:DCPIP OR) (EC 1.1.1.) ACOB_CUPNH Acetoin:2,6-dichlorophenolindophenol oxidoreductase acoB 0.42 <0.06 subunit beta (Acetoin:DCPIP oxidoreductase-beta) H16_B0145 (Ao:DCPIP OR) (EC 1.1.1.) (TPP-dependent acetoin dehydrogenase E1 subunit beta) ACOC_CUPNH Dihydrolipoyllysine-residue acetyltransferase acoC 0.29 <0.06 component of acetoin cleaving system (EC 2.3.1.12) H16_B0146 (Acetoin dehydrogenase E2 component) (Dihydrolipoamide acetyltransferase component of acetoin cleaving system) (Fast-migrating protein) (FMP) ACP_CUPNH Acyl carrier protein (ACP) acpP 0.33 0.42 H16_A2566 ACSA_CUPNH Acetyl-coenzyme A synthetase (AcCoA synthetase) acsA acoE 0.52 0.33 (Acs) (EC 6.2.1.1) (Acetate-CoA ligase) (Acyl- H16_A2525 activating enzyme) ATPB_CUPNH ATP synthase subunit beta (EC 3.6.3.14) (ATP atpD 0.42 0.46 synthase F1 sector subunit beta) (F-ATPase subunit H16_A3637 beta) BDHA_CUPNH D-beta-hydroxybutyrate dehydrogenase (BDH) (EC hbdH1 0.26 0.21 1.1.1.30) (3-hydroxybutyrate dehydrogenase) (3- H16_A1334 HBDH) BKTB_CUPNH Beta-ketothiolase BktB (EC 2.3.1.16) (EC 2.3.1.9) bktB 0.40 2.00 (Acetyl-CoA acetyltransferase) (Acetyl-CoA H16_A1445 acyltransferase) CH10_CUPNH 10 kDa chaperonin (GroES protein) (Protein Cpn10) groS groES 0.86 0.78 H16_A0705 CH60_CUPNH 60 kDa chaperonin (GroEL protein) (Protein Cpn60) groL groEL 4.38 4.22 H16_A0706 CLPP_CUPNH ATP-dependent Clp protease proteolytic subunit (EC clpP 0.20 0.20 3.4.21.92) (Endopeptidase Clp) H16_A1483 DAPD_CUPNH 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N- dapD 0.25 0.21 succinyltransferase (EC 2.3.1.117) H16_A2066 (Tetrahydrodipicolinate N-succinyltransferase) (THDP succinyltransferase) (THP succinyltransferase) (Tetrahydropicolinate succinylase) DLDH_CUPNH Dihydrolipoyl dehydrogenase (EC 1.8.1.4) odhL 0.72 0.79 (Dihydrolipoamide dehydrogenase) (E3 component of H16_A2323 2-oxoglutarate dehydrogenase complex) DNAK_CUPNH Chaperone protein DnaK (HSP70) (Heat shock 70 kDa dnaK 1.18 0.90 protein) (Heat shock protein 70) H16_A3089 EFP_CUPNH Elongation factor P (EF-P) efp 0.29 0.30 H16_A2549 EFTS_CUPNH Elongation factor Ts (EF-Ts) tsf 0.65 0.68 H16_A2054 EFTU_CUPNH Elongation factor Tu (EF-Tu) tuf1 tufA 7.24 6.51 H16_A3491; tuf2 tufB H16_A3505 ENO_CUPNH Enolase (EC 4.2.1.11) (2-phospho-D-glycerate hydrolyase) eno 0.80 0.78 (2-phosphoglycerate dehydratase) H16_A1188 GCST_CUPNH Aminomethyltransferase (EC 2.1.2.10) (Glycine gcvT 0.29 0.30 cleavage system T protein) H16_A3619 GLYA_CUPNH Serine hydroxymethyltransferase (SHMT) (Serine glyA 1.01 0.91 methylase) (EC 2.1.2.1) H16_A2834 ILVC_CUPNH Ketol-acid reductoisomerase (EC 1.1.1.86) ilvC 1.06 0.95 (Acetohydroxy-acid isomeroreductase) (Alpha-keto- H16_A1037 beta-hydroxylacyl reductoisomerase) KAD_CUPNH Adenylate kinase (AK) (EC 2.7.4.3) (ATP-AMP adk 0.16 0.15 transphosphorylase) (ATP:AMP phosphotransferase) H16_A0603 (Adenylate monophosphate kinase) KDSA_CUPNH 2-dehydro-3-deoxyphosphooctonate aldolase (EC kdsA 0.14 0.11 2.5.1.55) (3-deoxy-D-manno-octulosonic acid 8- H16_A1186 phosphate synthase) (KDO-8-phosphate synthase) (KDO 8-P synthase) (KDOPS) (Phospho-2-dehydro-3- deoxyoctonate aldolase) LPXA_CUPNH Acyl-[acyl-carrier-protein]--UDP-N-acetylglucosamine IpxA 0.20 0.22 O-acyltransferase (UDP-N-acetylglucosamine H16_A2043 acyltransferase) (EC 2.3.1.129) MDH_CUPNH Malate dehydrogenase (EC 1.1.1.37) mdh 1.50 1.51 H16_A2634 METE_CUPNH 5-methyltetrahydropteroyltriglutamate--homocysteine metE 1.17 1.20 methyltransferase (EC 2.1.1.14) (Cobalamin- H16_B1581 independent methionine synthase) (Methionine synthase, vitamin-B12 independent isozyme) METK_CUPNH S-adenosylmethionine synthase (AdoMet synthase) metK 0.41 0.39 (EC 2.5.1.6) (MAT) (Methionine adenosyltransferase) H16_A0230 NDK_CUPNH Nucleoside diphosphate kinase (NDK) (NDP kinase) ndk 0.40 0.34 (EC 2.7.4.6) (Nucleoside-2-P