Method for the enzymatic production of D-erythrose and acetyl phosphate

Abstract

Described is a method for the production of D-erythrose and acetyl phosphate comprising the enzymatic conversion of D-fructose into D-erythrose and acetyl phosphate by making use of a phosphoketolase. The produced D-erythrose can further be converted into glycolaldehyde by a method for the production of glycolaldehyde comprising the enzymatic conversion of D-erythrose into glycolaldehyde by making use of an aldolase, wherein aldolase is a 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4) or a fructose-bisphosphate aldolase (EC 4.1.2.13). The produced glycolaldehyde can finally be converted into acetyl phosphate by the enzymatic conversion of the thus produced glycolaldehyde into acetyl phosphate by making use of a phosphoketolase or a sulfoacetaldehyde acetyltransferase.

Claims

1. A method of producing D-erythrose and acetyl phosphate comprising enzymatically converting D-fructose and phosphate into D-erythrose and acetyl phosphate by a phosphoketolase (EC 4.1.2.9) or a fructose-6-phosphate phosphoketolase (EC 4.1.2.22) wherein the phosphoketolase or fructose-6-phosphate phosphoketolase is obtained from fungi or bacteria.

2. The method of claim 1, further comprising enzymatically converting D-erythrose into glycolaldehyde by an aldolase, wherein said aldolase is a 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4) or a fructose-bisphosphate aldolase (EC 4.1.2.13).

3. The method of claim 2, wherein the method further comprises: enzymatically converting glycolaldehyde into acetyl phosphate by a phosphoketolase (EC 4.1.2.9), a fructose-6-phosphate phosphoketolase (EC 4.1.2.22), or a sulfoacetaldehyde acetyltransferase (EC 2.3.3.15).

4. The method of claim 1, wherein the method further comprises enzymatically converting D-glucose into said D-fructose by a glucose-fructose isomerase.

5. The method of claim 4, wherein said glucose-fructose isomerase is a xylose isomerase (EC 5.3.1.5).

6. The method of claim 1, wherein said method is carried out in vitro.

7. The method of claim 2, wherein said method is carried out in vitro).

8. The method of claim 1, wherein the method is carried out in a microorganism expressing the phosphoketolase (EC 4.1.2.9) or the fructose-6-phosphate phosphoketolase (EC 4.1.2.22).

9. The method of claim 2, wherein the method is carried out in a microorganism expressing the 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4) or the fructose-bisphosphate aldolase (EC 4.1.2.13).

10. The method of claim 1, wherein the method further comprises enzymatically converting the acetyl phosphate into acetyl-CoA by a phosphotransacetylase in the presence of co-enzyme A (CoA).

11. The method of claim 2, wherein the method further comprises enzymatically converting the acetyl phosphate into acetyl-CoA by a phosphotransacetylase in the presence of co-enzyme A (CoA).

12. The method of claim 3, wherein the method further comprises enzymatically converting the acetyl phosphate into acetyl-CoA by a phosphotransacetylase in the presence of co-enzyme A (CoA).

13. The method of claim 6, wherein the method further comprises enzymatically converting the acetyl phosphate into acetyl-CoA by a phosphotransacetylase in the presence of co-enzyme A (CoA).

14. The method of claim 4, wherein the method further comprises enzymatically converting the acetyl phosphate into acetyl-CoA by a phosphotransacetylase in the presence of co-enzyme A (CoA).

15. The method of claim 5, wherein the method further comprises enzymatically converting the acetyl phosphate into acetyl-CoA by a phosphotransacetylase in the presence of co-enzyme A (CoA).

16. The method of claim 7, wherein the method further comprises enzymatically converting glycolaldehyde into acetyl phosphate by a phosphoketolase (EC 4.1.2.9) or the fructose-6-phosphate phosphoketolase (EC 4.1.2.22) or a sulfoacetaldehyde acetyltransferase (EC 2.3.3.15).

