Method for producing fructose-6-phosphate from dihydroxy acetone phosphate and glyceraldehyde-3-phosphate

Abstract

Described is a method for the production of fructose-6-phosphate (F6P) from dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) comprising the steps of: (a) enzymatically converting dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA); and (b) enzymatically converting the thus produced dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P) into fructose-6-phosphate (F6P); or
comprising the steps of: (a′) enzymatically converting glyceraldehyde-3-phosphate (G3P) into glyceraldehyde; and (b′) enzymatically converting the thus produced glyceraldehyde together with dihydroxyacetone phosphate (DHAP) into fructose-1-phosphate (F1P); and (c′) enzymatically converting the thus produced fructose-1-phosphate (F1P) into fructose-6-phosphate (F6P).

Claims

1. A method for the production of fructose-6-phosphate (F6P) from dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) (A) comprising the steps of: (a) enzymatically converting dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA) using a phosphoric monoester hydrolase (EC 3.1.3.-); and (b) enzymatically converting the thus produced dihydroxyacetone (DHA) together with glyceraldehyde-3-phosphate (G3P) into fructose-6-phosphate (F6P) using an aldehyde lyase (EC 4.1.2.-) or a transaldolase (EC 2.2.1.2); or (B) comprising the steps of: (a′) enzymatically converting glyceraldehyde-3-phosphate (G3P) into glyceraldehyde using a phosphoric monoester hydrolase (EC 3.1.3.-); and (b′) enzymatically converting the thus produced glyceraldehyde together with dihydroxyacetone phosphate (DHAP) into fructose-1-phosphate (F1P) using a fructose bisphosphate aldolase (EC 4.1.2.13); and (c′) enzymatically converting the thus produced fructose-1-phosphate (F1P) into fructose-6-phosphate (F6P) using a phosphoglucomutase (EC 5.4.2.2) or a phosphomannomutase (EC 5.4.2.8).

2. The method of claim 1 comprising the steps of: (a) enzymatically converting dihydroxyacetone phosphate (DHAP) into dihydroxyacetone (DHA) using a phosphoric monoester hydrolase (EC 3.1.3.-); and (b) enzymatically converting the thus produced dihydroxyacetone (DHA) together with glyceraldehyde-3-phosphate (G3P) into fructose-6-phosphate (F6P) using an aldehyde lyase (EC 4.1.2.-) or a transaldolase (EC 2.2.1.2).

3. The method of claim 2, wherein the phosphoric monoester hydrolase (EC 3.1.3.-) is selected from the group consisting of: (i) sugar phosphatase (EC 3.1.3.23); (ii) 6-phosphogluconate phosphatase (EC 3.1.3.-); (iii) Pyridoxal phosphate phosphatase (EC 3.1.3.74); (iv) Fructose-1-phosphate phosphatase (EC 3.1.3.-); (v) Dihydroxyacetone phosphatase (EC 3.1.3.-); (vi) Hexitol phosphatase (EC 3.1.3.-); (vii) Acid phosphatase (EC 3.1.3.2); (viii) Alkaline phosphatase (EC 3.1.3.1); (ix) Glycerol-1-phosphate phosphatase (EC 3.1.3.21); and (x) 3-phosphoglycerate phosphatase (EC 3.1.3.38).

4. The method of claim 2, wherein the conversion of dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P) into fructose-6-phosphate (F6P) according to step (b) is achieved by an aldehyde lyase (EC 4.1.2.-).

5. The method of claim 1 comprising the steps of: (a′) enzymatically converting glyceraldehyde-3-phosphate (G3P) into glyceraldehyde using a phosphoric monoester hydrolase (EC 3.1.3.-); and (b′) enzymatically converting the thus produced glyceraldehyde together with dihydroxyacetone phosphate (DHAP) into fructose-1-phosphate (F1P) using a fructose bisphosphate aldolase (EC 4.1.2.13); and (c′) enzymatically converting the thus produced fructose-1-phosphate (F1P) into fructose-6-phosphate (F6P) using a phosphoglucomutase (EC 5.4.2.2) or a phosphomannomutase (EC 5.4.2.8).

6. The method of claim 5, wherein the phosphoric monoester hydrolase (EC 3.1.3.-) is selected from the group consisting of: (i) Glyceraldehyde 3-phosphate phosphatase (EC 3.1.3.-); (ii) Alkaline phosphatase (EC 3.1.3.1); (iii) Acid phosphatase (EC 3.1.3.2); (iv) Sugar phosphatase (EC 3.1.3.23); and (v) Hexitol phosphatase (EC 3.1.3.-).

7. The method of claim 1 (B), wherein the conversion of fructose-1-phosphate (F1P) into fructose-6-phosphate (F6P) according to step (c′) is achieved by a Phosphoglucomutase (EC 5.4.2.2).

8. The method of claim 1 (B), wherein the conversion of fructose-1-phosphate (F1P) into fructose-6-phosphate (F6P) according to step (c′) is achieved by a Phosphomannomutase (EC 5.4.2.8).

9. The method of claim 1 which is carried out in vitro.

10. The method of claim 1(A) which is carried out in vivo in a recombinant microorganism which has been transformed with a nucleotide sequence which encodes an enzyme which can catalyze the conversion recited in step (a) of claim 1 and with a nucleotide sequence which encodes an enzyme which can catalyze the conversion recited in step (b) of claim 1.

11. The method of claim 1(B) which is carried out in vivo in a recombinant microorganism which has been transformed with a nucleotide sequence which encodes an enzyme which can catalyze the conversion recited in step (a′) of claim 1 (B) and with a nucleotide sequence which encodes an enzyme which can catalyze the conversion recited in step (b′) of claim 1 (B).

12. The method of claim 11, wherein the microorganism has furthermore been transformed with a nucleotide sequence which encodes an enzyme which can catalyze the conversion recited in step (c′) of claim 1 (C).

13. The method of claim 10, wherein the microorganism is furthermore characterized in that it a) has phosphoketolase activity; b) (i) has a diminished or inactivated Embden-Meyerhof-Parnas pathway (EMPP) by inactivation of the gene(s) encoding phosphofructokinase or by reducing phosphofructokinase activity as compared to a non-modified microorganism; or (ii) does not possess phosphofructokinase activity; and c) (i) has a diminished or inactivated oxidative branch of the pentose phosphate pathway (PPP) by inactivation of the gene(s) encoding glucose-6-phosphate dehydrogenase or by reducing glucose-6-phosphate dehydrogenase activity as compared to a non-modified microorganism; or (ii) does not possess glucose-6-phosphate dehydrogenase activity.

14. The method of claim 13, wherein the microorganism is furthermore characterized in that the EMPP is further diminished or inactivated by inactivation of the gene(s) encoding glyceraldehyde 3-phosphate dehydrogenase or by reducing glyceraldehyde 3-phosphate dehydrogenase activity as compared to a non-modified microorganism.

15. The method of claim 1, wherein said method is carried out in vivo in a recombinant microorganism, wherein said microorganism has been transformed with (a) a nucleotide sequence encoding a phosphoric monoester hydrolase (EC 3.1.3.-); and (b) a nucleotide sequence encoding an enzyme selected from the group consisting of (i) an aldehyde lyase (EC 4.1.2.-); and/or (ii) a transaldolase (EC 2.2.1.2).

16. The method of claim 1, wherein said method is carried out in vivo in a recombinant microorganism, wherein said microorganism has been transformed with (a) a nucleotide sequence encoding a phosphoric monoester hydrolase (EC 3.1.3.-); and (b) a nucleotide sequence encoding a fructose bisphosphate aldolase (EC 4.1.2.13); wherein said microorganism also possesses phosphoglucomutase (EC 5.4.2.2) or phosphomannomutase (EC 5.4.2.8) activity.

17. The method of claim 16, wherein said microorganism has been further transformed with a nucleotide sequence encoding an enzyme selected from the group consisting of: (i) Phosphoglucomutase (EC 5.4.2.2); and (ii) Phosphomannomutase (EC 5.4.2.8).

18. The method of claim 2, wherein the conversion of dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P) into fructose-6-phosphate (F6P) according to step (b) is achieved by a transaldolase (EC. 2.2.1.2).

Description

(1) FIG. 1 shows the impact of the AMP concentration on fructose bisphosphatase activity.

(2) FIG. 2 shows the impact of the AMP concentration on Fsa A129S.

(3) FIG. 3 shows the specific productivity of acetone and isopropanol for strains overexpressing the enzymes responsible for conversion of glyceraldehyde-3-phosphate (G3P) and dihydroxy-acetone phosphate (DHAP) into fructose-6-phosphate (F6P) (GBI 17553, solid line) and for strains which do not overexpress the enzymes responsible for conversion of glyceraldehyde-3-phosphate (G3P) and dihydroxy-acetone phosphate (DHAP) into fructose-6-phosphate (GBI 15847, dotted line).

(4) In this specification, a number of documents including patent applications are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

(5) The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.

EXAMPLES

(6) General Methods and Materials

(7) Procedure for ligations and transformations are well known in the art. Techniques suitable for use in the following examples may be found in Sambrook J., et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y., 1989, and Sambrook J., supra.

(8) Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Techniques suitable for use in the following examples may be found in Manual of Methods for General Bacteriology (Philipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Philips, eds).

(9) All reagents and materials used for the growth and maintenance of bacterial cells were obtained from Sigma-Aldrich Company (St. Louis, Mo.) unless otherwise specified.

(10) Enzymes Overexpression and Purification.

(11) a) Enzymes from E. coli

(12) Plasmids from the ASKA collection have been used (Kitagawa, M et al. DNA Res. 12:291-299 (2005)) for overexpression of enzymes from E. coli. Strain BL21(DE3) cells (Novagen) were cultivated in LB medium and were made electrocompetent. Electrocompetent BL21 cells were transformed with the corresponding plasmids for expression of the desired enzymes (see Table 1) and then plated on LB plates containing Chloramphenicol (25 ug/ml). Plates were incubated overnight at 30° C. The transformed cells were grown with shaking (160 rpm) using ZYM-5052 auto-induction medium (Studier F W, Prot. Exp. Pur. 41:207-234 (2005)) for 20 h at 30° C. The cells were collected by centrifugation at 4° C., 4,000 rpm for 20 min and the pellets were stored at −80° C.