kinase) H16_A2368 ODO1_CUPNH 2-oxoglutarate dehydrogenase E1 component (EC odhA 0.87 1.17 1.2.4.2) (Alpha-ketoglutarate dehydrogenase) H16_A2325 ODO2_CUPNH Dihydrolipoyllysine-residue succinyltransferase sucB odhB 0.46 0.49 component of 2-oxoglutarate dehydrogenase complex H16_A2324 (EC 2.3.1.61) (2-oxoglutarate dehydrogenase complex component E2) (OGDC-E2) (Dihydrolipoamide succinyltransferase component of 2-oxoglutarate dehydrogenase complex) ODP1_CUPNH Pyruvate dehydrogenase E1 component (PDH E1 pdhA 0.37 0.57 component) (EC 1.2.4.1) H16_A1374 PHBB_CUPNH Acetoacetyl-CoA reductase (EC 1.1.1.36) phbB phaB 1.14 0.85 H16_A1439 PNP_CUPNH Polyribonucleotide nucleotidyltransferase (EC 2.7.7.8) pnp 0.59 0.61 (Polynucleotide phosphorylase) (PNPase) H16_A1045 PUR7_CUPNH Phosphoribosylaminoimidazole-succinocarboxamide purC 0.19 0.17 synthase (EC 6.3.2.6) (SAICAR synthetase) H16_A0569 PUR9_CUPNH Bifunctional purine biosynthesis protein PurH purH 0.23 0.30 [Includes: H16_A0501 Phosphoribosylaminoimidazolecarboxamide formyltransferase (EC 2.1.2.3) (AICAR transformylase); IMP cyclohydrolase (EC 3.5.4.10) (ATIC) (IMP synthase) (Inosinicase)] Q0JY61_CUPNH Argininosuccinate synthase (EC 6.3.4.5) (Citrulline- argG 0.37 0.34 aspartate ligase) H16_B2531 Q0JZ13_CUPNH O-Acetylhomoserine sulfhydrylase (EC 2.5.1.49) metY2 0.24 0.48 H16_B2229 Q0JZI5_CUPNH Succinate-semialdehyde dehydrogenase (NADP+) gabD4 0.30 0.48 (EC 1.2.1.16) H16_B2057 Q0JZW1_CUPNH Isocitrate dehydrogenase [NADP] (EC 1.1.1.42) icd2 0.68 0.85 H16_B1931 Q0K0L9_CUPNH Fumarylacetoacetase hydrolase (EC 3.7.1.2) fahA 0.48 0.41 H16_B1670 Q0K1I7_CUPNH Putative peptidase, C56 family (EC 3.4..) H16_B1347 <0.06 0.45 Q0K1T6_CUPNH Bacterial DNA-binding protein, histone-like H16_B1248 0.16 0.19 Q0K1U4_CUPNH Dehydrogenase with different specificities (EC 1...) H16_B1240 <0.06 0.34 Q0K1U9_CUPNH DNA-binding protein HU family hupB3 0.13 0.13 H16_B1235 Q0K1Z2_CUPNH Methylmalonate-semialdehyde dehydrogenase (EC mmsA3 1.27 1.02 1.2.1.27) H16_B1191 Q0K1Z3_CUPNH 3-hydroxyisobutyrate dehydrogenase (HIBADH) (EC H16_B1190 0.72 0.47 1.1.1.31) Q0K1Z4_CUPNH 3-Hydroxybutyryl-CoA dehydratase (EC 4.2.1.55) crt 0.13 0.12 H16_B1189 Q0K2A0_CUPNH 4-Hydroxyphenylpyruvate dioxygenase (EC hpd 0.30 0.35 1.13.11.27) H16_B1083 Q0K2A2_CUPNH Tyrosine aminotransferase (EC 2.6.1.57) tyrB2 0.33 0.22 H16_B1081 Q0K3Q9_CUPNH Aconitate hydratase 2 (EC 4.2.1.3) (EC 4.2.1.99) (2- acnB <0.06 0.35 methylisocitrate dehydratase) H16_B0568 Q0K4P6_CUPNH H-NS-like DNA-binding protein H16_B0227 0.15 0.10 Q0K5B6_CUPNH Cold-shock protein, DNA-binding H16_B0002 0.19 0.16 Q0K5I0_CUPNH DNA-binding protein HU family hupB2 0.48 0.69 H16_A3684 Q0K5K4_CUPNH ABC-type transporter, periplasmic component H16_A3660 1.00 <0.06 Q0K5N4_CUPNH ABC-type transporter, periplasmic component: HAAT H16_A3630 0.96 0.72 family Q0K5P3_CUPNH Glycine dehydrogenase (decarboxylating) (EC gcvP 0.50 0.60 1.4.4.2) (Glycine cleavage system P-protein) (Glycine H16_A3621 decarboxylase) (Glycine dehydrogenase (aminomethyl-transferring)) Q0K611_CUPNH Elongation factor G (EF-G) fusA 2.93 2.89 H16_A3492 Q0K6A8_CUPNH Stringent starvation protein A (Glutathione S- sspA 0.27 0.29 transferase) H16_A3395 Q0K6J9_CUPNH ABC-type transporter, periplasmic component: PepT H16_A3298 0.26 <0.06 family Q0K6W8_CUPNH Probable histone H1-like protein (Alanine/lysin-rich H16_A3178 0.69 0.64 protein) Q0K6X1_CUPNH Probable thiol peroxidase (EC 1.11.1.) tpx 0.36 0.20 H16_A3175 Q0K6X4_CUPNH Biotin carboxylase (EC 6.3.4.14) accC2 0.14 0.18 H16_A3172 Q0K6Z3_CUPNH Malic enzyme (NAD-binding) (EC 1.1.1.38) maeA 0.74 0.87 H16_A3153 Q0K700_CUPNH Glyceraldehyde-3-phosphate dehydrogenase (EC gapA 0.52 0.77 1.2.1.) H16_A3146 Q0K729_CUPNH Response regulator H16_A3117 0.23 0.17 Q0K737_CUPNH Catalase (EC 1.11.1.6) katE1 