17. The method of claim 4, wherein said method is carried out in vitro.

18. The method of claim 9, wherein the method is carried out in a recombinant microorganism expressing: (a) a recombinant phosphoketolase (EC 4.1.2.9) or the fructose-6-phosphate phosphoketolase (EC 4.1.2.22) and/or a recombinant sulfoacetaldehyde acetyltransferase (EC 2.3.3.15); and (b) a recombinant 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4) or a recombinant fructose-bisphosphate aldolase (EC 4.1.2.13); and optionally (c) a recombinant glucose-fructose isomerase.

19. The method of claim 18, wherein the recombinant microorganism expresses both the recombinant phosphoketolase (EC 4.1.2.9) or the fructose-6-phosphate phosphoketolase (EC 4.1.2.22) and the recombinant sulfoacetaldehyde acetyltransferase (EC 2.3.3.15).

20. The method of claim 4, wherein said method is carried out in a microorganism expressing the recombinant glucose-fructose isomerase.

21. The method of claim 4, wherein said glucose-fructose isomerase is a xylose isomerase (EC 5.3.1.5).

22. The method of claim 18, wherein the recombinant microorganism is genetically modified to overexpress the recombinant phosphoketolase, the recombinant sulfoacetaldehyde acetyltransferase (EC 2.3.3.15), the recombinant 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4), the recombinant fructose-bisphosphate aldolase (EC 4.1.2.13) and/or the recombinant glucose-fructose isomerase, wherein the genetic modification is selected from: (a) operably associating a heterologous promoter with a polynucleotide encoding the phosphoketolase, the sulfoacetaldehyde acetyltransferase (EC 2.3.3.15), the 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4), the fructose-bisphosphate aldolase (EC 4.1.2.13) and/or the glucose-fructose isomerase; (b) transforming a heterologous polynucleotide encoding the phosphoketolase, the sulfoacetaldehyde acetyltransferase (EC 2.3.3.15), the 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4), the fructose-bisphosphate aldolase (EC 4.1.2.13) and/or the glucose-fructose isomerase into the recombinant microorganism; and/or (c) introducing a mutation in the promoter of a polynucleotide encoding the phosphoketolase, the sulfoacetaldehyde acetyltransferase (EC 2.3.3.15), the 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4), the fructose-bisphosphate aldolase (EC 4.1.2.13), and/or the glucose-fructose isomerase wherein said mutation results in overexpression of the polynucleotide.

23. The method of claim 18, wherein the recombinant microorganism is genetically modified to alter or improve the enzymatic activity of the recombinant phosphoketolase, the recombinant sulfoacetaldehyde acetyltransferase (EC 2.3.3.15), the recombinant 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4), the recombinant fructose-bisphosphate aldolase (EC 4.1.2.13) and/or the recombinant glucose-fructose isomerase, wherein the genetic modification is selected from: (a) transforming a heterologous polynucleotide encoding the phosphoketolase, the sulfoacetaldehyde acetyltransferase (EC 2.3.3.15), the 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4), the fructose-bisphosphate aldolase (EC 4.1.2.13), and/or the glucose-fructose isomerase into the recombinant microorganism; and/or (b) introducing a mutation into a polynucleotide encoding the phosphoketolase, the sulfoacetaldehyde acetyltransferase (EC 2.3.3.15), the 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4), the fructose-bisphosphate aldolase (EC 4.1.2.13), and/or the glucose-fructose isomerase wherein said mutation alters or improves enzymatic activity.

24. The method of claim 3, wherein the phosphoketolase is different than the phosphoketolase used in converting D-fructose into D-erythrose and acetyl phosphate.