(13) TABLE-US-00001 TABLE 1 Enzymes from E. coli overexpressed using plasmids from the ASKA collection and the corresponding coding sequence. Genes from E. coli for Enzymes overexpression Nucleotide sequence Protein encoded fsaA atgGAACTGTATCTGGATACTTCAGACGTTGTTGCGGTGAAGGCGC SEQ ID NO: 8 TGTCACGTATTTTTCCGCTGGCGGGTGTGACCACTAACCCAAGCAT Uniprot accession TATCGCCGCGGGTAAAAAACCGCTGGATGTTGTGCTTCCGCAACTT number P78055 CATGAAGCGATGGGCGGTCAGGGGCGTCTGTTTGCCCAGGTAATGG CTACCACTGCCGAAGGGATGGTTAATGACGCGCTTAAGCTGCGTTC TATTATTGCGGATATCGTGGTGAAAGTTCCGGTGACCGCCGAGGGG CTGGCAGCTATTAAGATGTTAAAAGCGGAAGGGATTCCGACGCTGG GAACCGCGGTATATGGCGCAGCACAAGGGCTGCTGTCGGCGCTGGC AGGTGCGGAATATGTTGCGCCTTACGTTAATCGTATTGATGCTCAG GGCGGTAGCGGCATTCAGACTGTGACCGACTTACACCAGTTATTGA AAATGCATGCGCCGCAGGCGAAAGTGCTGGCAGCGAGTTTCAAAAC CCCGCGTCAGGCGCTGGACTGCTTACTGGCAGGATGTGAATCAATT ACTCTGCCACTGGATGTGGCACAACAGATGATTAGCTATCCGGCGG TTGATGCCGCTGTGGCGAAGTTTGAGCAGGACTGGCAGGGAGCGTT TGGCAGAACGTCGATTtaa SEQ ID NO: 34 fsaA mutated ATGAGAGGATCTCACCATCACCATCACCATACGGATCCGGCCCTGA SEQ ID NO: 9 A129S GGGCCGAACTGTATCTGGATACTTCAGACGTTGTTGCGGTGAAGGC GCTGTCACGTATTTTTCCGCTGGCGGGTGTGACCACTAACCCAAGC ATTATCGCCGCGGGTAAAAAACCGCTGGATGTTGTGCTTCCGCAAC TTCATGAAGCGATGGGCGGTCAGGGGCGTCTGTTTGCCCAGGTAAT GGCTACCACTGCCGAAGGGATGGTTAATGACGCGCTTAAGCTGCGT TCTATTATTGCGGATATCGTGGTGAAAGTTCCGGTGACCGCCGAGG GGCTGGCAGCTATTAAGATGTTAAAAGCGGAAGGGATTCCGACGCT GGGAACCGCGGTATATGGCGCAGCACAAGGGCTGCTGTCGGCGCTG GCAGGTGCGGAATATGTTagcCCTTACGTTAATCGTATTGATGCTC AGGGCGGTAGCGGCATTCAGACTGTGACCGACTTACACCAGTTATT GAAAATGCATGCGCCGCAGGCGAAAGTGCTGGCAGCGAGTTTCAAA ACCCCGCGTCAGGCGCTGGACTGCTTACTGGCAGGATGTGAATCAA TTACTCTGCCACTGGATGTGGCACAACAGATGATTAGCTATCCGGC GGTTGATGCCGCTGTGGCGAAGTTTGAGCAGGACTGGCAGGGAGCG TTTGGCAGAACGTCGATTGGCCTATGCGGACGCTAA SEQ ID NO: 35 fsaB atgGAACTGTATCTGGACACCGCTAACGTCGCAGAAGTCGAACGTC SEQ ID NO: 16 TGGCACGCATATTCCCCATTGCCGGGGTGACAACTAACCCGAGCAT Uniprot accession TATCGCTGCCAGCAAGGAGTCCATATGGGAAGTGCTGCCGCGTCTG number P32669 CAAAAAGCGATTGGTGATGAGGGCATTCTGTTTGCTCAGACCATGA GCCGCGACGCGCAGGGGATGGTGGAAGAAGCGAAGCGCCTGCGCGA CGCTATTCCGGGTATTGTGGTGAAAATCCCGGTGACTTCCGAAGGT CTGGCAGCAATTAAAATACTGAAAAAAGAGGGTATTACTACACTTG GCACTGCTGTATATAGCGCCGCACAAGGGTTATTAGCCGCACTGGC AGGGGCAAAATACGTTGCTCCGTATGTTAACCGCGTAGATGCCCAG GGCGGAGACGGCATTCGTACGGTTCAGGAGCTGCAAACGCTGTTAG AAATGCACGCGCCAGAAAGCATGGTGCTGGCAGCCAGCTTTAAAAC GCCGCGTCAGGCGCTGGACTGTTTACTGGCAGGATGTGAATCCATC ACCCTGCCCTTAGATGTAGCGCAACAAATGCTCAACACCCCTGCGG TAGAGTCAGCTATAGAGAAGTTCGAACACGACTGGAATGCCGCATT TGGCACTACTCATCTCtaa SEQ ID NO: 36 talB atgACGGACAAATTGACCTCCCTTCGTCAGTACACCACCGTAGTGG SEQ ID NO: 17 CCGACACTGGGGACATCGCGGCAATGAAGCTGTATCAACCGCAGGA Uniprot accession TGCCACAACCAACCCTTCTCTCATTCTTAACGCAGCGCAGATTCCG number P0A870 GAATACCGTAAGTTGATTGATGATGCTGTCGCCTGGGCGAAACAGC AGAGCAACGATCGCGCGCAGCAGATCGTGGACGCGACCGACAAACT GGCAGTAAATATTGGTCTGGAAATCCTGAAACTGGTTCCGGGCCGT ATCTCAACTGAAGTTGATGCGCGTCTTTCCTATGACACCGAAGCGT CAATTGCGAAAGCAAAACGCCTGATCAAACTCTACAACGATGCTGG TATTAGCAACGATCGTATTCTGATCAAACTGGCTTCTACCTGGCAG GGTATCCGTGCTGCAGAACAGCTGGAAAAAGAAGGCATCAACTGTA ACCTGACCCTGCTGTTCTCCTTCGCTCAGGCTCGTGCTTGTGCGGA AGCGGGCGTGTTCCTGATCTCGCCGTTTGTTGGCCGTATTCTTGAC TGGTACAAAGCGAATACCGATAAGAAAGAGTACGCTCCGGCAGAAG ATCCGGGCGTGGTTTCTGTATCTGAAATCTACCAGTACTACAAAGA GCACGGTTATGAAACCGTGGTTATGGGCGCAAGCTTCCGTAACATC GGCGAAATTCTGGAACTGGCAGGCTGCGACCGTCTGACCATCGCAC CGGCACTGCTGAAAGAGCTGGCGGAGAGCGAAGGGGCTATCGAACG TAAACTGTCTTACACCGGCGAAGTGAAAGCGCGTCCGGCGCGTATC ACTGAGTCCGAGTTCCTGTGGCAGCACAACCAGGATCCAATGGCAG TAGATAAACTGGCGGAAGGTATCCGTAAGTTTGCTATTGACCAGGA AAAACTGGAAAAAATGATCGGCGATCTGCTGtaa SEQ ID NO: 37 talB mutated atgACGGACAAATTGACCTCCCTTCGTCAGTACACCACCGTAGTGG SEQ ID NO: 64 F178Y CCGACACTGGGGACATCGCGGCAATGAAGCTGTATCAACCGCAGGA TGCCACAACCAACCCTTCTCTCATTCTTAACGCAGCGCAGATTCCG GAATACCGTAAGTTGATTGATGATGCTGTCGCCTGGGCGAAACAGC AGAGCAACGATCGCGCGCAGCAGATCGTGGACGCGACCGACAAACT GGCAGTAAATATTGGTCTGGAAATCCTGAAACTGGTTCCGGGCCGT ATCTCAACTGAAGTTGATGCGCGTCTTTCCTATGACACCGAAGCGT CAATTGCGAAAGCAAAACGCCTGATCAAACTCTACAACGATGCTGG TATTAGCAACGATCGTATTCTGATCAAACTGGCTTCTACCTGGCAG GGTATCCGTGCTGCAGAACAGCTGGAAAAAGAAGGCATCAACTGTA ACCTGACCCTGCTGTTCTCCTTCGCTCAGGCTCGTGCTTGTGCGGA AGCGGGCGTGTTCCTGATCTCGCCGTaTGTTGGCCGTATTCTTGAC TGGTACAAAGCGAATACCGATAAGAAAGAGTACGCTCCGGCAGAAG ATCCGGGCGTGGTTTCTGTATCTGAAATCTACCAGTACTACAAAGA GCACGGTTATGAAACCGTGGTTATGGGCGCAAGCTTCCGTAACATC GGCGAAATTCTGGAACTGGCAGGCTGCGACCGTCTGACCATCGCAC CGGCACTGCTGAAAGAGCTGGCGGAGAGCGAAGGGGCTATCGAACG TAAACTGTCTTACACCGGCGAAGTGAAAGCGCGTCCGGCGCGTATC ACTGAGTCCGAGTTCCTGTGGCAGCACAACCAGGATCCAATGGCAG TAGATAAACTGGCGGAAGGTATCCGTAAGTTTGCTATTGACCAGGA AAAACTGGAAAAAATGATCGGCGATCTGCTGtaa SEQ ID NO: 38 ybiV atgAGCGTAAAAGTTATCGTCACAGACATGGACGGTACTTTTCTTA SEQ ID NO: 1 ACGACGCCAAAACGTACAACCAACCACGTTTTATGGCGCAATATCA Uniprot accession GGAACTGAAAAAGCGCGGCATTAAGTTCGTTGTTGCCAGCGGTAAT number P75792 CAGTATTACCAGCTTATTTCATTCTTTCCTGAGCTAAAGGATGAGA TCTCTTTTGTCGCGGAAAACGGCGCACTGGTTTACGAACATGGCAA GCAGTTGTTCCACGGCGAACTGACCCGACATGAATCGCGGATTGTT ATTGGCGAGTTGCTAAAAGATAAGCAACTCAATTTTGTCGCCTGCG GTCTGCAAAGTGCATATGTCAGCGAAAATGCCCCCGAAGCATTTGT CGCACTGATGGCAAAACACTACCATCGCCTGAAACCTGTAAAAGAT TATCAGGAGATTGACGACGTACTGTTCAAGTTTTCGCTCAACCTGC CGGATGAACAAATCCCGTTAGTGATCGACAAACTGCACGTAGCGCT CGATGGCATTATGAAACCCGTTACCAGTGGTTTTGGCTTTATCGAC CTGATTATTCCCGGTCTACATAAAGCAAACGGTATTTCGCGGTTAC TGAAACGCTGGGATCTGTCACCGCAAAATGTGGTAGCGATTGGCGA CAGCGGTAACGATGCGGAGATGCTGAAAATGGCGCGTTATTCCTTT GCGATGGGCAATGCTGCGGAAAACATTAAACAAATCGCCCGTTACG CTACCGATGATAATAATCATGAAGGCGCGCTGAATGTGATTCAGGC GGTGCTGGATAACACATCCCCTTTTAACAGCtga SEQ ID NO: 39 yieH atgTCCCGGATAGAAGCGGTATTTTTCGACTGCGACGGTACGCTGG SEQ ID NO: 3 TCGACAGTGAAGTCATTTGCTCTCGCGCATATGTAACGATGTTTCA Uniprot accession GGAATTTGGTATTACGCTCGATCCTGAAGAGGTATTCAAACGTTTC number P31467 AAAGGTGTAAAACTGTACGAAATTATCGATATTGTTTCCCTTGAAC ATGGTGTTACGTTAGCGAAAACAGAAGCTGAACACGTTTACCGTGC AGAAGTCGCTCGGCTGTTCGATTCAGAACTGGAAGCCATCGAAGGG GCTGGAGCGCTCCTGTCAGCGATCACTGCGCCAATGTGTGTGGTAT CTAACGGCCCAAATAACAAAATGCAGCATTCTATGGGCAAGCTGAA TATGTTGCACTACTTCCCGGATAAACTGTTCAGCGGCTACGATATT CAGCGCTGGAAGCCAGACCCGGCGTTAATGTTCCATGCGGCAAAAG CGATGAATGTAAATGTAGAAAACTGCATTCTGGTTGATGACTCAGT TGCCGGTGCACAATCTGGTATCGACGCAGGTATGGAAGTGTTCTAC TTCTGCGCCGACCCGCACAATAAGCCGATCGTTCACCCGAAAGTCA CCACCTTTACCCATCTTTCGCAGTTACCTGAACTGTGGAAAGCGCG TGGTTGGGATATTACGGCAtag SEQ ID NO: 40 yidA atgGCTATTAAACTCATTGCTATCGATATGGATGGCACCCTTCTGC SEQ ID NO: 2 TGCCCGATCACACCATTTCACCCGCCGTTAAAAATGCGATTGCCGC Uniprot accession AGCTCGCGCCCGTGGCGTGAATGTCGTGCTAACGACGGGTCGCCCG number P0A8Y5 TATGCAGGTGTGCACAACTACCTGAAAGAGCTGCATATGGAACAGC CGGGCGACTACTGCATTACTTATAACGGCGCGCTGGTACAGAAGGC CGCTGATGGTAGCACCGTGGCGCAAACTGCTCTCAGCTATGACGAC TATCGTTTCCTGGAAAAACTCTCTCGCGAAGTCGGTTCTCATTTCC ACGCCCTGGACCGCACCACGCTGTACACCGCCAACCGTGATATCAG CTACTACACGGTGCATGAATCCTTCGTTGCCACCATTCCGCTGGTG TTCTGCGAAGCGGAGAAAATGGACCCCAATACCCAGTTCCTGAAAG