CN114369633A - Preparation method of 6-allose phosphate, acetyl phosphate and acetyl coenzyme A - Google Patents

Preparation method of 6-allose phosphate, acetyl phosphate and acetyl coenzyme A Download PDF

Info

Publication number
CN114369633A
CN114369633A CN202111478556.8A CN202111478556A CN114369633A CN 114369633 A CN114369633 A CN 114369633A CN 202111478556 A CN202111478556 A CN 202111478556A CN 114369633 A CN114369633 A CN 114369633A
Authority
CN
China
Prior art keywords
phosphate
acetyl
allose
fructose
psicose
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202111478556.8A
Other languages
Chinese (zh)
Inventor
马红武
袁倩倩
毛雨丰
杨雪
成颖
罗家豪
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tianjin Institute of Industrial Biotechnology of CAS
Original Assignee
Tianjin Institute of Industrial Biotechnology of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tianjin Institute of Industrial Biotechnology of CAS filed Critical Tianjin Institute of Industrial Biotechnology of CAS
Priority to CN202111478556.8A priority Critical patent/CN114369633A/en
Publication of CN114369633A publication Critical patent/CN114369633A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P9/00—Preparation of organic compounds containing a metal or atom other than H, N, C, O, S or halogen
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01036—Acetoacetyl-CoA reductase (1.1.1.36)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01009—Acetyl-CoA C-acetyltransferase (2.3.1.9)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention discloses a method for further synthesizing acetyl coenzyme A by reacting glycolaldehyde with 4-erythrose phosphate under aldolase catalysis to generate 6-allose phosphate. The method has the advantages of high catalytic rate, high reaction efficiency and no carbon loss, the theoretical yield of carbon in the reaction route is 100 percent, and 4-erythrose phosphate, enzyme and coenzyme can be recycled. The method of the invention can play a more obvious advantage in the production process of in vitro continuous multi-enzyme catalysis, fed-batch fermentation, continuous fermentation and the like which can control the substrate level.

Description

Preparation method of 6-allose phosphate, acetyl phosphate and acetyl coenzyme A
Technical Field
The invention relates to the technical field of biotechnology and medicine, in particular to a method for synthesizing 6-allose phosphate, acetyl coenzyme A and derivatives thereof by using glycolaldehyde.
Background
Acetyl-coa (acacoa) is a key intermediate in the synthesis of essential biological compounds including polyketides, fatty acids, isoprenoids, alkaloids, vitamins, and amino acids, among others. Metabolites derived from acetyl-coa are primary and secondary metabolites, which include industrially useful compounds. Acetyl coenzyme A can be generated from acetyl phosphate (AcP) (for example, the acetyl coenzyme A can be obtained by catalyzing phosphate acetyltransferase Pta (EC 2.3.1.8)), and then a series of biological products taking acetyl coenzyme A as a platform are generated, so that the acetyl coenzyme A can be widely applied to the fields of biological catalysis and the like. The synthesis method of acetyl phosphate is, for example: patent application WO2015/144447a1 discloses a method for catalyzing formaldehyde to produce acetyl phosphate using phosphoketolase (EC 4.1.2.9, fructose-6-phosphate phosphoketolase EC4.1.2.22) or sulfoacetaldehyde acetyltransferase (EC 2.3.3.15), the reaction equation is as follows:
2CH2o + phosphoric acid → acetyl phosphoric acid + H2O。
The phosphoketolase used in the preparation reaction has poor affinity with formaldehyde, catalytic efficiency is not as high as one percent of that of the phosphoketolase used for converting the optimal substrate 5-xylulose phosphate, and microbial cells have generally poor tolerance to formaldehyde and have small application and expansion space.