25. The method of claim 10, wherein the method is carried out in vitro.

26. The method of claim 10, wherein the method is carried out in a recombinant microorganism.

27. The method of claim 3, wherein the method is carried out in vitro.

28. The method of claim 3, wherein the method is carried out in a recombinant microorganism.

Description

(1) FIG. 1: shows an artificial metabolic pathway for acetyl phosphate production from D-glucose (or D-fructose) via D-erythrose and glycolaldehyde by making use of a (naturally occurring) glucose-fructose isomerase (first enzymatic step), a phosphoketolase (second enzymatic step), an aldolase (third enzymatic step) and a phosphoketolase (fourth enzymatic step).

(2) FIG. 2: shows HPLC chromatograms a) of the reaction mixture in a phosphoketolase assay with a phosphoketolase from Bifidobacterium pseudolongum and D-fructose as substrate; b) of the reaction mixture without enzyme.

(3) FIG. 3: shows a plot of the velocity as a function of substrate concentration for the phoshoketolase reaction catalyzed by the phosphoketolase of Bifidobacterium pseudolongum. Initial rates were computed from the kinetics over 100 minutes of the reaction.

(4) FIG. 4: shows HPLC chromatograms a) of the reaction mixture in a phosphoketolase assay with the phosphoketolase from Bifidobacterium pseudolongum and glycolaldehyde as substrate; b) of the reaction mixture without enzyme.

(5) FIG. 5: shows the production of acetate by the hydrolysis of acetyl phosphate, which itself was produced from glycolaldehyde in the presence of phosphate catalyzed by sulfoacetaldehyde acetyltransferases (Xsc).

(6) FIG. 6: shows the production of glycolaldehyde produced from D-erythrose catalyzed by different 2-deoxyribose-5-phosphate aldolases as indicated.

(7) Other aspects and advantages of the invention will be described in the following examples, which are given for purposes of illustration and not by way of limitation. Each publication, patent, patent application or other document cited in this application is hereby incorporated by reference in its entirety.

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 Sites (MCS). The genes of interest were cloned at PacI and NotI restriction sites. 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.

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

(11) Protein Purification and Concentration

(12) 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 l 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 230 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. 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 Activity of Phosphoketolases with D-Fructose as Substrate

(13) Enzymatic Reactions

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

(15) 50 mM Tris-HCl pH 7.5

(16) 50 mM Sodium phosphate pH 7.5

(17) 5 mM Thiamine pyrophosphate (TPP)

(18) 5 mM MgCl.sub.2

(19) 23 mM Sodium fluoride

(20) 1.9 mM L-Cysteine hydrochloride

(21) 50 mM Fructose (Sigma)

(22) The pH was adjusted to 7.5

(23) Enzyme concentration ranged from 3 to 5 mg/ml.

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

(25) The ability of phosphoketolase to use D-fructose as substrate was confirmed through the use of up to three analytical methods: the detection of acetate and D-erythrose using HPLC-based analysis and the chemical determination of acetyl phosphate.

(26) HPLC-Based Method

(27) The formation of acetate and D-erythrose from D-fructose in the presence of phosphoketolase was monitored using HPLC-based method. Acetyl phosphate is particularly unstable to hydrolysis, releasing acetate. Therefore, the monitoring of the acetate was chosen as a part of analytical method.

(28) The enzymatic reactions (see description above) were run in total volume of 0.15 ml for 18 hours with shaking at 37 C. and stopped by a 5-min incubation at 80 C. The assays tubes were then centrifuged and 100 l of the clarified supernatant was transferred into a clean vial. Commercial sodium acetate, D-fructose and D-erythrose (Sigma-Aldrich) were used as references. HPLC analyses were performed using a 1260 Inifinity LC System (Agilent), equipped with a refractometer detector and a column heating module. 10 l sample was separated on Hi-Plex H column (3007.7 mm, 8 m particle size, column temp. 65 C.) equipped with a PL Hi-Plex H Guard Column (507.7 mm). The mobile phase consisted of aqueous sulfuric acid (5.5 mM) and was run with a flow rate of 0.6 ml/min. Retention time of D-fructose, D-erythrose and sodium acetate under these conditions was 12.5, 14.4 and 18.5 min, respectively. A typical chromatogram obtained with recombinant phosphoketolase from Bifidobacterium pseudolongum is shown in FIG. 2.