TGATGATGATTGATGAACCCGCCATCCTCGACCAGGCTATCGCGCG TATTCCGCAGGAAGTGAAAGAGAAATATACCGTGCTGAAAAGTGCG CCGTACTTCCTCGAAATCCTCGATAAACGCGTTAACAAAGGTACGG GGGTGAAATCACTGGCCGACGTGTTAGGTATTAAACCGGAAGAAAT CATGGCGATTGGCGATCAGGAAAACGATATCGCAATGATTGAATAT GCAGGCGTCGGTGTGGCGATGGATAACGCTATTCCTTCAGTGAAAG AAGTGGCGAACTTTGTCACCAAATCTAACCTTGAAGATGGCGTGGC GTTTGCTATTGAGAAGTATGTGCTGAATtaa SEQ ID NO: 41 yigL atgTACCAGGTTGTTGCGTCTGATTTAGATGGCACGTTACTTTCTC SEQ ID NO: 4 CCGACCATACGTTATCCCCTTACGCCAAAGAAACTCTGAAGCTGCT Uniprot accession CACCGCGCGCGGCATCAACTTTGTGTTTGCGACCGGTCGTCACCAC number P27848 GTTGATGTGGGGCAAATTCGCGATAATCTGGAGATTAAGTCTTACA TGATTACCTCCAATGGTGCGCGCGTTCACGATCTGGATGGTAATCT GATTTTTGCTCATAACCTGGATCGCGACATTGCCAGCGATCTGTTT GGCGTAGTCAACGACAATCCGGACATCATTACTAACGTTTATCGCG ACGACGAATGGTTTATGAATCGCCATCGCCCGGAAGAGATGCGCTT TTTTAAAGAAGCGGTGTTCCAATATGCGCTGTATGAGCCTGGATTA CTGGAGCCGGAAGGCGTCAGCAAAGTGTTCTTCACCTGCGATTCCC ATGAACAACTGCTGCCGCTGGAGCAGGCGATTAACGCTCGTTGGGG CGATCGCGTCAACGTCAGTTTCTCTACCTTAACCTGTCTGGAAGTG ATGGCGGGCGGCGTTTCAAAAGGCCATGCGCTGGAAGCGGTGGCGA AGAAACTGGGCTACAGCCTGAAGGATTGTATTGCGTTTGGTGACGG GATGAACGACGCCGAAATGCTGTCGATGGCGGGGAAAGGCTGCATT ATGGGCAGTGCGCACCAGCGTCTGAAAGACCTTCATCCCGAGCTGG AAGTGATTGGTACTAATGCCGACGACGCGGTGCCGCATTATCTGCG TAAACTCTATTTATCGtaa SEQ ID NO: 42 yqaB atgTACGAGCGTTATGCAGGTTTAATTTTTGATATGGATGGCACAA SEQ ID NO: 5 TCCTGGATACGGAGCCTACGCACCGTAAAGCGTGGCGCGAAGTATT Uniprot accession AGGGCACTACGGTCTTCAGTACGATATTCAGGCGATGATTGCGCTT number P77475 AATGGATCGCCCACCTGGCGTATTGCTCAGGCAATTATTGAGCTGA ATCAGGCCGATCTCGACCCGCATGCGTTAGCGCGTGAAAAAACAGA AGCAGTAAGAAGTATGCTGCTGGATAGCGTCGAACCGCTTCCTCTT GTTGATGTGGTGAAAAGTTGGCATGGTCGTCGCCCAATGGCTGTAG GAACGGGGAGTGAAAGCGCCATCGCTGAGGCATTGCTGGCGCACCT GGGATTACGCCATTATTTTGACGCCGTCGTCGCTGCCGATCACGTC AAACACCATAAACCCGCGCCAGACACATTTTTGTTGTGCGCGCAGC GTATGGGCGTGCAACCGACGCAGTGTGTGGTCTTTGAAGATGCCGA TTTCGGTATTCAGGCGGCCCGTGCAGCAGGCATGGACGCCGTGGAT GTTCGCTTGCTGtga SEQ ID NO: 43 hxpA gtgCGGTGCAAAGGTTTTCTGTTTGATCTTGATGGAACGCTGGTGG SEQ ID NO: 7 ATTCCCTGCCTGCGGTAGAACGGGCGTGGAGCAACTGGGCCAGACG Uniprot accession TCATGGGTTAGCGCCGGAAGAGGTGCTGGCTTTCATTCACGGTAAA number P77625 CAGGCGATCACCTCTCTGCGCCATTTTATGGCGGGCAAATCCGAGG CTGATATTGCCGCCGAGTTTACGCGTCTGGAGCACATCGAGGCCAC GGAAACCGAAGGTATTACCGCGCTTCCGGGGGCAATCGCCTTACTC AGTCATTTGAATAAAGCAGGTATTCCGTGGGCCATTGTGACTTCTG GCTCCATGCCGGTAGCGCGAGCGCGCCATAAAATAGCTGGGCTTCC CGCACCAGAGGTGTTTGTAACCGCTGAGCGAGTGAAGCGCGGAAAA CCAGAACCTGATGCGTATCTGTTAGGCGCGCAGCTGCTGGGGCTTG CGCCGCAGGAGTGTGTGGTGGTGGAAGATGCTCCCGCTGGCGTGCT TTCTGGCCTGGCGGCGGGTTGTCATGTCATTGCGGTTAACGCTCCG GCAGATACCCCGCGCCTGAATGAGGTCGATTTGGTCCTCCACAGTC TGGAGCAAATTACTGTGACCAAACAGCCAAATGGCGATGTTATTAT TCAGtga SEQ ID NO: 44 hxpB atgTCAACCCCGCGTCAGATTCTTGCTGCAATTTTTGATATGGATG SEQ ID NO: 23 GATTACTTATCGACTCAGAACCTTTATGGGATCGAGCCGAACTGGA Uniprot accession TGTGATGGCAAGCCTGGGGGTGGATATCTCCCGTCGTAACGAGCTG number P77247 CCGGACACCTTAGGTTTACGCATCGATATGGTGGTCGATCTTTGGT ACGCCCGGCAACCGTGGAATGGGCCAAGCCGTCAGGAAGTAGTAGA ACGGGTTATTGCCCGTGCCATTTCACTGGTTGAAGAGACACGTCCA TTATTACCAGGCGTGCGCGAAGCCGTTGCGTTATGCAAAGAACAAG GTTTATTGGTGGGACTGGCCTCCGCGTCACCACTACATATGCTGGA AAAAGTGTTGACCATGTTTGACTTACGCGACAGTTTCGATGCCCTC GCCTCGGCCGAAAAACTGCCTTACAGCAAGCCGCATCCGCAAGTAT ATCTCGACTGCGCAGCAAAACTGGGCGTTGACCCTCTGACCTGCGT AGCGCTGGAAGATTCGGTAAATGGCATGATCGCCTCTAAAGCAGCC CGCATGCGTTCCATCGTCGTTCCTGCGCCAGAAGCGCAAAATGATC CACGTTTTGTATTAGCAGACGTCAAACTTTCATCGCTGACAGAACT CACCGCAAAAGACCTTCTCGGTtaa SEQ ID NO: 45 fbaA atgTCTAAGATTTTTGATTTCGTAAAACCTGGCGTAATCACTGGTG SEQ ID NO: 24 ATGACGTACAGAAAGTTTTCCAGGTAGCAAAAGAAAACAACTTCGC Uniprot accession ACTGCCAGCAGTAAACTGCGTCGGTACTGACTCCATCAACGCCGTA number P0AB71 CTGGAAACCGCTGCTAAAGTTAAAGCGCCGGTTATCGTTCAGTTCT CCAACGGTGGTGCTTCCTTTATCGCTGGTAAAGGCGTGAAATCTGA CGTTCCGCAGGGTGCTGCTATCCTGGGCGCGATCTCTGGTGCGCAT CACGTTCACCAGATGGCTGAACATTATGGTGTTCCGGTTATCCTGC ACACTGACCACTGCGCGAAGAAACTGCTGCCGTGGATCGACGGTCT GTTGGACGCGGGTGAAAAACACTTCGCAGCTACCGGTAAGCCGCTG TTCTCTTCTCACATGATCGACCTGTCTGAAGAATCTCTGCAAGAGA ACATCGAAATCTGCTCTAAATACCTGGAGCGCATGTCCAAAATCGG CATGACTCTGGAAATCGAACTGGGTTGCACCGGTGGTGAAGAAGAC GGCGTGGACAACAGCCACATGGACGCTTCTGCACTGTACACCCAGC CGGAAGACGTTGATTACGCATACACCGAACTGAGCAAAATCAGCCC GCGTTTCACCATCGCAGCGTCCTTCGGTAACGTACACGGTGTTTAC AAGCCGGGTAACGTGGTTCTGACTCCGACCATCCTGCGTGATTCTC AGGAATATGTTTCCAAGAAACACAACCTGCCGCACAACAGCCTGAA CTTCGTATTCCACGGTGGTTCCGGTTCTACTGCTCAGGAAATCAAA GACTCCGTAAGCTACGGCGTAGTAAAAATGAACATCGATACCGATA CCCAATGGGCAACCTGGGAAGGCGTTCTGAACTACTACAAAGCGAA CGAAGCTTATCTGCAGGGTCAGCTGGGTAACCCGAAAGGCGAAGAT CAGCCGAACAAGAAATACTACGATCCGCGCGTATGGCTGCGTGCCG GTCAGACTTCGATGATCGCTCGTCTGGAGAAAGCATTCCAGGAACT GAACGCGATCGACGTTCTGtaa SEQ ID NO: 46 fbaB atgACAGATATTGCGCAGTTGCTTGGCAAAGACGCCGACAACCTTT SEQ ID NO: 25 TACAGCACCGTTGTATGACAATTCCTTCTGACCAGCTTTATCTCCC Uniprot accession CGGACATGACTACGTAGACCGCGTAATGATTGACAATAATCGCCCG number P0A991 CCAGCGGTGTTACGTAATATGCAGACGTTGTACAACACCGGGCGTC TGGCTGGCACAGGATATCTTTCTATTCTGCCGGTTGACCAGGGCGT TGAGCACTCTGCCGGAGCTTCATTTGCTGCTAACCCGCTCTACTTT GACCCGAAAAACATTGTTGAACTGGCGATCGAAGCGGGCTGTAACT GTGTGGCGTCAACTTACGGCGTGCTGGCGTCGGTATCGCGGCGTTA TGCGCATCGCATTCCATTCCTCGTCAAACTTAATCACAACGAGACG CTAAGTTACCCGAATACCTACGATCAAACGCTGTATGCCAGCGTGG AGCAGGCGTTCAACATGGGCGCGGTTGCGGTTGGTGCGACTATCTA TTTTGGCTCGGAAGAGTCACGTCGCCAGATTGAAGAAATTTCTGCG GCTTTTGAACGTGCGCACGAGCTGGGTATGGTGACAGTGCTGTGGG CCTATTTGCGTAACTCCGCCTTTAAGAAAGATGGCGTTGATTACCA TGTTTCCGCCGACCTGACCGGTCAGGCAAACCATCTGGCGGCAACC ATCGGTGCAGATATCGTCAAACAAAAAATGGCGGAAAATAACGGCG GCTATAAAGCAATTAATTACGGTTACACCGACGATCGTGTTTACAG CAAATTGACCAGCGAAAACCCGATTGATCTGGTGCGTTATCAGTTA GCTAACTGCTATATGGGTCGGGCTGGGTTGATAAACTCCGGCGGTG CTGCGGGCGGTGAAACTGACCTCAGCGATGCAGTGCGTACTGCGGT TATCAACAAACGCGCAGGCGGAATGGGGCTGATTCTTGGACGTAAA GCGTTCAAGAAATCGATGGCTGACGGCGTGAAACTGATTAACGCCG TGCAGGACGTTTATCTCGATAGCAAAATTACTATCGCCtga SEQ ID NO: 47 The expression of the recombinant enzymes was checked on a protein gel, after purification of the recombinant protein using a His trap (Protino Ni-IDA 1000 kit, Macherey Nagel). Purification was processed according to the manufacturer's recommendations.
b) Enzymes from Other Organisms than E. coli The target genes (see Table 2) from several organisms were codon-optimized by GeneArt® (Invitrogen) for optimal expression in Escherichia coli. In addition, a His-tag was added at the 5′ end of the gene and an additional stop codon was added at the 3′ end. The gene construction is flanked by NdeI and EcoRI restriction sites and provided within plasmid pET25b+ (Merckmillipore). Competent E. coli BL21(DE3) cells (Novagen) were transformed with these vectors according to standard heat shock procedure. The transformed cells were grown with shaking (160 rpm) using ZYM-5052 auto-induction medium (Studier F W, Prot. Exp. Pur. 41:207-234(2005)) for 20 h at 30° C. The cells were collected by centrifugation at 4° C., 4,000 rpm for 20 min and the pellets were stored at −80° C.

