Enzymatic production of an acyl phosphate from a 2-hydroxyaldehyde

Abstract

Described is a method for the enzymatic production of an acyl phosphate from a 2-hydroxyaldehyde using a phosphoketolase or a sulfoacetaldehyde acetyltransferase.

Claims

1. A method for the enzymatic production of an acyl phosphate, wherein 2-hydroxyaldehyde and phosphate is enzymatically converted to an acyl phosphate by a phosphoketolase, wherein said phosphoketolase is selected form EC 4.1.2.9, EC 4.1.2.22 or any one of SEQ ID NO:1-3 or 16-20, or a sulfoacetaldehyde acetyltransferase (EC 2.3.3.15) according to the following reaction scheme: ##STR00016## wherein R.sup.1 and R.sup.2 are selected independently from H, CH.sub.3, CH.sub.2OH and C.sub.2H.sub.5 and wherein if R.sup.1 is H, R.sup.2 cannot be H, wherein said method is carried out in an in vitro, cell-free system or by a recombinant microorganism or plant cell overexpressing said phophoketolase or sulfoacetaldehyde acetyltransferase.

2. The method of claim 1, wherein the acyl phosphate is recovered.

3. The method of claim 1 which further comprises converting the acyl phosphate into a carboxylic acid according to the following reaction scheme: ##STR00017## wherein R.sup.1 and R.sup.2 are selected independently from H, CH.sub.3, CH.sub.2OH and C.sub.2H.sub.5 and wherein if R.sup.1 is H, R.sup.2 cannot be H.

4. The method of claim 3, wherein the conversion of the acyl phosphate into the carboxylic acid is achieved by an acylphosphatase (EC 3.6.1.7).

5. The method of claim 1 which further comprises converting the acyl phosphate into a carboxylic acid according to the following reaction scheme: ##STR00018## wherein R.sup.1 and R.sup.2 are selected independently from H, CH.sub.3, CH.sub.2OH and C.sub.2H.sub.5 and wherein if R.sup.1 is H, R.sup.2 cannot be H.

6. The method of claim 5, wherein the conversion of the acyl phosphate into the carboxylic acid is achieved by an enzyme which is classified as a phosphotransferase with a carboxyl group as acceptor (EC 2.7.2).

7. The method of claim 6, wherein the phosphotransferase is an acetate kinase (EC 2.7.2.1), a butyrate kinase (EC 2.7.2.7), an acetate kinase (diphosphate) (EC 2.7.2.12), a branched-chain-fatty-acid kinase (EC 2.7.2.14) or of a propionate kinase (EC 2.7.2.15).

8. The method of claim 1 which further comprises enzymatically converting the acyl phosphate into an acyl-coenzyme A according to the following reaction scheme: ##STR00019## wherein R.sup.1 and R.sup.2 are selected independently from H, CH.sub.3, CH.sub.2OH and C.sub.2H.sub.5 and wherein if R.sup.1 is H, R.sup.2 cannot be H.

9. The method of claim 8, wherein the conversion of the acyl phosphate into the acyl-coenzyme A is achieved by a phosphate acetyltransferase (EC 2.3.1.8) or of a phosphate butyryltransferase (EC 2.3.1.19).

10. The method of claim 1, wherein the 2-hydroxyaldehyde is 2-hydroxypropanal.

11. The method of claim 1, wherein the 2-hydroxyaldehyde is 2,3-dihydroxypropanal.

12. A composition comprising (a) an in vitro cell free system comprising 2-hydroxyaldehyde and a phosphoketolase wherein said phosphoketolase is selected from EC 4.1.2.9, EC 4.1.2.22 or any one of SEQ ID NO:1-3 or 16-20 and/or a sulfoacetaldehyde acetyltransferase (EC 2.3.3.15); or (b) 2 hydroxyaldehyde and a recombinant microorganism or plant cell overexpressing a phosphoketolase wherein said phosphoketolase is selected from EC 4.1.2.9, EC 4.1.2.22 or any one of SEQ ID NO:1-3 or 16-20 and/or a sulfoacetaldehyde acetyltransferase (EC 2.3.3.15).

13. The method of claim 1, wherein the phosphoketolase and the sulfoacetaldehyde acetyltransferase is overexpressed by the recombinant microorganism or plant cell.