(29) The results of HPLC analysis are shown in Table 1. The yields of acetate and D-erythrose indicate the quantitative recovery of the carbon moiety of the D-fructose.

(30) TABLE-US-00001 TABLE 1 The products formed from the transformation of 50 mM D-fructose by phosphoketolase (PKT) from different sources. (The precision and accuracy of HPLC measurement were about 20% and 80-120%, respectively.) D-fructose, Acetate mM D-erythrose formed, Reaction (unconsumed) formed, mM mM Control without enzyme 48 mM 0 mM 0 mM In the presence of PKT from 11 mM 46 mM 35 mM Lactococcus lactis subsp. lactis (strain KF147) (Uniprot A9QST6) In the presence of PKT from 8 mM 44 mM 46 mM Bifidobacterium pseudolongum subsp. globosum (Uniprot Q6R2Q6) In the presence of PKT from 13 mM 45 mM 37 mM Clostridium acetobutylicum (strain ATCC 824) (Uniprot Q97JE3)
Kinetics Analysis of Acetyl Phosphate Formation from D-Fructose Using a Hydroxamate-Based Colorimetric Assay

(31) The composition of enzymatic reactions was identical to that described above. Kinetic parameters were determined using a range of D-fructose concentrations (0-500 mM) and a constant concentration of sodium phosphate (50 mM).

(32) Each enzymatic reaction was started by adding 3 mg/ml of purified phosphoketolase. Incubations were run for 20, 40, 60, 80, 100 min with shaking at 37 C. Acetyl phosphate concentration was determined through the detection of iron (III) acetyl-hydroxamate using the following procedure (Racker E., Methods Enzymol. 5, 1962, 276-280):

(33) 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 l of 30% trichloroacetic acid. 35 l of 8 M HCl and 35 l 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 acetyl-hydroxamate complex was measured at 505 nm. A calibration curve was prepared using commercial acetyl phosphate (Sigma-Aldrich). Kinetic parameters obtained for purified recombinant phosphoketolases are presented in Table 2.

(34) TABLE-US-00002 TABLE 2 Kinetic parameters of phosphoketolases from different sources with D- fructose as substrate. Phosphoketolase K.sub.m, mM k.sub.cat, s.sup.1 Lactococcus lactis subsp. lactis (strain 0.25M 0.11 0.04 KF147) (Uniprot A9QST6) Bifidobacterium pseudolongum subsp. higher than 0.3M 0.10 0.02 globosum (Uniprot Q6R2Q6)

(35) FIG. 3 shows an example of a Michaelis-Menten plot corresponding to the data collecting for phosphoketolase from Bifidobacterium pseudolongum.

Example 3: Analysis of Activity of Phosphoketolases with Glycolaldehyde as Substrate

(36) Enzymatic Reactions

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

(38) 50 mM Tris-HCl pH 7.5

(39) 50 mM Sodium phosphate pH 7.5

(40) 5 mM Thiamine pyrophosphate (TPP)

(41) 5 mM MgCl.sub.2

(42) 23 mM Sodium fluoride

(43) 1.9 mM L-Cysteine hydrochloride

(44) 50 mM Glycolaldehyde (Sigma)

(45) The pH was adjusted to 7.5

(46) Enzyme concentration ranged from 3 to 5 mg/ml.

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

(48) The ability of phosphoketolase to use glycolaldehyde as substrate was confirmed through the use of up to two analytical methods: the detection of acetate using HPLC-based analysis and the chemical determination of acetyl phosphate.

(49) HPLC-Based Method

(50) The enzymatic reactions (see description above) were run for 48 hours with shaking at 37 C. and stopped by a 5-min incubation at 80 C. The assays tubes were then centrifuged and 100 l of the clarified supernatant was transferred into a clean vial. HPLC analyses were performed on Hi-Plex H column according to the procedure described in Example 2. Commercial sodium acetate and glycolaldehyde (Sigma-Aldrich) were used as references. Retention time of glycolaldehyde under these conditions was 15.4 min.