(14) TABLE-US-00002 TABLE 2 Enzymes from several organisms and corresponding coding sequence (codon optimized for expression E. coli). Genes for Enzymes overexpression Nucleotide sequence Protein encoded hdpA ATGCATCATCATCACCATCACATGACCGTGAATATTAG SEQ ID NO: 6 (from Corynebarium CTATCTGACCGATATGGATGGCGTGCTGATTAAAGAAG Uniprot accession glutamicum (strain R) GTGAAATGATTCCGGGTGCCGATCGTTTTCTGCAAAGC number A4QFW4 CTGACAGATAATAACGTGGAATTTATGGTGCTGACCAA CAACAGCATTTTTACACCGCGTGATCTGAGCGCACGTC TGAAAACCAGCGGTCTGGATATTCCGCCTGAACGTATT TGGACCAGCGCAACCGCCACCGCACATTTTCTGAAAAG TCAGGTGAAAGAAGGCACCGCATACGTTGTTGGTGAAA GCGGTCTGACCACCGCACTGCATACCGCAGGTTGGATT CTGACAGATGCAAATCCGGAATTTGTTGTTCTGGGTGA AACCCGTACCTATAGCTTTGAAGCAATTACCACCGCCA TTAATCTGATTTTAGGTGGTGCACGTTTCATTTGTACC AATCCGGATGTTACCGGTCCGAGTCCGAGCGGTATTCT GCCTGCAACCGGTAGCGTTGCAGCACTGATTACCGCAG CAACCGGTGCAGAACCGTATTACATTGGTAAACCGAAT CCTGTGATGATGCGTAGCGCACTGAATACCATTGGTGC ACATAGCGAACATACCGTTATGATTGGTGATCGTATGG ATACCGATGTTAAAAGTGGTCTGGAAGCAGGTCTGAGT ACCGTTCTGGTTCGTAGCGGTATTTCAGATGATGCAGA AATTCGTCGTTATCCGTTTCGTCCGACACATGTGATTA ATAGCATTGCCGATCTGGCAGATTGTTGGGATGATCCG TTTGGTGATGGTGCATTTCATGTTCCGGATGAACAGCA GTTTACCGATTAA SEQ ID NO: 48 LMRG_00181 ATGCATCTGGATAGCGCAAATCTGGATGACGTGAAAAA SEQ ID NO: 11 (from Listeria AATCCAGGCAAGCAGCATCTTTAAAGGCATTACCACCA Uniprot accession monocytogenes serotype ATCCGAGCATTCTGGTTAAAGAAAAATGTAATCGTCAG number A0A0H3GHX1 1/2a (strain 10403S)) ACCGCCATTAACCGTATTCTGGAACTGACCGATAAACA GGTTTTTGTTCAGACCGTTGGCTTTACCTATGAAGAAA TTCTGGCAGATGCACGTATGCTGCTGACCATGTTTGGT AAAGACAAAATCGCAATCAAAATTCCGGCACATGAAGC AGGCACCAATGTTATTGATACCCTGAAAAAAGAGGACA AAACCATTCAGATTCTGGGCACCGCAATTTATAGCGCA GATCAGGCAATTACCGCAGCACTGGCAGGCGCAGATTT TGTTGCACCGTATGTTAATCGTATGAGCGCAGCAAATA TCGACCCGTTTAAAGAAATTACCCAGATGCGCCACTTC TTCGATAAAAAAGCACTGAAAACCCAGATTATGGCAGC CAGCTTTAAACATAGCGGTCAGGTTATGCAGGCCTATG AAAGCGGTGCAGATACCGTTACCATTCCGTATGAAATC TATAGCCAGATGACCAATAAAGTTCTGGCAGTTGAAGC CATTCGCGTGTTTAATGAAGATGCAGTTCTGTACGAGA AATGA SEQ ID NO: 49 mipB ATGGAATATATGCTGGATACCCTGGATCTGGAAGCAAT SEQ ID NO: 12 (from Streptococcus CAAAAAATGGCATCACATTCTGCCGCTGGCAGGCGTTA Uniprot accession pyogenes serotype M1) CCAGCAATCCGAGCATTGCAAAAAAAGAAGGCGAGATC number Q99XT4 GATTTTTTTGAACGCATTCGTGAAGTGCGTGCCATTAT TGGTGATAAAGCAAGCATTCATGTTCAGGTTATTGCCC AGGATTATGAAGGCATTCTGAAAGATGCAGCAGAAATT CGTCGTCAGTGTGGTGATAGCGTTTATGTTAAAGTTCC GGTTACCACCGAAGGTCTGGCAGCAATTAAAACCCTGA AAGCAGAAGGTTATCATATTACCGCAACCGCAATTTAT ACCACCTTTCAGGGCCTGCTGGCAATTGAAGCCGGTGC AGATTATCTGGCTCCGTATTATAACCGTATGGAAAATC TGAACATTGATCCGGAAGCAGTTATTGAACAGCTGGCC GAAGCAATTAATCGTGAAAATGCCAATAGCAAAATTCT GGCAGCCAGCTTTAAAAACGTTGCCCAGGTGAATAAAA GTTTTGCACTGGGTGCACAGGCAATTACCGCAGGTCCG GATGTTTTTGAAGCAGGTTTTGCCATGCCGAGCATTCA GAAAGCAGTTGATGATTTTGGTAAAGACTGGGAAGCAA TTCATCACCGCAAAAGCATCTGA SEQ ID NO: 50 SGO_1787 ATGGAATTTATGCTGGATACCCTGAACCTGGAAGAAAT SEQ ID NO: 10 (from Streptococcus CAAAAAATGGTCAGAAGTTCTGCCGCTGGCAGGCGTTA Uniprot accession gordonii) CCAGCAATCCGACCATTGCAAAAAAAGAAGGCAAAATC number A8AZ46 GACTTTTTCGAACGCATTAGCGCAGTGCGTGAAATTAT TGGTGAAGGTCCGAGCATTCATGTTCAGGTTGTTGCAA AAGATTATGAGGGCATTCTGAAAGATGCAGCCACCATT CGTAAAAAATGTGGTGATGCCGTGTATATCAAAATTCC GGTTACACCGGATGGTCTGGCAGCAATTAAAACCCTGA AAGCAGAAGGCTATAAAATCACCGCAACCGCAATTTAT ACCACCTTTCAGGGCCTGCTGGCAATTGAAGCAGAAGC AGATTATCTGGCACCGTATTATAACCGTATGGAAAATC TGAACATCGATTCCGATGCAGTTATTAGTCAGCTGGCA CAGGCCATTGAACGTGATCATAGCGATAGCAAAATTCT GGCAGCCAGCTTTAAAAACGTTGCACAGGTTAATCGTG CATTTGCAGATGGTGCACAGGCAGTTACCGCAGGTCCG GATGTTTTTGCAGCAGCATTTGCAATGCCGAGTATTGC AAAAGCAGTTGATGATTTTGCAACCGATTGGAGCGATA TTCACAGCCAAGAATATGTGTGA SEQ ID NO: 51 UMC_00018 ATGGAATTTATGCTGGACACCATTAACCTGGAAGCCAT SEQ ID NO: 18 (from Enterococcus TCGTAAATATCAGAAAATTCTGCCGCTGGCAGGCGTTA Uniprot accession faecalis EnGen0302) CCAGCAATCCGAGCATTGTTAAACAGGCAGGCAAAATT number A0A0M2AGL1 GATTTTTTTGCCCAGATGAAAGAAATCAAAAAGACCAT TGGTCAGGCAAGCCTGCATGTTCAGGTTGTTGGTCAGA CCACCGAAGAAATGCTGGAAGATGCACAGACCATTGTG CAGCAGCTGGGTCAAGAAACCTTTATCAAAATTCCGGT TAATGAAGCAGGTCTGGCAGCAATTAAACAGCTGAAAC AGGCAAATTATCGTATTACCGCAACCGCCATTTATACC GAATTTCAGGGTTATCTGGCAATTGCAGCCGGTGCAGA TTACCTGGCACCGTATTATAACCGTATGGAAAATCTGA CCATCGACAGCCAGAAAGTTATTGAACATCTGGCAGCC GAAATTAAACGTACCAATGCCAAAAGCAAAATTCTGGC AGCGAGCTTTAAAAACGTTGCGCAGATTAATCAGGCAT GTCAGATGGGTGCACAGGCAGTTACCATTGCACCGGAA CTGGTTACCCAAGGTCTGGCCATGCCTGCAATTCAGAA AGCAGTTACCGATTTTCAAGAAGATTGGGTTGCAGTTT TTGGTGTGGAAACCGTTAATGAACTGGCCTGA SEQ ID NO: 52 tal ATGCGCTTTTTTCTGGATACCGCCAACGTGGATCATAT SEQ ID NO: 13 (from Clostridium TAAAGAAGCAAATGAAATGGGCGTGATTTGTGGTGTTA Uniprot accession beijerinckii CCACCAATCCGAGCCTGGTTGCAAAAGAAGGTCGCGAT number A0A0B5QQ90 (Clostridium MP)) TTTAACGAAGTGATCAAAGAAATTACCGAGATTGTGGA TGGTCCGATTAGCGGTGAAGTTGTTGCCGAAGATGCAC AGGGTATGATTAAAGAGGGACGCGAAATTGCAGCCATC CATAAAAACATGATTGTGAAAATTCCGATGACCGCAGA AGGTCTGAAAGCAACCAAAGTTCTGAGCAGCGAAGGTA TTAAAACCAATGTGACCCTGATTTTTAGCGCAACCCAG AGCCTGCTGGCAGCAAATGCCGGTGCAACCTATGTTAG CCCGTTTCTGGGTCGTGTTGATGATATTAGCATGATTG GTATGGATCTGGTTCGTGATATTGCCGAAATTTTTGCC GTTCATGGTATCGAAACCGAAATCATTGCAGCAAGCGT TCGTAATCCGATTCATGTTATTGAAGCAGCAAAAGCGG GTGCCGATATTGCAACCATTCCGTATGCACTGGTTATG CAGATGCTGAATCATCCGCTGACCGATCAAGGTCTGGA AAAATTCAAAGCAGATTGGGCAGCAGCATTCGGCAAAT GA SEQ ID NO: 53 tal ATGCAGATTTTTCTGGATAGCACCGACACCAAAGTTAT SEQ ID NO: 14 (from Caulobacter