14. The method of claim 1, wherein the method is carried out in the recombinant microorganism or plant cell.

15. The method of claim 1, wherein the method is carried out in an in vitro, cell-free system.

16. The method of claim 3, wherein the method is carried out in the recombinant microorganism or plant cell.

17. The method of claim 4, wherein the acylphosphatase (EC 3.6.1.7) is overexpressed by the recombinant microorganism or plant cell.

18. The method of claim 5, wherein the method is carried out in the recombinant microorganism or plant cell.

19. The method of claim 6, wherein the phosphotransferase with a carboxyl group as acceptor (EC 2.7.2) is overexpressed by the recombinant microorganism or plant cell.

20. The method of claim 7, wherein the acetate kinase (EC 2.7.2.1), the butyrate kinase (EC 2.7.2.7), the acetate kinase (diphosphate) (EC 2.7.2.12), the branched-chain-fatty-acid kinase (EC 2.7.2.14) or the propionate kinase (EC 2.7.2.15) is overexpressed by the recombinant microorganism or plant cell.

21. The method of claim 1, wherein the recombinant microorganism or plant cell is genetically modified to overexpress the phosphoketolase or the sulfoacetaldehyde acetyltransferase, wherein the genetic modification is selected from: (a) operably associating a heterologous promoter with a polynucleotide encoding the phosphoketolase or the sulfoacetaldehyde acetyltransferase; (b) transforming a heterologous polynucleotide encoding the phosphoketolase or the sulfoacetaldehyde acetyltransferase into the recombinant microorganism or plant cell; and/or (c) introducing a mutation in the promoter of a polynucleotide encoding the phosphoketolase or sulfoacetaldehyde acetyltransferase wherein said mutation results in overexpression of the polynucleotide.

22. The composition of claim 12, wherein the composition further comprises an acylphosphatase (EC 3.6.1.7).

23. The composition of claim 22, wherein the acylphosphatase (EC 3.6.1.7) is overexpressed by the recombinant microorganism or plant cell.

24. The composition of claim 12, wherein the composition further comprises a phosphotransferase with a carboxyl group as acceptor (EC 2.7.2).

25. The composition of claim 24, wherein the phosphotransferase is an acetate kinase (EC 2.7.2.1), a butyrate kinase (EC 2.7.2.7), an acetate kinase (diphosphate) (EC 2.7.2.12), a branched-chain-fatty-acid kinase (EC 2.7.2.14) or of a propionate kinase (EC 2.7.2.15).

26. The composition of claim 24, wherein the phosphotransferase with a carboxyl group as acceptor (EC 2.7.2) is overexpressed by the recombinant microorganism or plant cell.

27. The composition of claim 26, wherein the acetate kinase (EC 2.7.2.1), the butyrate kinase (EC 2.7.2.7), the acetate kinase (diphosphate) (EC 2.7.2.12), the branched-chain-fatty-acid kinase (EC 2.7.2.14) or the propionate kinase (EC 2.7.2.15) is overexpressed by the recombinant microorganism or plant cell.

28. The composition of claim 12, wherein the composition further comprises a phosphate acetyltransferase (EC 2.3.1.8) or a phosphate butyryltransferase (EC 2.3.1.19).

29. The composition of claim 12, wherein the 2-hydroxyaldehyde is 2-hydroxypropanal.

30. The composition of claim 12, wherein the 2-hydroxyaldehyde is 2,3-dihydroxypropanal.

31. The composition of claim 12, wherein the composition is the in vitro cell free system.

32. The composition of claim 12, wherein the composition comprises the recombinant microorganism or plant cell.

33. The composition of claim 12, wherein the recombinant microorganism or plant cell is genetically modified to overexpress the phosphoketolase or the sulfoacetaldehyde acetyltransferase, wherein the genetic modification is selected from: (a) operably associating a heterologous promoter with a polynucleotide encoding the phosphoketolase or the sulfoacetaldehyde acetyltransferase; (b) transforming a heterologous polynucleotide encoding the phosphoketolase or the sulfoacetaldehyde acetyltransferase into the recombinant microorganism or plant cell; and/or (c) introducing a mutation in the promoter of a polynucleotide encoding the phosphoketolase or sulfoacetaldehyde acetyltransferase wherein said mutation results in overexpression of the polynucleotide.

Description

(1) FIG. 1 shows schematically the conversion of a 2-hydroxyaldehyde into the corresponding acyl phosphate.

(2) FIG. 2 shows schematically the conversion of acyl phosphate into the corresponding carboxylic acid.