(51) A significant amount of acetate was produced in the enzymatic assay in the presence of phosphoketolase, no acetate signal was detected in the enzyme-free control reaction.

(52) A typical chromatogram obtained with phosphoketolase from Bifidobacterium pseudolongum is showed in FIG. 4.

(53) Analysis of Kinetics of Acetyl Phosphate Formation from Glycolaldehyde Using a Hydroxamate-Based Colorimetric Assay

(54) The composition of enzymatic reactions was identical to that described above. Kinetic parameters were determined using a range of glycolaldehyde concentrations (0-100 mM) and a constant concentration of sodium phosphate (50 mM).

(55) Each assay was started by adding 3 mg/ml of purified phosphoketolase. Incubations were run for 20, 40, 60, 80, 100 min with shaking at 37 C. The concentration of acetyl phosphate was determined chemically through the detection of iron (III) acetyl-hydroxamate according to the procedure described in Example 2.

(56) Kinetic parameters obtained for purified recombinant phosphoketolases are presented in Table 3.

(57) TABLE-US-00003 TABLE 3 Kinetic parameters of phosphoketolases from different sources with glycolaldehyde as substrate. Phosphoketolase K.sub.m, mM k.sub.cat, s.sup.1 Clostridium acetobutylicum (strain ATCC 824) 30 mM 0.08 (Q97JE3) Bifidobacterium pseudolongum subsp. 20 mM 0.07 globosum (Q6R2Q6) Lactococcus lactis subsp. lactis (strain KF147) 25 mM 0.05 (Uniprot A9QST6)

Example 4: Expression, and Purification of E. coli 2-Deoxy-D-Ribose-5-Phosphate Aldolase

(58) Protein Expression

(59) The vector pCAN containing the gene coding for E. coli 2-deoxy-D-ribose-5-phosphate aldolase (Uniprot P0A6L0) was purchased from NAIST (Nara Institute of Science and Technology, Japan, ASKA collection). Provided vector contained a stretch of 6 histidine codons after the methionine initiation codon.

(60) Competent E. coli BL21(DE3) cells (Novagen) were transformed with this vector using standard heat shock procedure. The transformed cells were grown with shaking (160 rpm) on ZYM-5052 auto-induction medium (Studier F W, Prot. Exp. Pur. 41 (2005), 207-234), supplemented with chloramphenicol (25 g/ml) for 7 hours at 37 C. Protein expression was continued at 18 C. overnight (approximately 12 hours). The cells were collected by centrifugation at 4 C., 10,000 rpm for 20 min and the pellets were frozen at 80 C.

(61) Protein Purification

(62) The pellet from 200 ml of cultured cells was thawed on ice and resuspended in 6 ml of 50 mM Tris-HCl containing 0.5 M NaCl, 5 mM MgCl.sub.2, 1 mM DTT and 10 mM Imidazole. 10 l 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 230 seconds. The bacterial extracts were then clarified by centrifugation at 4 C., 10,000 rpm for 20 min.

(63) 2-deoxy-D-ribose-5-phosphate aldolase was purified on PROTINO-1000 Ni-TED column (Macherey-Nagel) according to the manufacturer's recommendations. Eluates, containing the enzyme of interest were then concentrated, desalted on Amicon Ultra-4 10 kDa filter unit (Millipore) and enzyme were resuspended in 50 mM Tris-HCl pH 7.5, complemented with 50 mM NaCl and 10% glycerol. Protein concentrations were quantified by direct UV 280 nm measurement on the NanoDrop 1000 spectrophotometer (Thermo Scientific). The purity of protein thus purified varied from 70% to 90%.