TGCCGATCTGGCAAGCACCGGTCTGATTGATGGTGTTA Uniprot accession vibriodes (strain CCACCAATCCGACACTGATTGCAAAAAGCGGTCGTCCG number Q9A2F1 ATCC 19089)) ATGCTGGAAGTGATTGCAGAAATTTGTGATATTGTTCC GGGTCCGATTAGCGCAGAAGTTGCAGCAACCACCGCAG ATGCAATGATTGCCGAAGGTCAGAAACTGGCAAAAATT GCACCGAATGTTGTTGTGAAAATTCCGCTGACACGTGA TGGCCTGATTGCATGTGCAGCATTTGCAGATGAAGAAA TCAAAACCAATGTGACCCTGTGTTTTAGCCCGACACAG GCACTGCTGGCAGCAAAAGCCGGTGCAACCTATATTAG CCCGTTTATTGGTCGTCTGGATGATTATGGCTTTGATG GTATGGATCTGATTCGTGATATTCGTGCCATCTATGAT AACTATGGCTATGAAACCGAAATTCTGGCAGCCAGCGT TCGTAATGCAGCACATGTTAAAGAAGCAGCAATTGTTG GCGCAGATGTTGTTACCATTCCTCCGGCAGTTTTTAGC GATCTGTATAAACATCCGCTGACCGATAAAGGTCTGGA ACAGTTCCTGAAAGATTGGGCATCAACCGGTCAGAGCA TTCTGTAA SEQ ID NO: 54 fsa_like ATGGAATTTATGCTGGACACCCTGAACATTGAAGAAAT SEQ ID NO: 19 (from Streptococcus TCGTAAATGGGCAGAAGTGCTGCCGCTGGCAGGCGTTA Uniprot accession suis) CCAGCAATCCGACCATTGCACGTAAAGAAGGTGACATA number A0A0E4C393 GATTTTTTTGAACGCCTGCATCTGATTCGCGATATTAT TGGTCCGAATGCAAGCCTGCATGTTCAGGTTGTTGCAA AAGATTATGAAGGTATTCTGGCCGACGCGAAAAAAATC CGTGAACTGGCACCGGAAAACATCTATATCAAAGTTCC GGTTACACCGGCAGGTCTGGCAGCAATGAAAACCCTGA AAGCACAGGGTTATCAGATTACCGCAACCGCAATTTAT ACCGTTTTTCAGGGTCTGCTGGCAATTGAAGCCGGTGC AGATTATCTGGCTCCGTATTATAACCGTATGGCCAACC TGAATATTGATAGCAATGCAGTTATTGCACAGCTGAGC GAAGCAATTGATCGTGAATGTAGCGAAAGCAAAATTCT GGCAGCCAGCTTTAAAAACGTTGATCAGGTTAATCAGG CCTTTGCAAATGGTGCACAGGCAATTACCGCAGGCGCA GATATTTTTGAAGCAGCATTTAGTATGCCGAGCATTGA AAAAGCCGTTAACGATTTTGCAGATGATTGGAGCGCAA TTCATGGTCGTTATACCATCTGA SEQ ID NO: 55 SMU_494 ATGGAATTTATGCTGGATACCCTGAACCTGGCCGATAT SEQ ID NO: 15 (from Streptococcus TGAAAAATGGGCAGCAATTCTGCCGCTGGCAGGCGTTA Uniprot accession mutans serotype c CCAGCAATCCGAGCATTGCAAAAAAAGAAGGCAAAATC number Q8DVJ4 (strain ATCC 700610) GACTTCTTTGAACAGGTTAAACGTGTGCGTGCAATTAT TGGTGAAGAACCGAGCATTCATGCACAGGTTGTTGCAG CAGATGTTGAAGGTATTATCAAAGATGCCCACAAACTG CAAGATGAATTAGGTGGTAATCTGTATGTTAAAGTTCC GGTTAGCCCGACCGGTCTGACCGCAATGAAACAGCTGA AAGAAGAAGGTTTTCAGATTACCGCAACCGCCATTTAT ACCGTTTTTCAGGGTCTGCTGGCAATTGAAGCCGGTGC AGATTATCTGGCTCCGTATTATAACCGTATGGAAAACC TGAACATTGATCCGATTGAAGTTATTGGTCAGCTGGCA CAGGCCATTGAATGTCAGCAGGCAAGCGCAAAAATTCT GGCAGCCAGCTTTAAAAACGTTACCCAGGTTGCAAAAG CACTGGCAGCCGGTGCCAAAGCAGTTACCGCAGGCGCA GATATTTTTGCAGCAGGTTTTGCAAATCCGAGTATTCA GAAAGCCGTTGATGATTTTGCAGCCGATTGGGAAAGCA CCCAGGGTCGTCCGTATATCTAA SEQ ID NO: 56 fsa_like ATGGAATTTCTGCTGGATACCCTGAATCTGGAAGCAAT SEQ ID NO: 31 (from Streptococcus CAAAAAATGGCATCACATTCTGCCGCTGGCAGGCGTTA Uniprot accession agalactiae serotype III CCAGCAATCCGACCATTGCAAAAAAAGAAGGCGACATC number Q8E738 (strain NEM316)) CATTTTTTTCAGCGCATTCGTGATGTGCGCGAAATTAT TGGTCGTGAAGCAAGCCTGCATGTTCAGGTTGTTGCAA AAGATTATCAGGGCATTCTGGATGATGCAGCCAAAATT CGTCAAGAAACCGATGATGACATCTACATTAAAGTTCC GGTTACACCGGATGGTCTGGCAGCAATTAAAACCCTGA AAGCAGAAGGTTATAACATTACCGCAACCGCCATTTAT ACCAGTATGCAGGGTCTGCTGGCAATTAGTGCCGGTGC AGATTATCTGGCTCCGTATTTTAACCGTATGGAAAACC TGGATATTGATGCGACCCAGGTTATTAAAGAACTGGCA CAGGCAATTGAACGTACCGGTAGCAGCAGCAAAATTCT GGCAGCCAGCTTTAAAAACGCAAGCCAGGTTACCAAAG CACTGAGCCAGGGTGCACAGAGTATTACCGCAGGTCCG GATATTTTTGAAAGCGTTTTTGCCATGCCGAGCATTGC CAAAGCAGTTAATGATTTTGCAGATGATTGGAAAGCCA GCCAGCATAGCGAACATATCTAA SEQ ID NO: 57 fsaA ATGGAATTTATGCTGGATACCCTGAACCTGGATGAAAT SEQ ID NO: 20 (from Streptococcus CAAAAAATGGTCAGAAATTCTGCCGCTGGCAGGCGTTA Uniprot accession pneumoniae) CCAGCAATCCGACCATTGCAAAACGTGAAGGTAGCATC number A0A0D6J3Z8 AACTTTTTCGAACGCATTAAAGATGTGCGCGAACTGAT TGGTAGCACCCCGAGCATTCATGTTCAGGTTATTAGCC AGGATTTTGAGGGCATTCTGAAAGATGCACATAAAATT CGTCGTCAAGCCGGTGATGACATCTTTATCAAAGTTCC GGTTACACCGGCAGGTCTGCGTGCAATTAAAGCACTGA AAAAAGAAGGCTATCATATTACCGCAACCGCCATTTAT ACCGTTATTCAGGGTCTGCTGGCAATTGAAGCCGGTGC AGATTATCTGGCTCCGTATTATAACCGTATGGAAAATC TGAACATCGACAGCAATAGCGTTATTCGTCAGCTGGCA CTGGCCATTGATCGTCAGAATAGCCCGAGCAAAATTCT GGCAGCCAGCTTTAAAAACGTTGCCCAGGTTAATAATG CACTGGCAGCGGGTGCACATGCAGTTACCGCAGGCGCA GATGTTTTTGAAAGCGCATTTGCAATGCCGAGTATTCA GAAAGCAGTGGATGATTTTTCCGATGATTGGTTTGTTA CCCAGAATAGTCGCAGCATCTGA SEQ ID NO: 58 PH1655 ATGCATCATCATCATCATCACATGGTGAAAGTGATCTT SEQ ID NO: 21 (from Pyrococcus TTTCGATCTGGATGATACCCTGGTTGATACCAGCAAAC Uniprot accession horikoshii (strain ATCC TGGCAGAAATTGCACGTAAAAATGCCATCGAAAATATG number O59346 700860)) ATTCGTCATGGTCTGCCGGTTGATTTTGAAACCGCATA TAGTGAACTGATCGAGCTGATTAAAGAATACGGTAGCA ACTTTCCGTATCACTTCGATTATCTGCTGCGTCGTCTG GATCTGCCGTATAATCCGAAATGGATTAGTGCCGGTGT TATCGCATATCACAATACCAAATTTGCCTATCTGCGTG AAGTTCCGGGTGCGCGTAAAGTTCTGATTCGTCTGAAA GAACTGGGTTATGAACTGGGCATTATTACCGATGGTAA TCCGGTTAAACAGTGGGAAAAAATTCTGCGTCTGGAAC TGGATGATTTTTTTGAACATGTGATCATCAGCGATTTC GAGGGTGTTAAAAAACCGCATCCGAAAATCTTCAAAAA AGCCCTGAAAGCCTTTAACGTGAAACCGGAAGAGGCAC TGATGGTTGGTGATCGTCTGTATAGCGATATTTATGGT GCAAAACGTGTGGGTATGAAAACCGTTTGGTTTCGCTA TGGTAAACATAGTGAACGCGAACTGGAATATCGTAAAT ATGCCGATTATGAGATCGACAATCTGGAAAGCCTGCTG GAAGTTCTGGCACGTGAAAGCAGCAGCAACAAAAAAGT TCATCCGCCTCGTCAGCAGATTTGA SEQ ID NO: 59 MJ1437 ATGCATCATCATCACCATCACATGATTAAAGGCATCCT SEQ ID NO: 22 (from Methanocaldococcus GTTTGATCTGGATGATACCCTGTATAACAGCAGCGAAT Uniprot accession jannaschii (strain ATCC TTGTTGAAATTGCACGTCGTGAAGCAGTGAAAAGCATG number Q58832 43067)) ATTGATGCAGGTCTGAACATCGATTTTGAAGAAGCCAT GAACATCCTGAACAAGATCATCAAAGATAAGGGCAGCA ACTATGGCAAACATTTCGATGATCTGGTTAAAGCCGTT CTGGGTAAATATGATCCGAAAATTATCACCACCGGCAT TATCACCTATCACAATGTGAAAGTTGCACTGCTGCGTC CGTATCCGCATACCATTAAAACCCTGATGGAACTGAAA