0.65 0.30 H16_A3109 Q0K738_CUPNH Metalloregulation DNA-binding stress protein H16_A3108 0.25 0.28 Q0K790_CUPNH Isocitrate dehydrogenase [NADP] (EC 1.1.1.42) icd1 0.55 0.42 H16_A3056 Q0K7C1_CUPNH Acetylornithine aminotransferase (ACOAT) (EC argD 0.31 0.30 2.6.1.11) H16_A3025 Q0K7C4_CUPNH ABC-type transporter, periplasmic component H16_A3022 0.19 <0.06 Q0K7C9_CUPNH Urocanate hydratase (Urocanase) (EC 4.2.1.49) hutU1 hutU 0.22 <0.06 (Imidazolonepropionate hydrolase) H16_A3017 Q0K7D7_CUPNH L-Aspartate decarboxylase (EC 4.1.1.12) asdA 0.53 0.13 H16_A3009 Q0K7G8_CUPNH Predicted periplasmic or secreted protein H16_A2977 0.22 0.28 Q0K7X5_CUPNH Glutaryl-CoA dehydrogenase (EC 1.3.99.7) gcdH 0.22 0.12 H16_A2818 Q0K840_CUPNH UTP--glucose-1-phosphate uridylyltransferase (EC galU 0.32 0.26 2.7.7.9) (UDP-glucose pyrophosphorylase) H16_A2752 Q0K8F1_CUPNH Aconitate hydratase (Aconitase) (EC 4.2.1.3) acnA 1.24 1.17 H16_A2638 Q0K8F2_CUPNH Hypothetical membrane associated protein H16_A2637 <0.06 0.13 Q0K8G2_CUPNH Citrate synthase (EC 2.3.3.16) cisY 1.73 1.74 H16_A2627 Q0K8G3_CUPNH ABC-type transporter, periplasmic component: HAAT livK1 1.52 1.28 family H16_A2626 Q0K8H1_CUPNH Aspartate-semialdehyde dehydrogenase (ASA asd 0.49 0.38 dehydrogenase) (ASADH) (EC 1.2.1.11) (Aspartate- H16_A2618 beta-semialdehyde dehydrogenase) Q0K8M0_CUPNH Malonyl CoA-acyl carrier protein transacylase (EC fabD 0.33 0.29 2.3.1.39) H16_A2568 Q0K8M3_CUPNH 3-oxoacyl-[acyl-carrier-protein] synthase 2 (EC fabF 0.24 0.22 2.3.1.179) H16_A2565 Q0K8P8_CUPNH Uncharacterized protein, possibly involved in H16_A2536 0.22 0.17 utilization of glycolate and propanediol Q0K8X8_CUPNH Carbamoyl-phosphate synthase small chain (EC carA 0.23 <0.06 6.3.5.5) (Carbamoyl-phosphate synthetase glutamine H16_A2454 chain) Q0K8Y0_CUPNH Carbamoyl-phosphate synthase (glutamine- carB2 0.42 0.56 hydrolyzing) (EC 6.3.5.5) H16_A2452 Q0K919_CUPNH Enoyl-[acyl-carrier-protein] reductase [NADH] (EC fabl1 0.22 0.22 1.3.1.9) H16_A2410 Q0K931_CUPNH Transcription termination factor Rho (EC 3.6.4.) rho <0.06 0.13 (ATP-dependent helicase Rho) H16_A2395 Q0K932_CUPNH Thioredoxin H16_A2394 0.13 0.10 Q0K972_CUPNH Adenylosuccinate synthetase (AMPSase) (AdSS) (EC purA 0.32 0.26 6.3.4.4) (IMP--aspartate ligase) H16_A2354 Q0K990_CUPNH Glutamine synthetase (EC 6.3.1.2) glnA1 1.11 1.15 H16_A2335 Q0K9F8_CUPNH Homoserine dehydrogenase (EC 1.1.1.3) thrA 0.27 0.26 H16_A2266 Q0K9K7_CUPNH Malate synthase (EC 2.3.3.9) aceB 0.70 0.58 H16_A2217 Q0K9L1_CUPNH Non-heme haloperoxidase H16_A2213 0.65 0.27 Q0K9U7_CUPNH ABC-type transporter, periplasmic component: H16_A2125 0.23 0.25 MUTfamily Q0K9X2_CUPNH ABC-type transporter, periplasmic component: PepT dppA1b 0.56 0.39 family H16_A2100 Q0KA33_CUPNH Phosphoenolpyruvate synthase (PEP synthase) (EC ppsA 0.32 0.50 2.7.9.2) (Pyruvate, water dikinase) H16_A2038 Q0KA41_CUPNH Inosine-5-monophosphate dehydrogenase (IMP guaB 0.43 0.59 dehydrogenase) (IMPD) (IMPDH) (EC 1.1.1.205) H16_A2030 Q0KA97_CUPNH Acetyl/propionyl-CoA carboxylase, carboxyltransferase H16_A1973 0.56 0.41 subunit (EC 6.4.1.) Q0KA99_CUPNH Acyl-CoA synthetase (AMP-forming)/AMP-acid H16_A1971 <0.06 0.15 ligase II (EC 6.2.1.) Q0KAF1_CUPNH Putative Lactaldehyde dehydrogenase (EC 1.2.1.22) H16_A1919 0.21 <0.06 Q0KAG0_CUPNH Glutaminase-asparaginase (Amidohydrolase) (EC ansA 1.56 0.62 3.5.1.38) H16_A1910 Q0KAG3_CUPNH Aconitate hydratase (Aconitase) (EC 4.2.1.3) acnM <0.06 0.64 H16_A1907 Q0KAG4_CUPNH 2-Methylcitrate synthase 1 (EC 2.3.3.5) prpC1 0.49 1.82 H16_A1906 Q0KBG1_CUPNH Acetyl-CoA acetyltransferase (EC 2.3.1.9) H16_A1528 <0.06 0.30 Q0KBG3_CUPNH Enoyl-CoA hydratase/Delta(3)-cis-delta(2)-trans- H16_A1526 <0.06 0.72 enoyl-CoA isomerase Q0KBM6_CUPNH Peroxiredoxin (EC 1.11.1.) H16_A1460 1.73 1.51 Q0KBM7_CUPNH Alkyl hydroperoxide reductase AhpD (EC 