Example 5: Study of Enzymatic Production of Glycolaldehyde from D-Erythrose

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

(65) 50 mM Tris-HCl pH 7.5

(66) 50 mM NaCl

(67) 10 mM MgCl.sub.2

(68) 1 mM DTT

(69) 50 mM D-erythrose (Sigma-Aldrich)

(70) The pH was adjusted to 7.5

(71) 1 mg of purified 2-deoxy-D-ribose-5-phosphate aldolase was added to 0.2 ml of reaction mixture. Control assays were performed in which either no enzyme was added, or no substrate was added. The reaction mixtures were incubated for overnight (approximately 18 hours) at 37 C. and the reaction was stopped by a 10-min incubation at 80 C.

(72) The assays tubes were then centrifuged and filtered and 100 l of the clarified supernatant was transferred into a clean vial. HPLC analyses were performed on Hi-Plex H column according to the procedure described in Example 2.

(73) No formation of glycolaldehyde was observed without substrate. The HPLC analysis of reaction without enzyme showed only traces of glycolaldehyde, probably resulted from the spontaneous decomposition of the D-erythrose. The catalytic tests showed a significant increase of glycolaldehyde production in the presence of purified 2-deoxy-D-ribose-5-phosphate aldolase from E. coli. The ratio of glycolaldehyde produced after 18 hours incubation in the presence of enzyme versus glycolaldehyde produced in the absence of enzyme is about 9 fold judging from glycolaldehyde peak areas (Table 4). These results clearly indicate that a 2-deoxy-D-ribose-5-phosphate aldolase catalyzes the conversion of D-erythrose to glycolaldehyde.

(74) TABLE-US-00004 TABLE 4 Production of glycolaldehyde from D-erythrose. Glycolaldehyde peak Assay area, arbitrary units Without enzyme 2 10.sup.3 Enzymatic assay in the presence of 2- 18 10.sup.3 deoxy-D-ribose-5-phosphate aldolase from E. coli

Example 6: Study of the Activity of Sulfoacetaldehyde Acetyltransferases with Glycolaldehyde as a Substrate

(75) Gene Cloning and Protein Expression

(76) The sequences of sulfoacetaldehyde acetyltransferases (Xsc) inferred from the genome 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).

(77) 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.

(78) Protein Purification

(79) Sulfoacetaldehyde acetyltransferases were purified using PROTINO-1000 Ni-TED column (Macherey-Nagel) according to the procedure specified in Example 1 and using 50 mM sodium phosphate pH 7.5 instead of Tris-HCl buffer. 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 75% to 90% as estimated by SDS-PAGE analysis.

(80) Enzymatic Assays

(81) The enzymatic assays were carried out under the following conditions:

(82) 50 mM Sodium phosphate pH 7.5

(83) 1 mM Thiamine pyrophosphate (TPP)

(84) 5 mM MgCl.sub.2

(85) 50 mM Glycolaldehyde (Sigma-Aldrich)

(86) The pH was adjusted to 7.5

(87) Each assay was started by adding 5 mg/ml of purified enzyme. Incubations were run for 1 h with shaking at 37 C. Control assays were performed in which either no enzyme was added, or no substrate was added.

(88) The ability of sulfoacetaldehyde acetyltransferases (Xsc) to use glycolaldehyde as a substrate was confirmed through the use of two analytical methods: the chemical determination of acetyl phosphate and the detection of acetate using HPLC-based analysis.

(89) Hydroxamate-Based Colorimetric Assay

(90) Acetyl phosphate was determined through the detection of iron acetyl-hydroxamate according to the procedure described in Example 2.

(91) The concentration of acetyl phosphate produced in enzymatic assays with different sulfoacetaldehyde acetyltransferases is shown in Table 5.