GCAATGGGTCTGAAACTGGGTGTTATTACCGATGGTCT GACCATTAAACAGTGGGAAAAACTGATTCGTCTGGGCA TTCATCCGTTTTTTGATGATGTGATTACCAGCGAAGAA TTTGGTCTGGGCAAACCGCATCTGGAATTTTTCAAATA TGGCCTGAAACGTATGGGCCTGAAAGCCGAAGAAACCG TTTATGTTGGTGATCGTGTGGACAAAGATATTAAGCCT GCAAAAGAACTGGGCATGATTACCGTTCGTATTCTGAA AGGCAAATACAAAGACATGGAAGATGATGAGTATAGCG ACTACACCATTAATAGCCTGCAAGAGCTGGTTGACATT GTGAAAAACCTGAAAAAGGATTAA SEQ ID NO: 60 ALDOB ATGCATCATCATCACCATCACATGGCACATCGTTTTCC SEQ ID NO: 26 (from Homo sapiens GGCACTGACCCAAGAACAGAAAAAAGAACTGAGCGAAA Uniprot accession (Human)) TTGCCCAGAGCATTGTTGCAAATGGTAAAGGTATTCTG number P05062 GCAGCAGATGAAAGCGTTGGTACAATGGGTAATCGTCT GCAACGTATTAAAGTGGAAAACACCGAAGAAAATCGTC GTCAGTTTCGTGAAATTCTGTTTAGCGTTGATAGCAGC ATTAATCAGAGTATTGGTGGCGTGATTCTGTTCCATGA AACCCTGTATCAGAAAGATAGCCAGGGTAAACTGTTTC GCAACATCCTGAAAGAAAAAGGTATTGTGGTGGGCATC AAACTGGATCAAGGTGGTGCACCGCTGGCAGGCACCAA TAAAGAAACCACCATTCAAGGTCTGGATGGTCTGAGCG AACGTTGTGCACAGTACAAAAAAGATGGTGTGGATTTT GGTAAATGGCGTGCAGTTCTGCGTATTGCAGATCAGTG TCCGAGCAGCCTGGCAATTCAAGAAAATGCAAATGCAC TGGCACGTTATGCAAGCATTTGTCAGCAGAATGGTCTG GTTCCGATTGTTGAACCGGAAGTTATTCCGGATGGTGA CCATGATCTGGAACATTGTCAGTATGTTACCGAAAAAG TGCTGGCAGCCGTTTATAAAGCACTGAATGATCATCAT GTTTACCTGGAAGGCACCCTGCTGAAACCGAATATGGT TACCGCAGGTCATGCATGTACCAAAAAATACACACCGG AACAGGTTGCAATGGCAACCGTTACCGCACTGCATCGT ACCGTTCCGGCAGCAGTTCCGGGTATTTGTTTTCTGAG CGGTGGTATGAGCGAAGAAGATGCAACCCTGAATCTGA ATGCAATTAATCTGTGTCCGCTGCCGAAACCGTGGAAA CTGAGCTTTAGCTATGGTCGTGCACTGCAAGCAAGCGC ACTGGCAGCATGGGGTGGTAAAGCAGCAAATAAAGAAG CAACCCAAGAGGCCTTTATGAAACGTGCAATGGCCAAT TGTCAGGCAGCAAAAGGCCAGTATGTTCATACCGGTAG CAGCGGTGCCGCAAGCACCCAGAGCCTGTTTACCGCAT GTTATACCTATTGA SEQ ID NO: 61 pgm ATGGCACAGCATAGCCATGCAGGTCAGCCTGCACGTCT SEQ ID NO: 29 (from Aeromonas GAGCGATCTGACCAATATTCCGCGTCTGGTTAGCGCAT Uniprot accession hydrophila subsp. ATTATCTGAATAAACCGGATATGAGCCGTCCGGAACAG number A0KIH4 hydrophila (strain ATCC CGTGTTGCATTTGGCACCAGCGGTCATCGTGGTAGCGC 7966)) ACTGCATAATGCATTTACCGAAAGCCATATTCTGGCAG TTACCCAGGCACTGGTTGAATATCGTCAGCAGGCAGGT ATTACCGGTCCGCTGTTTGTTGGTATGGATACCCATGC ACTGAGCGAAAGCGCATTTGCAAGCGCAGTTGAAGTTC TGGCAGCAAATGGTGTTGAAACCCGTATTCAGGCAGGT CTGGGTTTTACCCCGACACCGGTTATTAGCCATGCCAT TCTGCGTCATAATGCAGGTAAACCGGCAGCACGTGCAG ATGGTGTTGTTATTACCCCGAGCCATAATCCGCCTGAA GATGGTGGCTTTAAATACAATCCGCCTCATGGTGGTCC TGCCGAAGGTGAAATTACAAAATGGGTTGAAGATCGTG CCAATGCAATTCTGGAAGCCGGTCTGGCAGGCGTTAAA CGTATGGCATTTGCAGAAGCACTGAAAAGCCCGTTTGT TGCACTGCATGATTATGTTACCCCGTATGTTGATGATC TGAAAAACGTTCTGGATATGGATGCCATTAAACAGGCA GGCATTAAAATCGGTGTTGATCCGTTAGGTGGTAGCGG TGTTGCCTATTGGGATGTTATTGCAAAAACCTATGGCC TGAATATCGAGGTGGTGAACTATAAAGTTGATCCGACC TTTAGCTTTATGACCCTGGATAAAGATGGCAAAATTCG TATGGATTGTAGCAGTCCGTTTGCAATGGCAAGCCTGA TTGCACTGAAAGACAAATTTGATATTGCGCTGGGTAAC GATCCGGATTATGATCGTCATGGTATTGTTACCAAAAG CGGTCTGATGAATCCGAATCATTATCTGGCCGTTGCAA TTCAGTACCTGTTTACCCATCGTACCGGTTGGAGCAAA GAAAGCGCTGTTGGCAAAACCCTGGTTAGCAGCAGCAT GATTGATCGTGTTGCCGGTGAAATTGGTCGTACCCTGA AAGAAGTTCCGGTTGGTTTTAAATGGTTTGTGGATGGT CTGTATAGCGGTGAATTTGGTTTTGGTGGTGAAGAAAG TGCCGGTGCCAGCTTTCTGCGTAAAGATGGTACAGTTT GGACCACCGATAAAGACGGTTTTATTCTGGCCCTGCTG GCAGCAGAAATTCTGGCCGTGACCGGTAAAGATCCGCA GACACATTATGATGCACTGGAAGCAAAATTTGGTCGTA GCAGCTATCGTCGTATTGATGCACCGGCAAATAGCGCA CAGAAAGCAGTTCTGAGCAAATTAGATCCGGCACTGGT GGAAGCAAGCACCTTAGCCGGTGAACCGATTATTGCCA AACTGACCAAAGCACCGGGTAATGATGCAGCAATTGGT GGTCTGAAAGTTGTTACCGAAAATGGTTGGTTTGCAGC ACGTCCGAGCGGCACCGAAAGCATCTATAAAATCTATA TGGAATCCTTCAAAGGCGAAGCACATCTGGATCTGATT CAGCAAGAAGCACAGCAGATTGTTAGCGCAGCACTGGC AAAAGCCGGTGTTTAATAA SEQ ID NO: 62 AHA_2903 ATGAATCTGACCTGTTTCAAAGCCTATGACATTCGTGG SEQ ID NO: 30 (from Aeromonas TAAACTGGGTGATGAACTGAATATCGAAATTGCCTATC Uniprot accession hydrophila subsp. GTATTGGTCGTGCAACCGCACAGTATCTGAAAGCAACC number A0KMA6 hydrophila (strain ATCC CGTATTGCAGTTGGTGGTGATGTTCGTCTGACCAGCGA 7966)) AGGTCTGAAACAGGCACTGGCAAATGGTATTCTGGATG CAGGTTGTGATGTTATTGATCTGGGTGTTACCGGCACC GAAGAAACCTATTTCGCAGCATTTACCCTGGATATTGA TGGTGCAATTGAAGTTACCGCAAGCCATAATCCGATGG ATTACAATGGTATGAAACTGGTTGGTCGTGATGCATGT CCGATTAGCGGTGATAGCGGTCTGAATGATATTCGTGC ACTGGCAGAAAAAGGTGATTTTAGCGTTAGCTTTCGTC GTGGCACCCTGAGCAAAAAAAGCATCCTGGATGCCTAT GTTGATCATCTGCTGACCTATATCAAACCGCATCAGCT GCGTCCGCTGAAATTAGTTGTTAATGCAGGTAATGGTG CAGCCGGTCATGTTATCGATGTGATTGAACAGCGTTTT AACATTCTGAACATCCCGGTGGAATTTATCAAAATCCA TCATGAAGAAAACGGCAACTTTCCGAATGGCATTCCGA ATCCGCTGCTGCCGGAAAATCGTGATGTTACCAGTGAA GCAGTTAAACTGCATCATGCAGATATGGGTATTGCATG GGATGGTGATTTTGATCGCTGTTTTCTGTTTGATGAGA ACGGCATTTTTATCGAGGGCTATTATATCGTTGGTCTG CTGGCAGAAGCATTTCTGGTTGAAAATCCGCATGAACG CATTATTCATGATCCGCGTCTGACCTGGAATACCATCG ATATTGTTGAAAAAAGCGGTGGTATTCCGGTTCAGTCA AAAACCGGTCATGCCTTTATCAAAGAACGTATGCGTAG CGAAAATGCCATTTATGGTGGTGAAATGAGCGCACATC ATTATTTTCGCGATTTTGGTTATTGCGATAGCGGTATG ATTCCGTGGCTGCTGGTTATTAATCTGCTGAGCCTGAA AAATAGCACCCTGTCAAGCCTGGTTGCAGAACGTGTTA AAGCATATCCGTGTAGCGGTGAAATTAACTATCGTGTT GATAACGCCCTGGAAATCATCAAAAAACTGGAAGAGGT TTATGTTCCGCTGGCCGTTAAAGTTGAATATGTTGATG GTCTGAGCATCGAGATGAATGATTGGCGTTTTAATGTG CGCATTAGCAATACAGAACCTCTGCTGCGTCTGAATGT TGAAAGCAAAAACAACATTAGCAAACTGACCAGTGGTC TGAATAGCCTGCATAAGATGATTAACAACATCTAA SEQ ID NO: 63 The expression of the recombinant enzymes was checked on a protein gel, after purification of the recombinant protein using a His trap (Protino Ni-IDA 1000 kit, Macherey Nagel). Purification was processed according to the manufacturer's recommendations.