1.11.1.15) ahpD 0.06 0.08 (Alkylhydroperoxidase AhpD) H16_A1459 h16_A1459 Q0KBT6_CUPNH ABC-type transporter, periplasmic component H16_A1399 0.50 0.33 Q0KBV4_CUPNH Phasin (PHA-granule associated protein) phaP1 0.14 0.09 H16_A1381 Q0KBX0_CUPNH Uncharacterized protein H16_A1365 0.07 <0.06 Q0KBY8_CUPNH Phenylalanine--tRNA ligase beta subunit (EC pheT 0.24 0.39 6.1.1.20) (Phenylalanyl-tRNA synthetase beta H16_A1344 subunit) Q0KBZ9_CUPNH Succinyl-CoA:3-ketoacid-coenzyme A transferase H16_A1332 0.26 0.76 subunit B (EC 2.8.3.5) Q0KC00_CUPNH Succinyl-CoA:3-ketoacid-coenzyme A transferase H16_A1331 0.25 0.95 subunit A (EC 2.8.3.5) Q0KCA5_CUPNH Aspartokinase (EC 2.7.2.4) lysC 0.30 0.31 H16_A1225 Q0KCB2_CUPNH Peptidyl-prolyl cis-trans isomerase (EC 5.2.1.8) ppiB 0.38 0.38 H16_A1218 Q0KCC6_CUPNH 4-hydroxy-tetrahydrodipicolinate synthase (HTPA dapA dapA1 0.30 0.29 synthase) (EC 4.3.3.7) H16_A1204 Q0KCH9_CUPNH Tyrosine aminotransferase (EC 2.6.1.57) tyrB1 0.27 0.27 H16_A1151 Q0KCJ3_CUPNH Protein GrpE (HSP-70 cofactor) grpE 0.14 0.12 H16_A1137 Q0KCN9_CUPNH FadE2-like Acyl-CoA dehydrogenase (ACAD) H16_A1091 <0.06 3.16 Q0KCR2_CUPNH Acyl-CoA dehydrogenase (EC 1.3.99.3) H16_A1068 <0.06 0.12 Q0KCX7_CUPNH Malic enzyme (NADP) (EC 1.1.1.40) maeB 1.10 0.90 H16_A1002 Q0KD01_CUPNH Uncharacterized protein H16_A0977 0.11 0.07 Q0KDF9_CUPNH Electron transfer flavoprotein alpha subunit fixB 0.74 0.96 H16_A0815 Q0KDG0_CUPNH Electron transfer flavoprotein beta-subunit fixA 0.51 0.69 H16_A0814 Q0KDH6_CUPNH 30S ribosomal protein S1 rpsA 0.44 0.51 H16_A0798 Q0KDI4_CUPNH Uncharacterized protein H16_A0790 0.41 0.34 Q0KDI6_CUPNH Outer membrane protein or related peptidoglycan- H16_A0788 0.13 0.12 associated (Lipo)protein Q0KDL4_CUPNH ABC-type transporter, periplasmic component: H16_A0759 0.55 0.32 FeTfamily Q0KDM0_CUPNH Thioredoxin reductase (EC 1.8.1.9) H16_A0753 0.28 0.26 Q0KDM3_CUPNH Nitrogen regulatory protein PII H16_A0750 0.09 0.07 Q0KDM7_CUPNH Inorganic pyrophosphatase (EC 3.6.1.1) ppa 0.12 0.14 (Pyrophosphate phospho-hydrolase) H16_A0746 Q0KE13_CUPNH Superoxide dismutase (EC 1.15.1.1) sodA 1.09 0.94 H16_A0610 Q0KE21_CUPNH Short-chain alcohol dehydrogenase/3-hydroxyacyl- H16_A0602 0.27 0.22 CoA dehydrogenase (EC 1.1.1.) Q0KE31_CUPNH Probable extra-cytoplasmic solute receptor H16_A0592 0.67 0.31 Q0KE56_CUPNH Phosphoglycerate kinase (EC 2.7.2.3) pgk 0.26 0.24 H16_A0566 Q0KE74_CUPNH Succinyl-CoA ligase [ADP-forming] subunit alpha (EC sucD 0.64 0.60 6.2.1.5) H16_A0548 Q0KEC5_CUPNH Uncharacterized protein H16_A0496 0.46 0.13 Q0KEE9_CUPNH ABC-type transporter, periplasmic component: PAAT H16_A0472 2.03 1.19 family Q0KEF0_CUPNH Glutamate dehydrogenase (EC 1.4.1.3) gdhA1 0.83 0.69 H16_A0471 Q0KEL9_CUPNH Single-stranded DNA-binding protein H16_A0402 0.15 0.15 Q0KEP8_CUPNH Ribose-phosphate pyrophosphokinase (RPPK) (EC prsA prs 0.98 0.29 2.7.6.1) (5-phospho-D-ribosyl alpha-1-diphosphate) H16_A0372 (Phosphoribosyl diphosphate synthase) (Phosphoribosyl pyrophosphate synthase) Q0KF06_CUPNH Glutamine--fructose-6-phosphate glmS <0.06 0.39 aminotransferase [isomerizing] (EC 2.6.1.16) (D- H16_A0263 fructose-6-phosphate amidotransferase) (GFAT) (Glucosamine-6-phosphate synthase) (Hexosephosphate aminotransferase) (L- glutamine--D-fructose-6-phosphate amidotransferase) Q0KF17_CUPNH Glutathione S-transferase (EC 2.5.1.18) H16_A0252 0.14 0.13 Q0KF49_CUPNH N-acetyl-gamma-glutamyl-phosphate reductase argC1 argC 0.17 0.15 (AGPR) (EC 1.2.1.38) (N-acetyl-glutamate H16_A0220 semialdehyde dehydrogenase) Q0KF71_CUPNH Probable extra-cytoplasmic solute receptor H16_A0198 0.64 <0.06 Q0KF75_CUPNH RNA polymerase-binding transcription factor DksA dksA1 dksA 0.23 0.18 H16_A0194 Q0KF85_CUPNH Biotin carboxylase (EC 6.3.4.14) accC1 0.40 0.40 H16_A0184 Q0KF90_CUPNH Enoyl-CoA hydratase/carnithine racemase (EC H16_A0179 