WO2015/181074a1 discloses a method for producing D-erythrose and acetyl phosphate by catalyzing D-fructose with phosphoketolase (EC 4.1.2.9, fructose-6-phosphate phosphoketolase EC4.1.2.22), which further comprises converting D-erythrose into glycolaldehyde and then further into acetyl phosphate, wherein the reaction process is as follows:
Figure BDA0003394526780000021
the reaction rate of the preparation method of the acetyl phosphate is low, and the transformation number K of phosphoketolase to fructose iscatOnly 0.1/s, very low efficiency and long reaction times (18h) were challenging for enzyme and product stability. In addition, the reaction for cracking D-erythrose into glycolaldehyde in the third step is a reversible process, and the reaction rate is influenced by the concentration of the cracked glycolaldehyde, so that high-concentration glycolaldehyde is difficult to accumulate in a system, and the generation rate of acetyl phosphate is difficult to ensure. The acetyl phosphate formed by the second enzymatic reaction is likely to affect the yield of the final enzymatic reaction due to product inhibition, and if the acetyl phosphate formed by the second enzymatic reaction is removed immediately after the formation of acetyl phosphate, the operation of the whole production process is complicated.
Therefore, there is a need for a more efficient, simpler, and less costly process for preparing acetyl phosphate.
Disclosure of Invention
In order to overcome the defects of the prior art, a new algorithm comb-FBA [ PMID: 31491544], 15,225 pathway search calculations were performed with a network model containing 6566 MetaCyc known reactions as the main reaction set, with 28 theoretically feasible non-natural aldolase new reactions and 12 natural formaldehyde utilization reactions artificially designed as the combinatorial subset. Finally, 1 new synthesis way of acetyl phosphate/acetyl coenzyme A with short steps, no carbon loss and no carbon loss due to no consumption of reducing power is predicted and verified through practical experiments (figure 1).
In one aspect, the invention provides a method for preparing 6-allose phosphate (A6P), which comprises the reaction of glycolaldehyde and 4-erythrose phosphate under aldolase catalysis to generate the 6-allose phosphate. Aldolases can be derived from various microbial sources and artificially modified isozymes and mutant enzymes. Preferably, the aldolase is Escherichia coli-derived deoxyribose aldolase (EC 4.1.2.4, NP-418798.1).
Another aspect of the present invention provides a method for preparing acetyl phosphate, comprising the further step of converting the allose-6-phosphate prepared as described above into acetyl phosphate; preferably, the step of converting the allose 6-phosphate to fructose 6-phosphate (F6P) and further to acetyl phosphate is carried out.
Preferably, the step of converting the allose 6-phosphate into fructose 6-phosphate comprises the step of converting the allose 6-phosphate into psicose 6-phosphate (Au5P), and then further converting the psicose 6-phosphate into fructose 6-phosphate.
Preferably, the reaction for converting fructose-6-phosphate into acetyl phosphate comprises the step of converting fructose-6-phosphate and phosphoric acid into acetyl phosphate under the catalysis of phosphoketolase; more preferably, the phosphoketolase is fructose-6-phosphate phosphoketolase (EC 4.1.2.22) or phosphoketolase (EC 4.1.2.9); the phosphoketolase may be obtained through microbial expression, artificial synthesis and purification of various species, and especially fructose-6-phosphoketolase (BAF39468.1) derived from microbes, preferably from Bifidobacterium adolescentis strain, may exert good catalytic effect in the above reaction.
Preferably, the reaction system for converting fructose-6-phosphate into acetyl phosphate further comprises a coenzyme of phosphoketolase, such as thiamine pyrophosphate.
Preferably, the enzyme catalyzing the production of fructose-6-phosphate from allose-6-phosphate is a combination of allose-6-phosphate isomerase and psicose-6-phosphate 3-epimerase.
The stability of the acetyl phosphate is poor, and the acetyl phosphate is often made into the form of acetyl phosphate in practical application, and the invention provides a preparation method of the acetyl phosphate in another aspect, wherein the preparation method comprises the steps of the preparation method of the acetyl phosphate, wherein relevant metal ions are added into a buffer solution, so that the acetyl phosphate is specifically prepared into acetyl phosphate dilithium salt, acetyl phosphate disodium salt and acetyl phosphate diammonium salt.
Wherein the preparation method of acetyl coenzyme A comprises the step of acetyl phosphate; and a step of converting acetyl phosphate into acetyl-CoA by acetylphosphotransferase (Pta).