Example 1: Fructose-6-Phosphate Aldolase and Fructose Bisphosphatase Activity Inhibition Tests

(15) A series of tests was conducted in order to determine if AMP has an inhibitory effect on the enzymatic activity of fructose-6-phosphate aldolase and/or fructose bisphosphatase. The protocol used to test the enzymatic activities was adapted from C. Guérard-Hélaine, V. De Berardinis, M. Besnard-Gonnet, E. Darii, M. Debacker, et al. Genome Mining for Innovative Biocatalysts: New Dihydroxyacetone Aldolases for the Chemist's Toolbox. Chem Cat Chem, Wiley, 7:1871-1879 (2015).

(16) a) Impact of AMP Concentration on Fructose Bisphosphatase Activity

(17) 120 μl of each kinetic assay contained Tris HCl buffer (50 mM; pH 7.5), 20 mM NaCl, 10 mM MgCl2, 1 mM NADP+, AMP (several concentrations tested), 1 mM Fructose 1,6-bisphosphate (F1,6bisP), 0.2 mg/ml FBP enzyme, and the auxiliary enzymes (glucose-6-phosphate isomerase (PGI) and NADP.sup.+-dependent glucose-6-phosphate dehydrogenase (zwf) (0.5 mg/ml each)). The mix was incubated at 30° C. for up to 20 minutes and the reaction was monitored by spectrophotometry at 340 nm (measuring NADPH formation), assuming that 1 reduced NADPH molecule was produced per Fructose-6-Phosphate molecule. Results are shown in FIG. 1. A strong inhibitory impact of AMP on fructose bisphosphatase activity was observed.
b) Impact of AMP Concentration on Fructose-6-Phosphate Aldolase Activity of FsaA A129S 120 μl of each kinetic assay contained Tris HCl buffer (50 mM; pH 8.5), 1 mM NADP+, AMP (several concentrations tested), 200 mM DHA, 3 mM D,L-G3P, 0.4 mg/ml FsaA A129S, and the auxiliary enzymes (glucose-6-phosphate isomerase (PGI) and NADP.sup.+-dependent glucose-6-phosphate dehydrogenase (zwf) (0.5 mg/ml each)). The mix was incubated at 30° C. for up to 20 minutes and the reaction was monitored by spectrophotometry at 340 nm (measuring to NADPH formation), assuming that 1 reduced NADPH molecule was produced per Fructose-6-Phosphate molecule. Results are shown in FIG. 2. No inhibitory effect of AMP could be observed with FSAA A129S.

Example 2: In Vitro Conversion of Glyceraldehyde-3-Phosphate (G3P) and Dihydroxy-Acetone Phosphate (DHAP) into Fructose-6-Phosphate (F6P) Through a Dihydroxy-Acetone (DHA) Intermediate

(18) A series of tests were conducted in order to determine the best enzyme combinations to convert G3P and DHAP into F6P. These enzyme combinations should perform the 2 steps: 1) DHAP.fwdarw.DHA 2) DHA+G3P.fwdarw.F6P
a) Enzyme Catalyzing the Conversion of DHA and G3P into F6P The protocol used to test the enzymatic activities was adapted from C. Guérard-Hélaine, V. De Berardinis, M. Besnard-Gonnet, E. Darii, M. Debacker, et al., Genome Mining for Innovative Biocatalysts: New Dihydroxyacetone Aldolases for the Chemist's Toolbox. Chem Cat Chem, Wiley, 7:1871-1879 (2015). 120 μl of each kinetic assay contained Tris HCl buffer (50 mM pH 8.5), 3 mM D,L-G3P, 200 mM DHA, 1 mM NADP+, 0.4 mg/ml of enzyme, and the auxiliary enzymes (glucose-6-phosphate isomerase (PGI) and NADP.sup.+-dependent glucose-6-phosphate dehydrogenase (zwf) (0.5 mg/ml each)). The mix was incubated at 30° C. and the reaction was monitored by spectrophotometry at 340 nm (measuring NADPH formation), assuming that 1 reduced NADPH molecule was produced per F6P molecule. Results are shown in Table 3.

(19) TABLE-US-00003 TABLE 3 Production of F6P from DHA and G3P, with different enzymes Gene coding for SEQ Uniprot the enzyme ID NO number Activity LMRG_00181 49 A0A0H3GHX1 ++++ mipB 50 Q99XT4 ++++ SGO_1787 51 A8AZ46 ++++ fsaA A129S 35 — +++ fsa-like 57 Q8E738 +++ UMC_00018 52 A0A0M2AGL1 ++ SMU_494 56 Q8DVJ4 ++ fsa-like 55 A0A0E4C393 ++ fsa 58 A0A0D6J3Z8 ++ fsaB 36 P32669 ++ talB F178Y 38 — ++ tal 53 A0A0B5QQ90 + tal 54 Q9A2F1 + Control (no substrate) − Control (no enzyme) −
a) Enzyme Catalyzing the Conversion of DHAP into DHA The protocol used to test the enzymatic activities was adapted from C. Guérard-Hélaine, V. De Berardinis, M. Besnard-Gonnet, E. Darii, M. Debacker, et al., Genome Mining for Innovative Biocatalysts: New Dihydroxyacetone Aldolases for the Chemist's Toolbox. Chem Cat Chem, Wiley, 7:1871-1879 (2015)). 120 μl of each kinetic assay contained Tris HCl buffer (50 mM pH 8.5), 10 mM MgCl2, 100 mM DHAP, 0.8 mM NADP+, 0.6 mg/ml of enzyme, 0.8 mg/ml fructose-6-phosphate aldolase 1 from E. coli MG1655 (FSAA mutated A129S) and the auxiliary enzymes (glucose-6-phosphate isomerase (PGI) and NADP.sup.+-dependent glucose-6-phosphate dehydrogenase (zwf) (0.5 mg/ml each)). The mix was incubated at 30° C. and the reaction was monitored by spectrophotometry at 340 nm (measuring NADPH formation), assuming that 1 reduced NADPH molecule was produced per F6P molecule. Results are shown in Table 3.

(20) TABLE-US-00004 TABLE 4 Production of DHA from DHAP with different enzymes Gene coding for SEQ Uniprot the enzyme ID NO Number Activity ybiV 39 P75792 +++ yieH 40 P31467 +++ yidA 41 P0A8Y5 ++ yigL 42 P27848 ++ yqaB 43 P77475 + hdpA 48 A4QFW4 + hxpA 44 P77625 + Control (no substrate) − Control (no enzyme) −

Example 3: In Vitro Conversion of Glyceraldehyde-3-Phosphate (G3P) and Dihydroxy-Acetone-Phosphate (DHAP) into Fructose-6-Phosphate (F6P) Through a Glyceraldehyde Intermediate

(21) A series of tests were conducted in order to determine the best enzyme combinations to convert G3P and DHAP into F6P. The best enzymes combination should perform the 3 steps: 1) G3P.fwdarw.Glyceraldehyde 2) Glyceraldehyde+DHAP.fwdarw.F1P 3) F1P.fwdarw.F6P
a) Enzymes Catalyzing the Conversion of G3P into Glyceraldehyde 200 μl of each kinetic assay contained Tris HCl buffer (50 mM pH 7.5), 100 mM NaCl, 10 mM MgCl2, G3P (1-10-50 mM) and 2 mg/ml of the tested enzyme (see table 7). The mix was incubated overnight at 30° C. and the reaction was quenched with 1 volume acetonitrile. The final products were analysed by LCMS. LC-MS analyses were performed on an Ultimate 3000 (Dionex, Thermo Fisher Scientific) coupled to a Q-Orbitrap mass spectrometer (Thermo Fisher Scientific) fitted with an electrospray (ESI) source and operating in negative ion mode. The chromatographic separations were performed using a HILIC amide (1.9 μm, 2.1×150 mm) column maintained at 25° C. (Waters) operated under gradient elution, as follows. Mobile phases were: (A) 10 mM ammonium formiate pH 9.45 (adjusted with ammonium hydroxide), while mobile phase (B) was 100% acetonitrile and the flow rate was 500 μL/min. Elution started with an isocratic step of 1.5 min at 95% B, followed by a linear gradient from 95 to 55% of phase B in 7 min. The chromatographic system was then rinsed for 2 min at 55% B, and the run was ended with an equilibration step of 8.5 min.