(3) FIG. 3 shows schematically the conversion of acyl phosphate into the corresponding acyl-CoA.

(4) FIG. 4 shows schematically the conversion of 2-hydroxypropanal into propanoic acid or propionyl-CoA.

(5) FIG. 5 shows schematically the conversion of 2,3-dihydroxypropanal into 3-hydroxypropanoic acid or 3-hydroxypropionyl-CoA.

(6) 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.

(7) 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

Example 1

Cloning, Expression and Purification of Phosphoketolases

(8) Gene Synthesis, Cloning and Expression of Recombinant Enzymes

(9) The sequences of phosphoketolases inferred from the genomes of prokaryotic organisms were generated by oligonucleotide concatenation to fit the codon usage of E. coli (genes were commercially synthesized by GeneArt®). A stretch of 6 histidine codons was inserted after the methionine initiation codon to provide an affinity tag for purification. The genes thus synthesized were cloned in a modified pUC18 expression vector (New England Biolabs) containing a modified Multiple Cloning Site (MCS). The genes of interest were cloned at PacI and NotI restriction sites.

(10) Competent MG1655 E. coli cells were transformed with these vectors using standard heat shock procedure. The transformed cells were grown in LB-ampicillin medium for 24 h at 30° C., 160 rpm shaking.

(11) The cells were collected by centrifugation at 4° C., 10,000 rpm for 20 min and the pellets were stored at −80° C.

(12) Protein Purification and Concentration

(13) The pellets from 200 ml of cultured cells were thawed on ice and resuspended in 3 ml of 50 mM Tris-HCl pH 7.5 containing 300 mM NaCl, 5 mM MgCl.sub.2, 1 mM DTT and 10 mM Imidazole. 10 μl of lysonase (Merck) was added. Cells were incubated 10 minutes at room temperature and then returned to ice for 20 minutes. Cell lysis was completed by sonication for 2×30 seconds. The bacterial extracts were then clarified by centrifugation at 4° C., 10,000 rpm for 20 min. The clarified bacterial lysates were loaded on PROTINO-1000 Ni-TED column (Macherey-Nagel) allowing adsorption of 6-His tagged proteins. Columns were washed and the enzymes of interest were eluted with 4 ml of 50 mM Tris-HCl pH 7.5 containing 300 mM NaCl, 5 mM MgCl.sub.2, 1 mM DTT, 250 mM Imidazole. Eluates were then concentrated, desalted on Amicon Ultra-4 10 kDa filter unit (Millipore) and enzymes were resuspended in 50 mM Tris-HCl pH 7.5. The enzyme preparation was complemented with 10% glycerol prior to long-term storage. Protein concentrations were quantified by direct UV 280 nm measurement on the NanoDrop 1000 spectrophotometer (Thermo Scientific). The purity of proteins thus purified varied from 70% to 90%.

Example 2

Study of the Formation of 3-Hydroxypropionyl Phosphate From 2,3-Dihydroxypropanal, Catalyzed by Phosphoketolase

(14) Enzymatic Reactions

(15) The enzymatic reactions were carried out under the following conditions:

(16) 50 mM Tris-HCl pH 7.5

(17) 25 mM Potassium phosphate pH 7.5

(18) 5 mM Thiamine pyrophosphate (TPP)

(19) 5 mM MgCl.sub.2

(20) 23 mM Sodium fluoride

(21) 8 mM Sodium iodoacetate

(22) 1.9 mM L-Cysteine hydrochloride

(23) 50 mM 2,3-Dihydroxypropanal (D,L-glyceraldehyde) (Sigma)

(24) The pH was adjusted to 7.5

(25) Each enzymatic reaction was started by adding 3 mg/ml of purified recombinant phosphoketolase (PKT).

(26) Control assays were performed in which either no enzyme was added, or no substrate was added.

(27) Incubations were run overnight with shaking at 37° C. 3-hydroxypropionyl phosphate formation was studied through the detection of iron (III) 3-hydroxypropionyl-hydroxamate using the following procedure (Racker E., Methods Enzymol. 5, 1962, 276-280): 0.1 ml of hydroxylamine hydrochloride (2 M, pH 6.5) was added to 0.1 ml of reaction mixture. After 10 min of incubation at room temperature the samples were acidified with 35 μl of 30% trichloroacetic acid. 35 μl of 8 M HCl and 35 μl of FeCl.sub.3 reagent (10% FeCl.sub.3 in 0.1 M HCl) were then added. The samples were further clarified by centrifugation and the absorbance of ferric 3-hydroxypropionyl-hydroxamate complex was measured at 505 nm.