The present invention further provides a method for producing an acetyl-coa derivative compound, which comprises the above-mentioned steps of the method for producing acetyl-coa; further preparing the acetyl-CoA into an acetyl-CoA derivative compound, specifically selected from: acetone, isopropanol, acetic acid, L-glutamic acid, L-glutamine, L-proline, L-arginine, L-leucine, L-cysteine, succinate, and polyhydroxyalkanoates; more preferably, the acetyl-CoA derived compound is poly-3-hydroxybutyrate (PBH).
The invention further provides that the above method is carried out in a whole cell manner; preferably, it is carried out by whole cells of Escherichia coli; more specifically, it is prepared by introducing aldolase gene, and further, the gene synthesizing poly-3-hydroxybutyrate, more specifically PhaA, PhaB and PhaC into E.coli to prepare whole cells, and at the time, these genes are preferably controlled by, for example, IPTG inducible promoter.
The erythrose 4-phosphate described in the preparation method of the allose 6-phosphate, acetyl coenzyme A and acetyl coenzyme A derivative compound can be obtained by a commercial method or a method known in the prior art, such as the preparation method by using glucose, xylose, erythrose and the like, for example, erythrose can be converted into erythrose 4-phosphate under the action of glucokinase; the preparation reactant of 4-erythrose phosphate can be added into the reaction system of the preparation method to realize online preparation; the source of erythrose 4-phosphate is not intended to limit the scope of the present invention.
The glycolaldehyde in the preparation method of the allose 6-phosphate, the acetyl coenzyme A and the acetyl coenzyme A derivative compound can be prepared by methods known in the prior art, such as acetaldehyde halogenation reaction, saccharide cracking reaction and the like, wherein the cost for preparing the glycolaldehyde by using formaldehyde is low. Preferably, the glycolaldehyde is prepared by utilizing formaldehyde according to the prior art, preferably by utilizing the preparation method and the process of the glycolaldehyde (Xinkun, Liqingsong, Jiabing, and the like, research on the synthesis of the glycolaldehyde by the formaldehyde and the application progress thereof, natural gas chemical industry, C1 chemical industry, 2016 (41): 88-94) recorded in the research on the synthesis of the glycolaldehyde by the formaldehyde and the application progress thereof.
The invention also provides application of the microorganism expressing the aldolase or the isozyme and the mutant enzyme thereof in preparing 6-allose phosphate, acetyl coenzyme A and acetyl coenzyme A derivative compounds.
Finally, the present invention provides a recyclable reaction process using glycolaldehyde, characterized by comprising the steps of:
1) reacting glycolaldehyde with 4-erythrose phosphate under aldolase catalysis to generate 6-allose phosphate; preferably, the aldolase is Escherichia coli derived deoxyribose aldolase (EC 4.1.2.4); preferably, the catalytic reaction condition is 35-40 ℃ for 20-60 min; preferably, the glycolaldehyde is prepared from formaldehyde.
2) The allose-6-phosphate isomerase catalyzes the conversion of the allose-6-phosphate into the psicose-6-phosphate;
3) then the psicose-6-phosphate 3-epimerase converts the psicose-6-phosphate into fructose-6-phosphate; preferably, the method comprises the steps of converting the 6-psicose phosphate into the 6-psicose phosphate and further converting the 6-psicose phosphate into the 6-fructose phosphate; more preferably, by combination catalysis of a psicose-6-phosphate isomerase and a psicose-6-phosphate 3-epimerase.
4) Finally, converting fructose-6-phosphate into erythrose-4-phosphate and acetyl phosphate by using fructose-6-phosphate phosphoketolase;
5) repeating the step 1), thereby realizing the cyclic reaction.