0.22 0.25 4.2.1.17) Q0KF99_CUPNH Acetyl-CoA acetyltransferase (EC 2.3.1.9) H16_A0170 1.06 0.96 Q0KFA0_CUPNH Carbonic anhydrase (EC 4.2.1.1) can <0.06 0.16 H16_A0169 Q0KFA2_CUPNH Isovaleryl-CoA dehydrogenase (EC 1.3.99.10) ivd1 0.43 0.39 H16_A0167 Q0KFA8_CUPNH Thiol:disulfide interchange protein H16_A0161 0.30 0.22 Q0KFB1_CUPNH Uncharacterized protein H16_A0158 0.07 <0.06 Q0KFR7_CUPNH DNA polymerase III subunit beta (EC 2.7.7.7) dnaN 0.29 0.26 H16_A0002 RECA_CUPNH Protein RecA (Recombinase A) recA 0.17 0.13 H16_A0544 RL1_CUPNH 50S ribosomal protein L1 rplA 0.32 0.39 H16_A3500 RL11_CUPNH 50S ribosomal protein L11 rplK 0.15 0.14 H16_A3501 RL15_CUPNH 50S ribosomal protein L15 rplO 0.12 0.13 H16_A3465 RL16_CUPNH 50S ribosomal protein L16 rplP 0.08 0.10 H16_A3477 RL17_CUPNH 50S ribosomal protein L17 rplQ 0.37 0.41 H16_A3457 RL18_CUPNH 50S ribosomal protein L18 rplR 0.08 0.33 H16_A3468 RL2_CUPNH 50S ribosomal protein L2 rplB 0.22 0.31 H16_A3481 RL20_CUPNH 50S ribosomal protein L20 rplT 0.28 0.47 H16_A1342 RL21_CUPNH 50S ribosomal protein L21 rplU 0.13 0.19 H16_A3252 RL22_CUPNH 50S ribosomal protein L22 rplV 0.10 0.09 H16_A3479 RL23_CUPNH 50S ribosomal protein L23 rplW 0.13 0.19 H16_A3482 RL24_CUPNH 50S ribosomal protein L24 rplX 0.14 0.14 H16_A3473 RL25_CUPNH 50S ribosomal protein L25 (General stress protein rplY ctc 0.96 1.01 CTC) H16_A0371 RL29_CUPNH 50S ribosomal protein L29 rpmC 0.07 0.06 H16_A3476 RL3_CUPNH 50S ribosomal protein L3 rplC 0.19 0.23 H16_A3484 RL31B_CUPNH 50S ribosomal protein L31 type B rpmE2 0.16 0.13 H16_A2397 RL4_CUPNH 50S ribosomal protein L4 rplD 0.13 0.22 H16_A3483 RL7_CUPNH 50S ribosomal protein L7/L12 rplL 0.93 0.89 H16_A3498 RL9_CUPNH 50S ribosomal protein L9 rplI 0.16 0.18 H16_A2276 RPIA_CUPNH Ribose-5-phosphate isomerase A (EC 5.3.1.6) rpiA 0.11 0.13 (Phosphoriboisomerase A) (PRI) H16_A2345 RPOA_CUPNH DNA-directed RNA polymerase subunit alpha (RNAP rpoA 1.12 1.07 subunit alpha) (EC 2.7.7.6) (RNA polymerase subunit H16_A3458 alpha) (Transcriptase subunit alpha) RPOB_CUPNH DNA-directed RNA polymerase subunit beta (RNAP rpoB 2.55 2.85 subunit beta) (EC 2.7.7.6) (RNA polymerase subunit H16_A3497 beta) (Transcriptase subunit beta) RPOC_CUPNH DNA-directed RNA polymerase subunit beta (RNAP rpoC 1.75 1.69 subunit beta) (EC 2.7.7.6) (RNA polymerase subunit H16_A3496 beta) (Transcriptase subunit beta) RRF_CUPNH Ribosome-recycling factor (RRF) (Ribosome- frr 0.27 0.20 releasing factor) H16_A2052 RS10_CUPNH 30S ribosomal protein S10 rpsJ 0.11 <0.06 H16_A3490 RS13_CUPNH 30S ribosomal protein S13 rpsM 0.13 <0.06 H16_A3461 RS15_CUPNH 30S ribosomal protein S15 rpsO 0.17 0.13 H16_A1044 RS16_CUPNH 30S ribosomal protein S16 rpsP 0.16 0.11 H16_A0894 RS4_CUPNH 30S ribosomal protein S4 rpsD <0.06 0.14 H16_A3459 RS7_CUPNH 30S ribosomal protein S7 rpsG 0.32 0.38 H16_A3493 RS8_CUPNH 30S ribosomal protein S8 rpsH 0.18 0.20 H16_A3470 RS9_CUPNH 30S ribosomal protein S9 rpsI 0.13 0.17 H16_A0483 SAHH_CUPNH Adenosylhomocysteinase (EC 3.3.1.1) (S-adenosyl-L- ahcY 0.51 0.41 homocysteine hydrolase) (AdoHcyase) H16_A0244 SUCC_CUPNH Succinyl-CoA ligase [ADP-forming] subunit beta (EC sucC 1.19 1.38 6.2.1.5) (Succinyl-CoA synthetase subunit beta) H16_A0547 (SCS-beta) SYDND_CUPNH Aspartate--tRNA(Asp/Asn) ligase (EC 6.1.1.23) aspS 0.41 0.29 (Aspartyl-tRNA synthetase) (AspRS) (Non- H16_A0453 discriminating aspartyl-tRNA synthetase) (ND-AspRS) SYT_CUPNH Threonine--tRNA ligase (EC 6.1.1.3) (Threonyl-tRNA thrS 0.55 0.28 synthetase) (ThrRS) H16_A1339 THIL_CUPNH Acetyl-CoA acetyltransferase (EC 2.3.1.9) phbA 1.77 1.46 (Acetoacetyl-CoA thiolase) (Beta-ketothiolase PhbA) H16_A1438 TIG_CUPNH Trigger factor (TF) (EC 5.2.1.8) (PPlase) tig 0.51 0.56 H16_A1482 TPIS_CUPNH Triosephosphate isomerase (TIM) (EC 5.3.1.1) tpiA 0.19 0.14 (Triose-phosphate isomerase) H16_A1047

