ENZYMATIC PRODUCTION OF ACETYL PHOSPHATE FROM FORMALDEHYDE

Abstract

Described is a method for the enzymatic production of acetyl phosphate from formaldehyde using a phosphoketolase or a sulfoacetaldehyde acetyltransferase.

Claims

1. A method for the enzymatic production of acetyl phosphate from formaldehyde and phosphate in which the conversion of acetyl phosphate from formaldehyde and phosphate is achieved by the use of a phosphoketolase or of a sulfoacetaldehyde acetyltransferase (EC 2.3.3.15) according to the following reaction scheme:
2CH.sub.2O+phosphate.fwdarw.acetyl phosphate+H.sub.2O.

2. The method of claim 1, wherein the phosphoketolase is (a) a phosphoketolase (EC 4.1.2.9), or (b) a fructose-6-phosphate phosphoketolase (EC 4.1.2.22).

3. The method of claim 1 which further comprises the step of converting the produced acetyl phosphate into acetate.

4. The method of claim 3, wherein the conversion of acetyl phosphate into acetate is achieved by making use of an acetate kinase (EC 2.7.2.1) or of a butyrate kinase (EC 2.7.2.7) or of an acetate kinase (diphosphate) (EC 2.7.2.12) or of a propionate kinase (EC 2.7.2.15) or of an acylphosphatase (EC 3.6.1.7).

5. The method of claim 1 which further comprises the step of enzymatically converting the produced acetyl phosphate into acetyl-coenzyme A.

6. The method of claim 5, wherein the conversion of acetyl phosphate into acetyl-coenzyme A is achieved by making use of a phosphate acetyltransferase (EC 2.3.1.8).

7. The method of claim 1 further comprising the step of providing the formaldehyde to be converted into acetyl phosphate by enzymatically converting methanol into formaldehyde.

8. The method of claim 7 wherein the enzymatic conversion of methanol into formaldehyde is achieved by making use of a methanol dehydrogenase (EC 1.1.1.244) or a methanol dehydrogenase (cytochrome c) (EC 1.1.2.7) or an alcohol oxidase (EC 1.1.3.13).

9. A composition containing (a) formaldehyde and a phosphoketolase and/or a sulfoacetaldehyde acetyltransferase; or (b) formaldehyde and a microorganism expressing a phosphoketolase and/or a sulfoacetaldehyde acetyltransferase.

10. Use of a phosphoketolase or of a sulfoacetaldehyde acetyltransferase for the production of acetyl phosphate from formaldehyde.

11. Use of a microorganism expressing a phosphoketolase and/or a sulfoacetaldehyde acetyltransferase for the production of acetyl phosphate from formaldehyde.

Description

[0127] FIG. 1 shows a mass spectrum of an enzymatic reaction for the production of acetyl phosphate from formaldehyde using a phosphoketolase from L. lactis (A) and a mass spectrum of a control reaction without enzyme (B).

[0128] FIG. 2 shows the intensity of peak of acetate formed from the transformation of formaldehyde by phosphoketolases in the presence of phosphate.

[0129] 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.

[0130] 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

Gene Synthesis, Cloning and Expression of Recombinant Enzymes

[0131] 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 Pacl and Notl 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.

[0132] The cells were collected by centrifugation at 4 C., 10,000 rpm for 20 min and the pellets were stored at 80 C.

[0133] Protein Purification and Concentration

[0134] 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 Enzyme-Catalyzed Production of Acetyl Phosphate from Formaldehyde

[0135] Acetyl phosphate is particularly unstable to hydrolysis, releasing acetate. Therefore, the enzyme-catalyzed production of acetate from formaldehyde was monitored using Mass Spectrometry (MS) and HPLC analysis.

[0136] Mass Spectrometry (MS) Analysis

[0137] The enzymatic reactions were carried out under the following conditions: 50 mM Sodium phosphate pH 7.5

[0138] 1 mM Thiamine pyrophosphate (TPP)

[0139] 10 mM MgCl.sub.2

[0140] 50 mM Formaldehyde (Sigma)

[0141] The pH was adjusted to 7.5

[0142] Phosphoketolase (PKT) concentration was 10 mg/ml.

[0143] Control assays were performed in which either no enzyme was added, or no formaldehyde was added. The enzymatic reactions were run in total volume of 0.2 ml for 40 hours with shaking at 37 C. Typically, an aliquot of 200 l reaction was removed, centrifuged and the supernatant was transferred into a clean vial. The MS spectra were obtained on Ion Trapp Mass Spectrometer (Esquire 3000, Bruker) in negative ion mode by direct injection of sample using a syringe pump operated at a flow rate of 2 ml/h. The presence of acetate was evaluated. MS analysis showed an [M-H].sup. ion at m/z 59.4, corresponding to acetate, from the enzymatic sample but not from the controls (FIGS. 1A and B, FIG. 2).