(28) A low signal of absorbance was observed in the control assays in which either no phosphoketolase was added, or no 2,3-dihydrpoxypropanal was added. Absorbance values of the enzymatic samples corrected by subtraction of the control assay without enzyme, are shown in Table 1.

(29) TABLE-US-00001 TABLE 1 Enzymatic assay with Uniprot Accession Absorbance phosphoketolase (PKT) Number at 505 nm PKT from Q6R2Q6 0.024 Bifidobacterium pseudolongum (SEQ ID NO: 1) subsp. globosum PKT from Lactococcus lactis A9QST6 0.012 subsp. lactis strain KF147 (SEQ ID NO: 3) PKT from Clostridium Q97JE3 0.120 acetobutylicum strain ATCC 824 (SEQ ID NO: 2) PKT from D1NS90 0.020 Bifidobacterium gallicum (SEQ ID NO: 16) DSM 20093 PKT from B1MWV8 0.014 Leuconostoc citreum (SEQ ID NO: 17) (strain KM20) PKT from Streptococcus A8AV21 0.088 gordonii (SEQ ID NO: 18) (strain Challis/ATCC 35105/ CH1/DL1/V288)

Example 3

Study of the Formation of Propionyl Phosphate From 2-Hydroxypropanal Catalyzed by Phosphoketolase

(30) The enzymatic assays were carried out according to the protocol described in Example 2. 2-hydroxypropanal (lactaldehyde) was used as substrate instead of 2,3 dihydroxypropanal.

(31) Hydroxamate-Based Colorimetric Assay

(32) Propionyl phosphate formation was studied through the detection of iron (III) propionyl-hydroxamate using the procedure described in Example 2. A low signal of absorbance was observed in the control assays in which either no phosphoketolase was added, or no 2-hydroxypropanal was added. Absorbance values of the enzymatic samples corrected by subtraction of the control assay without enzyme, are shown in Table 2.

(33) TABLE-US-00002 TABLE 2 Enzymatic assay with Uniprot Accession Absorbance phosphoketolase (PKT) Number at 505 nm PKT from Q6R2Q6 0.006 Bifidobacterium pseudolongum (SEQ ID NO: 1) subsp. globosum PKT from Clostridium Q97JE3 0.004 acetobutylicum strain ATCC 824 (SEQ ID NO: 2) PKT from Thiobacillus denitrificans Q3SKJ7 0.008 (strain ATCC 25259) (SEQ ID NO: 19)

(34) Thus, different phosphoketolases were shown to catalyze the conversion of a 2-hydroxyaldehyde into the corresponding acyl phosphate.

Example 4

Cloning, Expression and Purification of Sulfoacetaldehyde Acetyltransferases

(35) Gene Synthesis, Cloning and Expression of Recombinant Proteins

(36) The sequences of the studied enzymes inferred from the genomes of prokaryotic organisms were generated by oligonucleotide concatenation to fit the codon usage of E. coli (genes were commercially synthesized by GeneArt®). A stretch of 6 histidine codons was inserted after the methionine initiation codon to provide an affinity tag for purification. The genes thus synthesized were cloned in a pET-25b(+) expression vector (vectors were constructed by GeneArt®).

(37) 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, (2005), 207-234) for 7 h at 30° C. and protein expression was continued at 18° C. overnight (approximately 16 h). The cells were collected by centrifugation at 4° C., 10,000 rpm for 20 min and the pellets were stored at −80° C.