(26) As shown in Table 6, several proteins of particular relevance were found to be up-regulated in response to levulinic acid: Q0KA99_CUPNH: an acyl-CoA synthetase (AMP-forming)/AMP-acid ligase II (EC 6.2.1.-), one of the putative candidates listed above for reaction A1; Q0KBZ9_CUPNH and Q0KC00_CUPNH: 2 subunits of a succinyl-CoA:3-ketoacid-coenzyme A transferase (EC 2.8.3.5), one of the putative candidates listed above for reaction A2; BKTB_CUPNH: a beta-ketothiolase (EC 2.3.1.16/EC 2.3.1.9), one of the putative candidates listed above for reaction B; Q0KBG1_CUPNH: an acetyl-CoA acetyltransferase (EC 2.3.1.9), one of the putative candidates listed above for reaction B; as well as proteins involved in the 2-methylcitric acid cycle: Q0KAG4_CUPNH, a 2-methylcitrate synthase (PrpC), and Q0K3Q9_CUPNH, a 2-methylisocitrate dehydratase (AcnB); and proteins involved in fatty acid metabolism: Q0KBG3_CUPNH, an enoyl-CoA hydratase, and Q0KCN9_CUPNH and Q0KCR2_CUPNH, two acyl-CoA dehydrogenases.