[0144] HPLC-Based Analysis

[0145] The enzymatic reactions were carried out under the following conditions:

[0146] 50 mM Sodium phosphate pH 7.5

[0147] 5 mM Thiamine pyrophosphate (TPP)

[0148] 5 mM MgCl.sub.2

[0149] 1.9 mM L-cysteine hydrochloride

[0150] 23 mM Sodium fluoride

[0151] 50 mM Formaldehyde (Sigma)

[0152] The pH was adjusted to 7.5

[0153] Phosphoketolase (PKT) was added at concentration of 5 mg/ml.

[0154] Control reactions consisting of (a) formaldehyde and phosphate without enzyme, (b) formaldehyde and enzyme without phosphate were run in parallel.

[0155] The enzymatic reactions were run in total volume of 0.3 ml 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. Commercial sodium acetate (Sigma-Aldrich) was used as reference. 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) was run with a flow rate of 0.6 ml/min. Retention time of acetate under these conditions was 18.4 min.

[0156] The results of HPLC analysis are shown in Table 1.

TABLE-US-00001 TABLE 1 Formation of acetate from formaldehyde as function of presence of phosphate in reaction mixture. Acetate peak area, arbitrary units In the presence of 50 mM sodium Phosphoketolase (PKT) No phosphate phosphate No PKT 0 PKT from Lactococcus lactis below the detection 0.26 subsp. lactis (strain KF147; limit Uniprot A9QST6) PKT from Bifidobacterium below the detection 0.23 pseudolongum subsp. limit globosum (Uniprot Q6R2Q6) PKT from Clostridium below the detection 0.16 acetobutylicum (strain ATCC limit 824; Uniprot Q97JE3)

[0157] These data indicate that production of acetate from formaldehyde takes place in the presence of phosphate and phosphoketolase proceeding through the formation of acetyl phosphate.

Example 3: Study of Production of Acetyl Phosphate from Formaldehyde, Catalyzed by Sulfoacetaldehyde Acetyltransferases

[0158] Acetyl phosphate is particularly unstable to hydrolysis, releasing acetate. Therefore, the enzyme-catalyzed production of acetate from formaldehyde is monitored using HPLC analysis.

[0159] HPLC-Based Analysis

[0160] The enzymatic reactions are carried out under the following conditions:

[0161] 50 mM sodium phosphate pH 7.5

[0162] 5 mM thiamine pyrophosphate (TPP)

[0163] 5 mM MgCl.sub.2

[0164] 1.9 mM L-cysteine hydrochloride

[0165] 23 mM sodium fluoride

[0166] 50 mM formaldehyde (Sigma-Aldrich)

[0167] 1-10 mg/ml of enzyme

[0168] The following enzymes are used in this study:

[0169] Sulfoacetaldehyde acetyltransferase from Alcaligenes xylosoxydans xylosoxydans (Uniprot Accession number: Q84H41)

[0170] Sulfoacetaldehyde acetyltransferase from Roseovarius nubinhibens ISM (Uniprot Accession number: A3SR25)

[0171] Sulfoacetaldehyde acetyltransferase from Castellaniella defragans (Uniprot Accession Number: Q84H44)

[0172] Sulfoacetaldehyde acetyltransferase from Desulfonispora thiosulfatigenes (Uniprot Accession Number: Q93PS3)

[0173] Control reactions consisting of (a) formaldehyde and phosphate without enzyme, (b) formaldehyde and enzyme without phosphate are run in parallel.

[0174] The enzymatic reactions are conducted in a total volume of 0.3 ml for 48 hours with shaking at 37 C. and stopped by a 5-min incubation at 80 C. The assays tubes are then centrifuged and 100 l of the clarified supernatant is transferred into a clean vial. Commercial sodium acetate (Sigma-Aldrich) is used as reference. HPLC analyses are performed using a 1260 Inifinity LC System (Agilent), equipped with a refractometer detector and a column heating module. 10 l sample is separated on Hi-flex H column (3007.7 mm, 8 m particle size, column temp. 65 C.) equipped with a PL Hi-flex H Guard Column (507.7 mm). The mobile phase, consisting of aqueous sulfuric acid (5.5 mM) is run with a flow rate of 0.6 ml/min. Retention time of acetate under these conditions is 18.4 min.