(38) Protein Purification and Concentration

(39) The pellets from 200 ml of culture cells were thawed on ice and resuspended in 5 ml of 50 mM Tris-HCl buffer pH 7.5 containing 300 mM NaCl, 10 mM MgCl.sub.2, 10 mM imidazole and 1 mM DTT. Twenty microliters of lysonase (Novagen) were added. Cells were incubated 10 minutes at room temperature and then returned to ice for 20 minutes. Cell lysis was completed by sonication for 2×30 seconds. The bacterial extracts were then clarified by centrifugation at 4° C., 4000 rpm for 40 min. The clarified bacterial lysates were loaded onto a PROTINO-2000 Ni-TED column (Macherey-Nagel) allowing adsorption of 6-His tagged proteins. Columns were washed and the enzymes of interest were eluted with 6 ml of 50 mM Tris-HCl buffer pH 7.5 containing 300 mM NaCl, 5 mM MgCl2, 1 mM DTT and 250 mM imidazole. Eluates were then concentrated, desalted on Amicon Ultra-4 10 kDa filter unit (Millipore) and enzymes were resuspended in 50 mM Tris-HCl buffer pH 7.5. The purity of proteins thus purified varied from 70% to 90% as estimated by SDS-PAGE analysis. Protein concentrations were determined by direct UV 280 nm measurement on the NanoDrop 1000 spectrophotometer (Thermo Scientific) or by Bradford assay (BioRad).

Example 5

HPLC-Based Analysis of the Conversion of D,L-Lactaldehyde (2-Hydroxypropanal) into Propionyl Phosphate and Further into Propionic Acid

(40) The phosphoketolases were expressed and purified as described in Example 1.

(41) The sulfoacetaldehyde acetyltransferases were expressed as described in Example 4.

(42) Enzyme Reaction

(43) The enzymatic reactions were carried out under the following conditions:

(44) 50 mM Tris-HCl pH 7.5

(45) With or without 25 mM sodium phosphate pH 7.5

(46) 0.6 mM thiamine pyrophosphate (TPP)

(47) 1 mM MgCl.sub.2

(48) 1.9 mM L-cysteine hydrochloride

(49) 50 mM D, L-lactaldehyde (Sigma-Aldrich)

(50) 2.8 mg/ml purified enzyme

(51) Total volume 150 μl.

(52) A control assay was performed in which no enzyme was added. Enzymatic assays were conducted overnight at 37° C. The formation of propionic acid was studied using HPLC-based analysis.

(53) HPLC-Based Method

(54) The enzymatic reactions were stopped by a 5-min incubation at 80° C. Then, 150 μl MeCN was added in the medium, and the assay tubes were centrifuged. 100 μl of the clarified supernatant was filtered, and transferred into a clean vial.

(55) HPLC analyses were performed using a 1260 Infinity LC System (Agilent), equipped with a refractometer detector and a column heating module. 5 μl sample was separated on Hi-Plex H column (100×7.7 mm, 8 μm particle size, column temp. 65° C.) equipped with a PL Hi-Plex H Guard Column (50×7.7 mm). The mobile phase consisted of aqueous sulfuric acid (1 mM) and was run with a flow rate of 0.8 ml/min. Retention time of D,L-lactaldehyde and propionic acid under these conditions were 4.94 and 6.62 min, respectively.

(56) Several phosphoketolases or sulfoacetaldehyde acetyltransferases were able to catalyze the conversion of D,L-lactaldehyde into propionic acid (Table 3). The formation of propionic acid was improved in the presence of inorganic phosphate, indicating that the conversion takes place through an acyl-phosphate as intermediate. The acyl phosphate which is rather unstable is converted into propionic acid by way of spontaneous hydrolysis.

(57) TABLE-US-00003 TABLE 3 uniprot accession propionic enzyme organism number acid mM With 50 Sulfoacetaldehyde Castellaniella 084H44 1.1 mM acetyltransferase defragans phopshate (SEQ ID NO: 4) Sulfoacetaldehyde Alcaligenes Q84H41 0.5 acetyltransferase xyloxydans (SEQ ID NO: 5) Sulfoacetaldehyde Roseovarius A35R25 1.8 acetyltransferase nubinhibens (SEQ ID NO: 8) Sulfoacetaldehyde Desulfonispora Q93PS3 0.4 acetyltransferase thiosulfatigenes (SEQ ID NO: 6) Phosphoketolase Streptococcus A8AV21 5.1 gordonii (SEQ ID NO: 18) Phosphoketolase Lactococcus A9QST6 6.9 lactis (SEQ ID NO: 3) Phosphoketolase Lactococcus D5H215 0.8 crispatus (SEQ ID NO: 20) control without enzyme 0.0 Without Sulfoacetaldehyde Castellaniella Q84H44 0.2 50 mM acetyltransferase defragans phosphate Sulfoacetaldehyde Alcaligenes Q84H41 0.2 acetyltransferase xyloxydans Sulfoacetaldehyde Roseovarius A3SR25 2.1 acetyltransferase nubinhibens Sulfoacetaldehyde Desulfonispora Q93PS3 0.3 acetyltransferase thiosulfatigenes Phosphoketolase Streptococcus A8AV21 0.6 gordonii Phosphoketolase Lactococcus A9QST6 1.6 lactis Phosphoketolase Lactococcus D5H215 0.4 crispatus control without enzyme 0.0

Example 6

HPLC-Based Analysis of the Conversion of D,L-Glyceraldehyde (2,3-Hydroxypropanal) into 3-Hydroxypropionyl Phosphate and Further into 3-Hydroxypropionic Acid

(58) All the phosphoketolases were expressed and purified as described in Example 1.