(22) TABLE-US-00005 TABLE 5 Enzymes catalysing the conversion of G3P into glyceraldehyde. Gene coding for SEQ Uniprot the enzyme ID NO Number Activity PH1655 59 O59346 − MJ1437 60 Q58832 − hxpB 45 P77247 + Control (no substrat) − Control (no enzyme) −
a) Enzymes Catalyzing the Conversion of Glyceraldehyde and DHAP into F1P, and the Further Conversion of F1P into F6P 200 μl of each kinetic assay contained Tris HCl buffer (50 mM pH 7.5), 50 mM NaCl, 5 mM MgCl.sub.2, 1 mM NADP.sup.+, 10 mM DHAP, 10 mM Glyceraldehyde, 1 mg/ml AldoB, 1 mg/ml PGM and PMM and the auxiliary enzymes (glucose-6-phosphate isomerase (PGI) and NADP.sup.+-dependent glucose-6-phosphate dehydrogenase (zwf) (1 mg/ml each)). The mix was incubated at 30° C. and the reaction was monitored by spectrophotometry at 340 nm (measuring NADPH formation), assuming that 1 reduced NADPH molecule was produced per F6P molecule. Results are shown in Table 6.

(23) TABLE-US-00006 TABLE 6 Enzymes catalyzing the conversion of glyceraldehyde and DHAP into F1P and the further conversion of F1P into F6P. The enzyme encoded by ALDOB was incubated together with the enzymes encoded by pgm (assay 1), with the enzymes encoded by AHA_2903 (assay 2) or with both (assay 3). SEQ Uniprot Assay Genes ID NO Number Activity 1 ALDOB 61 P05062 + pgm 62 A0KIH4 2 ALDOB 61 P05062 + AHA_2903 63 A0KMA6 3 ALDOB 61 P05062 ++ pgm 62 A0KIH4 AHA_2903 63 A0KMA6 4 Control (no substrate) − 5 Control (no enzyme) −

Example 4: Construction of a New E. coli Chassis for the Production of Acetone and Isopropanol

(24) Like most organisms, E. coli converts glucose to acetyl-CoA. A modified E. coli chassis in which the yield of acetyl-CoA production is optimized has been described previously (WO 2013/007786). A bacterial chassis, strain A, was constructed with the following genotype:

(25) MG1655 ptsHI zwf_edd_eda pfkA pfkB

(26) Plasmid-based overexpression of a PKT gene from phosphoketolase YP 003354041.1 from Lactococcus lactis into strain A resulted in strain B, a strain with a rewired central carbon metabolism, wherein a new phosphoketolase-based carbon catabolic pathway replaced the inactivated Embden-Meyerhoff-Parnas pathway (EMPP), the pentose phosphate pathway (PPP), and the Entner Doudoroff pathway (EDP). Upon introduction of an acetone pathway into strain B, superior acetone yields were observed, as compared with wild type MG1655 strain expressing the same acetone pathway.

(27) In order to construct a strain having a PKT pathway and capable of robust growth on sucrose as carbon source, strain A was further engineered as described below.

(28) A PKT gene was introduced into the chromosome of strain A, at the kdgk locus (kdgK::P1_RBST7_pkt). The resulting strain had the following genotype:

(29) MG1655 ptsHI zwf_edd_eda pfkA pfkB kdgK::P1_RBST7_pkt

(30) This strain was passaged for several months on minimal medium supplemented with glucose as the carbon source, while continuously selecting for clones or populations having the highest growth rate, until a doubling time of less than 5 hours was reached.

(31) Several gene deletions were performed in order to increase acetone and isopropanol production: hemA fsaA fsaB.

(32) To further increase isopropanol production, pntAB (pyridine nucleotide transhydrogenase subunits alpha and beta, Uniprot P07001 and P0AB67, NCBI Reference Sequences: NP_416120.1 and NP_416119.1) genes from E. coli were overexpressed by inserting a strong constitutive promotor at the pntAB locus.

(33) The resulting strain is referred to as strain C hereafter.

Example 5: Construction of E. coli Strains for the Production of Acetone and Isopropanol from Acetyl-CoA

(34) This working example shows the production of acetone and isopropanol by recombinant E. coli strains, expressing the genes constituting the acetone and isopropanol pathway.

(35) The enzymes used in this study to convert acetyl-CoA into acetone and isopropanol are listed in Table 7.

(36) TABLE-US-00007 TABLE 7 Enzymes catalyzing the conversion of acetyl- CoA into acetone and isopropanol Uniprot Accession Step Enzyme Gene NCBI reference number I Acetyl-CoA THLA WP_010966157.1 P45359 transferase from Clostridium acetobulyticum II Acetate ATOD NP_416725.1 P76458 CoA-transferase from ATOA NP_416726.1 P76459 Escherichia coli III Acetoacetate ADC NP_149328.1 P23670 decarboxylase from Clostridium acetobutylicum IV NADP-dependent ADH AF_157307.2 P25984 isopropanol dehydrogenase from Clostridium beijerinckii
Expression of Acetone/Isopropanol Biosynthetic Pathway in E. coli.

(37) Strain C as described in Example 4 was used as a host microorganism.

(38) All the listed genes were codon optimized for expression in E. coli and synthesized either by GeneArt® (Thermofisher), except the genes atoD and atoA. The last ones were directly amplified from the genomic DNA of E. coli MG1655.

(39) An expression vector containing the origin of replication pSC and a spectinomycin resistance marker was used for the expression of the genes thlA, atoD, atoA, adc and adh. The constructed vector was named pGB5344.

(40) Expression in E. coli of the Enzymes Responsible for Conversion of Glyceraldehyde-3-Phosphate (G3P) and Dihydroxy-Acetone Phosphate (DHAP) into Fructose-6-Phosphate (F6P).

(41) The modified version of pUC18 (New England Biolabs), containing a modified Multiple Cloning Site (pUC18 MCS) (WO 2013/007786), and an ampicilline resistance gene (plasmid pGB 271), was used for the overexpression of the genes listed in Table 8.

(42) TABLE-US-00008 TABLE 8 Enzymes catalyzing the conversion of DHAP and G3P into F6P. Uniprot Accession Constructed Enzyme Gene NCBI reference number plasmid Transaldolase from FSAA_SS WP_011922247.1 A0A0E4C393 PGB 12689 Streptococcus suis 6-phosphogluconate YIEH WP_000086486.1 P31467 phosphatase from Escherichia coli

(43) The different combinations of the plasmids were transformed by electroporation into strain C. The strains produced in this way are summarized in Table 9.

(44) TABLE-US-00009 TABLE 9 Strains generated for in vivo conversion of glucose into acetone + isopropanol. Strain Vectors STRAIN GBI 15847: PGB 5344 + Strain C, expressing the whole PGB 271 Acetone/Isopropanol metabolic pathway, without overexpression of enzymes responsible for conversion of Glyceraldehyde-3-Phosphate (G3P) and Dihydroxy-Acetone Phosphate (DHAP) into Frutose-6-Phosphate (F6P) STRAIN GBI 17553: PGB 5344 + Strain C, expressing the whole PGB 12689 Acetone/Isopropanol metabolic pathway, +overexpression of enzymes responsible for conversion of Glyceraldehyde-3-Phosphate (G3P) and Dihydroxy-Acetone Phosphate (DHAP) into Frutose-6-Phosphate (F6P)

Example 6: Growth of E. coli Strains and Production of Acetone/Isopropanol from Acetyl-CoA

(45) Pre-Culture Conditions

(46) The transformed cells were then plated on LB plates, supplied with ampicillin (100 μg/ml) and spectinomycin (100 μg/ml). Plates were incubated for 2 days at 30° C. Isolated colonies were used to inoculate LB medium, supplemented with ampicillin and spectinomycin. These pre-cultures were grown at 30° C. to reach an optical density of 0.6.

(47) Growth Conditions

(48) The fermentation was performed in a 1 liter bioreactor with pH and temperature control (Multifors 2, Infors HT). Cells of pre-cultures were used to inoculate 500 ml of the fermentation medium (Table 10), complemented with ampicillin (100 μg/ml), spectinomycin (100 μg/ml), thiamine (0.6 mM), glucose (1 g/l) and glycerol (5 g/L), to achieve an initial optical density (OD.sub.600) of 0.05. During the growth phase temperature (T=32° C.), pH=6.5 and pO.sub.2=5% were maintained constant. The feed of glucose was increased from 0.1 g/g DCW/h to 0.35 g/g DCW/h. The pulses of the addition of 5 g/L of yeast extract were done when OD.sub.600 reached 2, 8 and 20.

(49) TABLE-US-00010 TABLE 10 Fermentation medium composition (derived from ZYM-5052 medium (Studier FW, Prot. Exp. Pur. 41, (2005), 207-234)). Final concentration Products in bioreactor Yeast Extract 5 g/L Tryptone 10 g/L Sodium sulfate, Na.sub.2SO.sub.4 0.71 g/L Ammonium sulfate, (NH.sub.4).sub.2SO.sub.4 1.34 g/L Potassium phosphate monobasic, KH.sub.2PO.sub.4 3.4 g/L Sodium phosphate dibasic, Na.sub.2HPO.sub.4 4.45 g/L Magnesium sulfate, MgSO.sub.4 4 mM 5000X Trace elements solution 1X Antifoam Struktol ® J 673 A (Struktol) 80 μl/L
Acetone/Isopropanol Production Phase

(50) During this phase temperature, T=34° C., pH 6.5, and pO.sub.2=5% were maintained constant. Glucose feed was started at 0.50 g sucrose/g DCW/h and then adjusted according to the strain consumption. Glycerol concentration was maintained superior to 2 g/l.

(51) The acetone/isopropanol production by the strains was analyzed continuously using a Gas Chromatograph 7890A (Agilent Technology), equipped with Flame Ionization Detector (FID) to measure acetone and isopropanol. Volatile organic compounds were chromatographically separated on Hi-Plex H USP L17, 100×7.7 mm (Agilent) using Agilent 1260 InfinityII chromatographer. acetone/isopropanol were quantified using standards (Sigma).

(52) FIG. 3 shows the comparison between the observed specific productivity of acetone and isopropanol for a production strain expressing the enzymes responsible for the conversion of glyceraldehyde-3-phosphate (G3P) and dihydroxy-acetone phosphate (DHAP) into fructose-6-phosphate (F6P) or, as a control, for a strain which does not express the enzymes responsible for the conversion of glyceraldehyde-3-phosphate (G3P) and dihydroxy-acetone phosphate (DHAP) into fructose-6-phosphate (F6P).

(53) When the enzymes responsible for conversion of glyceraldehyde-3-phosphate (G3P) and dihydroxy-acetone phosphate (DHAP) into fructose-6-phosphate (F6P) are overexpressed (strain GBI 17553), acetone and isopropanol specific productivity (moles produced per unit of cell weight per unit of time) is higher compared to the strain GBI 15847.