In the preparation method of 6-allose phosphate, aldolase does not depend on coenzyme, and the catalytic efficiency is higher (V)maxMore than 60U/mg), and the aldolases are diverse in kind, and the optimization space is large. Catalytic substrate of fructose-6-phosphate phosphoketolase in the inventionIs fructose-6-phosphate as the most suitable substrate, and has high affinity and enzyme activity (K)mAbout 2-25mM, VmaxIs 120-1000U/mg), and as the last step of reaction, the catalytic process is irreversible, so that the system is not limited by the reaction balance, the aldolase speed can be pulled, the yield of the acetyl phosphate can be improved to the maximum extent, and the anaplerosis of the 4-phosphoerythrose can be promoted. The preparation method of the acetyl phosphate provided by the invention has the advantages that the catalytic rate is higher, the theoretical carbon yield of the reaction route is 100%, no carbon loss exists, both 4-erythrose phosphate and enzyme can be recycled, the reaction efficiency is higher, and the cost is reduced. In addition, the auxiliary enzymes allose-6-phosphate isomerase and psicose-6-phosphate 3-epimerase from the pentose phosphate pathway have strong affinity with the substrate, good stability and high catalytic activity. The method of the preparation method can play a more obvious advantage in the production process of feeding fermentation, continuous fermentation and the like which can control the substrate level.
Drawings
FIG. 1 is a schematic diagram of the biosynthetic pathway provided in example 2 of the present invention.
FIG. 2 shows the GC-MS detection results provided in example 4 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The biosynthetic pathway of acetyl phosphate using glycolaldehyde according to the embodiment of the present invention is shown in fig. 1, and the information of the reactants, intermediates and enzymes involved in the reaction process is detailed in tables 1 and 2.
TABLE 1 metabolite information Table referred to in the present invention
Figure BDA0003394526780000051
Figure BDA0003394526780000061
TABLE 2 enzyme information Table referred to in the present invention
Figure BDA0003394526780000062
EXAMPLE 1 preparation of 6-Phosphoallose
6-Phosphoallose was prepared by adding 2.5mM erythrose 4-phosphate to 10mM glycolaldehyde GALD at an initial concentration of 10mM, under the action of Escherichia coli-derived deoxyriboaldolase (EC 4.1.2.4, NP-418798.1), and reacting at 37 ℃ for 0.5 hour.
Detection of Au 6P: taking 50 mu L of reaction liquid, freeze-drying the reaction liquid, respectively deriving for 1h by utilizing 30 mu L of methoxylamine hydrochloride and 90 mu L of trimethylsilyl trifluoroacetamide, wherein the deriving temperature is 37 ℃, and detecting the content of 6-allose phosphate by utilizing GC-MS. The content of allose 6-phosphate detected quantitatively by GC-MS was 1.5mM, and the average synthesis rate of allose 6-phosphate was 50. mu. mol (allose 6-phosphate)/min/mg (enzyme protein).
Example 2 preparation of acetyl phosphate
Glycolaldehyde GALD at 10mM initial concentration, thiamine pyrophosphate ThDP concentration of 1mM, PO4 3+At a concentration of 2mM, fructose-6-phosphate phosphoketolase Fpk (EC 4.1.2.22) derived from Bifidobacterium adolescentis, deoxyribose aldolase DeoC (EC 4.1.2.4) derived from Escherichia coli, allose-6-phosphate isomerase RpiB (EC 5.3.1.6), psicose-6-phosphate 3-epimerase AlsE (EC 5.1.3.-) and 2.5mM erythrose 4-phosphate were added to carry out a reaction for producing acetyl phosphate, and the production of acetyl phosphate and the consumption of glycolaldehyde were examined.
Detection of acetyl phosphate: 40 mu L of reaction sample and hydroxylamine solution (2M, pH 6.5) are mixed uniformly and reacted for 10min, then trichloroacetic acid solution (15g/100mL), hydrochloric acid solution 1(4M) and ferric trichloride solution (5g/100mL hydrochloric acid solution 2, wherein the concentration of the hydrochloric acid solution 2 is 0.1M) are added into each 40 mu L, the mixture is centrifuged at 12000r/min for 5min to remove denatured enzyme, and the maximum absorption peak of the acetyl phosphate appears at 505 nm.
Acetyl phosphate was detected at a concentration of 5.60mM and a glycolaldehyde conversion rate of 31.1. mu. mol (glycolaldehyde)/min/mg (enzyme protein). The data above is the average rate of 3h of reaction.