(59) The sulfoacetaldehyde acetyltransferases were expressed as described in Example 4.

(60) Enzyme Reaction

(61) The enzymatic reactions were carried out under the following conditions:

(62) 50 mM Tris-HCl pH 7.5

(63) With or without 25 mM sodium phosphate pH 7.5

(64) 0.6 mM thiamine pyrophosphate (TPP)

(65) 1 mM MgCl.sub.2

(66) 1.9 mM L-cysteine hydrochloride

(67) 50 mM D,L-glyceraldehyde (Sigma)

(68) 2.8 mg/ml purified enzyme

(69) Total volume of the reaction was 150 μl.

(70) A control assay was performed in which no enzyme was added. Enzymatic assays were conducted overnight at 37° C. The formation of 3-hydroxypropionic acid was studied using HPLC-based analysis.

(71) HPLC-Based Method

(72) The enzymatic reactions were stopped by a 5-min incubation at 80° C. Then, 150 μl MeCN was added in the medium, and the assay tubes were centrifuged. 100 μl of the clarified supernatant was filtered, and transferred into a clean vial.

(73) The amount of 3-hydroxypropionic acid produced was measured using a HPLC-based procedure. HPLC analysis was performed using a 1260 Infinity LC System Agilent, equipped with column heating module, and refractometer. 5 μl of samples were separated using 3 columns connected in series as follows:

(74) 1. Hi-Plex guard column (50×7.7 mm, 8 μm particle size) (Agilent)

(75) 2. Hi-Plex column (100×7.7 mm, 8 μm particle size) (Agilent)

(76) 3. Zorbax SB-Aq column (250×4.6 mm, 5 μm particle size, column temp. 65° C.) (Agilent).

(77) The mobile phase consisted of aqueous sulfuric acid (1 mM), mobile phase flow rate was 0.5 ml/min. Retention time of D,L-glyceraldehyde and 3-hydroxypropionic acid under these conditions were 12.12 and 14.67 min, respectively.

(78) Several phosphoketolases or sulfoacetaldehyde acetyltransferases were able to catalyze the conversion of D,L-glyceraldehyde into 3-hydroxypropionic acid (Table 4). The conversion is considered to take place via the intermediate 3-hydroxypropionyl phosphate which is rather unstable and is spontaneously hydrolyzed to 3-hydroxypropionic acid.

(79) TABLE-US-00004 TABLE 4 uniprot accession 3-hydroxypropionic enzyme organism number acid mM with 50 mM Sulfoacetaldehyde Castellaniella Q84H44 0.2 phopshate acetyltransferase defragans Sulfoacetaldehyde Alcaligenes Q84H41 0.1 acetyltransferase xyloxydans Sulfoacetaldehyde Roseovarius A3SR25 0.3 acetyltransferase nubinhibens Sulfoacetaldehyde Desulfonispora Q93PS3 0.1 acetyltransferase thiosulfatigenes Phosphoketolase Streptococcus A8AV21 2.1 gordonii Phosphoketolase Lactococcus A9QST6 0.8 lactis Phosphoketolase Lactococcus D5H215 0.3 crispatus control without enzyme 0.2 without 50 Sulfoacetaldehyde Castellaniella Q84H44 0.1 mM acetyltransferase defragans phosphate Sulfoacetaldehyde Alcaligenes Q84H41 0.1 acetyltransferase xyloxydans Sulfoacetaldehyde Roseovarius A3SR25 0.4 acetyltransferase nubinhibens Sulfoacetaldehyde Desulfonispora Q93PS3 0.2 acetyltransferase thiosulfatigenes Phosphoketolase Streptococcus A8AV21 1.1 gordonii Phosphoketolase Lactococcus A9QST6 1.3 lactis Phosphoketolase Lactococcus D5H215 1.2 crispatus control without enzyme 0.1