(27) To the contrary, the putative candidates listed in Table 2-5 were not overexpressed: ACSA_CUPNH (putative candidate for reaction A1); SUCC_CUPNH/Q0KE74_CUPNH (putative candidate for reaction A1); and Q0KF99_CUPNH (putative candidate for reaction B).

Example 3: In Vitro Validation of the Candidates Identified by Proteomics

(28) To validate the activity of the candidates identified in Example 2 above with regard to reactions A1, A2 and B, their corresponding genes were cloned into the expression plasmid pPAL7 (Biorad). In the case of QOKBZ9_CUPNH and Q0KC00_CUPNH, the corresponding genes were cloned in operon into the pPAL7. The resulting plasmids were transformed into E. coli BL21(DE3) strain.

(29) TABLE-US-00007 TABLE 7 Proteomics candidates tested SEQ SEQ ID Protein names ID NO: Gene names NO: Q0KA99_CUPNH 1 H16_A1971 2 Q0KBZ9_CUPNH 3 H16_A1332 4 Q0KC00_CUPNH 5 H16_A1331 6 Q0KBG1_CUPNH 7 H16_A1528 8

(30) The resulting strains were cultivated as described in patent application WO 2010/076324, incorporated herein by reference. Cells were collected by centrifugation and resuspended in potassium phosphate buffer 100 mM pH 7.6. Proteins were extracted by sonication and crude extracts were clarified by centrifugation. Recombinant protein purification was carried out using Biorad PROfinity EXact cartridges according to the manufacturer's instructions.

(31) Validation of the enzymatic activities of the different candidates was achieved using LC-MS/MS for identifying levulinyl-CoA after incubation of the purified proteins with their respective substrates: 50 mM levulinic acid, 0.4 mM ATP and 0.4 mM coenzyme A for QOKA99_CUPNH (reaction A1); 50 mM levulinic acid and 0.3 mM succinyl-coA/acetoacetyl-coA/acetyl-coA for Q0KBZ9_CUPNH/Q0KC00_CUPNH (reaction A2 with either succinyl-coA, acetoacetyl-coA or acetyl-coA as coA donor); 0.3 mM acetyl-coA and 0.3 mM propionyl-coA for Q0KBG1_CUPNH (reaction B in the condensation direction, Modis & Wierenga, 1999).

(32) Levulinyl-CoA was detected by reverse phase UPLC coupled to negative ion MS/MS, by searching for the transition from mass 864 (mass of levulinyl-CoA minus 1) to 408 (ion corresponding to the adenine part according to the protocol described by Zirrolli et al. 1994) as shown on FIG. 3.

(33) Levulinyl-CoA was detected in all samples, while no significant signal was detected when either the purified proteins or the substrates were omitted from the reaction mixture.

Example 4: Overexpression of Ralstonia eutropha Genes for the Assimilation of Levulinic Acid in Other Microorganisms

(34) Since proteomics showed that Q0KBZ9_CUPNH/Q0KC00_CUPNH and BKTB_CUPNH (SEQ ID NO: 3, 5 and 9) were more abundant than their respective counterparts identified for reactions A and B, those were chosen to be overexpressed in different microbial strains not able to assimilate levulinic acid. Genes H16_A1331 and H16_A1332 were renamed respectively scoA and scoB.

(35) Construction of Strain 1

(36) The scoA (SEQ ID NO: 4) and scoB (SEQ ID NO: 6) operon together with the bktB (SEQ ID NO: 10) gene were cloned in operon into the pBBR1MCS3 plasmid (Kovach et al. 1995). The resulting plasmid named pBBR1MCS3-scoABre-bktBre was then transformed into E. coli MG1655 K12 strain, resulting in strain 1.