(92) TABLE-US-00005 TABLE 5 Production of acetyl phosphate from glycolaldehyde and phosphate catalyzed by different sulfoacetaldehyde acetyltransferases. Acetyl Enzyme phosphate, mM Sulfoacetaldehyde acetyltransferase from Castellaniella 0.75 defragrans (Uniprot Acession Number: Q84H44) Sulfoacetaldehyde acetyltransferase from Alcaligenes 1.03 xylosoxydans xylosoxydans (Uniprot Acession Number: Q84H41) Sulfoacetaldehyde acetyltransferase from Roseovarius 0.85 nubinhibens ISM (Uniprot Acession Number: A3SR25)
HPLC-Based Method

(93) The enzymatic reactions were run for 1 hour with shaking at 37 C. (see description above) and stopped by a 5-min incubation at 80 C. The assays tubes were then centrifuged and an aliquot of the clarified supernatant was transferred into a clean vial. HPLC analyses were performed on Hi-Plex H column according to the procedure described in Example 2. A significant amount of acetate was produced in the assays with sulfoacetaldehyde acetyltransferases (Xsc) (FIG. 5), no acetate signal was observed neither in the enzyme-free control nor in the assays without substrate.

(94) Overall, these data indicate that sulfoacetaldehyde acetyltransferases from different origins were able to catalyze the formation of acetyl phosphate from glycolaldehyde and phosphate.

Example 7: Conversion of D-Erythrose into Glycolaldehyde Catalyzed by Different 2-Deoxy-D-Ribose-5-Phosphate Aldolases

(95) A library of 11 genes encoding representatives of the 2-deoxyribose-5-phosphate aldolases (DeoC, DERA) family from various prokaryotic and eukaryotic organisms was constructed and tested.

(96) Gene Cloning, Protein Expression and Purification

(97) The genes encoding 2-deoxy-D-ribose-5-phosphate aldolases EC 4.1.2.4 were synthesized and cloned in the pET-25b(+) expression vector (vectors were constructed by GeneArt) as described in Example 6.

(98) The corresponding enzymes were expressed in E. coli and purified as specified in Example 4. Each enzyme was tested for its ability to catalyze the production of glycolaldehyde from D-erythrose using the following assay:

(99) 50 mM Tris-HCl pH 7.5

(100) 50 mM D-Erythrose (Sigma-Aldrich)

(101) 10 mM MgCl.sub.2

(102) 50 mM NaCl

(103) 1 mM DTT

(104) Enzyme 5 mg/ml

(105) Control assays were performed in which either no enzyme was added, or no substrate was added. The assays were run for 4 h at 37 C. (see description above) and stopped by a 10-min incubation at 95 C. The assay tubes were then centrifuged and an aliquot of clarified supernatant was transferred into a clean vial. HPLC analyses were performed using 1260 Infinity LC System (Agilent), equipped with a refractometer detector and a column heating module. 10 l of samples were separated using 3 columns connected in series as follows: 1. Hi-Plex guard column (507.7 mm, 8 m particle size) (Agilent) 2. Hi-Plex column (1007.7 mm, 8 m particle size) (Agilent) 3. Hi-Plex column (3007.7 mm, 8 m particle size, column temp. 70 C.) (Agilent).

(106) The mobile phase consisted of aqueous sulfuric acid (8.4 mM) and was run at 0.5 ml/min. The analyses were performed at 70 C.

(107) Commercial glycolaldehyde (Sigma-Aldrich) was used as a reference. Retention time of erythrose and glycolaldehyde under these conditions were 20.2 and 22 min, respectively.

(108) No glycolaldehyde signal was observed in the assays without substrate. A certain amount of glycolaldehyde was found in commercially provided D-erythrose (see, e.g., FIG. 6, bar no enzyme). The 2-deoxy-D-ribose-5-phosphate aldolases assays showed an enzyme-dependent increase of glycolaldehyde production from erythrose judging from glycolaldehyde peak areas (FIG. 6). These data indicate that the cleavage of D-erythrose into glycolaldehyde can be catalyzed by 2-deoxy-D-ribose-5-phosphate aldolases.