(37) Construction of Strain 2

(38) The codon optimized genes scoA (SEQ ID NO: 11), scoB (SEQ ID NO: 12) and bktB (SEQ ID NO: 13) were cloned into the p426-hphMX4 plasmid. The p426-hphMX4 plasmid is a pRS426 plasmid where the URA3 promoter and gene were replaced by the hphMX4 resistance gene (hygromycin resistance) under the tef1 promoter (SEQ ID NO: 14). The scoA, scoB and bktB optimized genes were respectively cloned under the hxt7, tef2 and pgk1 promoters (SEQ ID NO: 15 to 17). Transcriptional terminators cyc1, tef1 and pdcl (SEQ ID NO: 18 to 20) were respectively added after the scoA, scoB and bktB optimized genes. The resulting plasmid named p426-hph MX4-Phxt7-scoAreO1 sc-TTcyc1-Ptef2-scoBreO1 sc-TTtef1-Ppg k1-bktBreO1 sc-TTpdc1 was then transformed into Saccharomyces cerevisiae CEN.PK2-1C strain, resulting in strain 2.

(39) Construction of Strain 3

(40) The scoA and scoB operon together with the bktB gene were cloned in operon into the pEC-XT99A plasmid. The resulting plasmid named pEC-XT99A-scoABre-bktBre was then transformed into Corynebacterium glutamicum ATCC 13032 strain, resulting in strain 3.

(41) Construction of Strain 4

(42) The scoA and scoB operon together with the bktB gene were cloned in operon into the pSOS95 plasmid digested by the BamHI and SfoI restriction enzymes. The resulting plasmid named pSOS95-scoABre-bktBre was then transformed into Clostridium acetobutylicum ATCC 824 strain, resulting in strain 4.

(43) When cultivated in a medium containing levulinic acid at the IC50 determined for their parent strains, strains 1 to 4 exhibited at least 20% better growth.

Example 5: Overexpression of Ralstonia eutropha Genes for the Assimilation of Levulinic Acid in Industrial Production Strains

(44) The plasmids bearing scoA, scoB and bktB genes were then transformed into industrial production strains.

(45) Construction of Strain 5 Producing 1,2-propanediol

(46) The adh gene from Clostridium beijerinckii (Hanai et al. 2007) was cloned into the pME101VB01 plasmid described in patent application WO 2008/116853, incorporated herein by reference, resulting in plasmid named pME101VB01-sadH. To inactivate the fumarate reductase flavoprotein complex encoded by the frdABCD operon and the glucose phophotransferase Enzyme IIBC(Glc) encoded by the ptsG gene, the homologous recombination strategy was used (according to Protocols 1 and 3). Oligonucleotides for DfrdABCD (SEQ ID NO: 21 and 22) and DptsG (SEQ ID NO: 23 and 24), were used to PCR amplify the resistance cassettes. The strains retained were designated MG1655 DfrdABCD::Cm and MG1655 DptsG::Km. Finally, the DfrdABCD::Cm and the DptsG::Km deletions were transferred by P1 phage transduction (according to Protocol 2) into the evolved 1,2-propanediol production strain MG1655 lpd*DtpiA DpflAB DadhE DldhA DgloA DaldA DaldB Dedd DarcA Dndh described in patent application WO 2008/116852, incorporated herein by reference, The resistance genes were removed according to Protocol 3. Plasmids pME101VB01-sadH and pBBR1MCS3-scoABre-bktBre were transformed into the strain, resulting in strain 5.

(47) Construction of Strain 7 Producing Glycolic Acid

(48) The plasmid pBBR1MCS3-scoABre-bktBre was transformed into the glycolic acid production strain described in the Example 2 of patent application WO 2011/157728, incorporated herein by reference, resulting in strain 7.

(49) Construction of Strain 8 Producing 1,4-Butanediol

(50) The plasmid pBBR1MCS3-scoABre-bktBre was transformed into the 1,4-butanediol production strain described in patent application WO 2010/141920, incorporated herein by reference, resulting in strain 8.

(51) Construction of Strain 9 Producing Ethanol

(52) The plasmid p426-hphMX4-Phxt7-scoAreO1sc-TTcyc1-Ptef2-scoBreO1sc-TTtef1-Ppgk1-bktBreO1sc-TTpdc1 was transformed into the ethanol production strain described in patent application US 2015/104822, incorporated herein by reference, resulting in strain 9.

(53) Construction of Strain 10 Producing Succinic Acid

(54) The plasmid p426-hphMX4-Phxt7-scoAreO1sc-TTcyc1-Ptef2-scoBreO1sc-TTtef1-Ppgk1-bktBreO1sc-TTpdc1 was transformed into the succinic acid production strain described in patent application WO 2010/003728, incorporated herein by reference, resulting in strain 10.

(55) Construction of Strain 11 Producing Lysine

(56) The plasmid pEC-XT99A-scoABre-bktBre was transformed into the lysine production strain described in patent application U.S. Pat. No. 9,109,242, incorporated herein by reference, resulting in strain 11.

(57) Construction of Strain 13 Producing Butanol

(58) The plasmid pSOS95-Pthl-scoABre-bktBre was transformed into the butanol producing strain described in patent application WO 2008/052973, incorporated herein by reference resulting in strain 13.

(59) When cultivated in a medium containing levulinic acid at the IC50 determined for their parent strains, strains 5 to 13 exhibited at least 20% better growth and 20% better production.

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