Example 3 construction of recombinant bacterium for synthesizing Poly-3-hydroxybutyrate
The gene for synthesizing phosphoketolase was integrated into the genome of Escherichia coli K-12MG1655, and then a plasmid containing the gene for synthesizing poly-3-hydroxybutyrate (PHB) (PhaA, PhaB and PhaC) and the gene for aldolase DeoC was transferred into the above strain. The IPTG-inducible promoter (the remaining required enzymes, all already present in E.coli) was chosen for the above plasmid. Culturing the above strain in LB culture medium for 2.5 hr to obtain OD600When the value reached 0.8 to 1.0, IPTG was added to a final concentration of 0.5mM, and expression of the target enzyme protein on the above plasmid was induced (16 ℃ C., 12 h). The cells were collected by centrifugation (4 ℃ C., 8000r/min, 30min), suspended in M9 medium without carbon and nitrogen sources, and collected by centrifugation again, and the suspension and centrifugation were repeated three times to remove the residual carbon source from the cells. And (3) resuspending the finally collected thalli by using the M9 culture medium, dividing into 3 parts, adding 0.0g, 0.5g and 1.0g of glycolaldehyde (the thalli contains an intermediate metabolite, namely 4-erythrose phosphate, and no extra addition), uniformly metering to 30mL by using the M9 culture medium, fermenting for 20h, collecting the thalli, and detecting the content of the poly-3-hydroxybutyrate in the fermentation liquid.
Detection of poly-3-hydroxybutyrate: and centrifuging the fermentation liquor to obtain thalli, and freeze-drying and weighing the thalli to obtain 0.030g, 0.088g and 0.141g of dry weight of the thalli respectively. Deriving the lyophilized thallus powder for 4 hours at 100 ℃ by using 4mL of mixed solution of chloroform and an esterification solution (the main component is methanol) in a volume ratio of 1:1, then adding 2mL of ultrapure water, standing for layering, removing the methanol, cell debris and the like, taking a lower-layer chloroform solution of 3-hydroxybutyrate methyl ester, quantitatively detecting the 3-hydroxybutyrate methyl ester in the chloroform by using a gas chromatograph-mass spectrometer GC-MS, and comparing with a standard product to obtain that the total content of the 3-hydroxybutyrate in a sample is respectively 0.001g, 0.074g and 0.101g, and the carbon source conversion rate is between 10% and 20%. The result shows that the thalli can utilize glycolaldehyde and 4-erythrose phosphate contained in the thalli to synthesize acetyl phosphate and acetyl coenzyme A, and poly-3-hydroxybutyrate is continuously generated to be used for storing a carbon source of the thalli, the glycolaldehyde does not show obvious cytotoxicity in the fermentation process, and the normal metabolic consumption of the thalli can be maintained.
Example 4 comparison of different routes of preparation of acetyl-CoA
Acetyl phosphate and acetyl coenzyme A are prepared by different ways (namely four ways of E4P + GALD, E4P + GALD + DeoC, E4P + GALD + DeoC + RpiB + AlsE, E4P + GALD + DeoC + RpiB + AlsE + Fpk), reaction is carried out for 2h, the content of 6-allose phosphate and acetyl coenzyme A is detected after the reaction is finished, the content of acetyl coenzyme A can also indirectly represent the synthesis amount of acetyl phosphate, and reactants, enzymes and the addition amount thereof are shown in Table 3.
Detection of Au 6P: taking 50 mu L of reaction liquid, freeze-drying the reaction liquid, respectively deriving for 1h by utilizing 30 mu L of methoxylamine hydrochloride and 90 mu L of trimethylsilyl trifluoroacetamide, wherein the deriving temperature is 37 ℃, and detecting the content of 6-allose phosphate by utilizing GC-MS.
Detection of acetyl-CoA: after the reaction was terminated with a 10% sulfuric acid solution in 50. mu.L of the reaction mixture, impurities were removed with a 0.22 μm filter, and then liquid phase detection was carried out, wherein mobile phase A was a 0.2M, pH ═ 5 sodium dihydrogenphosphate solution, mobile phase B was acetonitrile, and the maximum absorption peak of acetyl-CoA appeared at 254 nm.
The results of the product content measurements are shown in Table 3. The results in Table 3 show that, when no aldolase was added to the reaction system, no allose-6-phosphate was produced, the amount of acetyl-CoA produced was very small, and the reaction conversion rate was very low; after aldolase and 4-erythrose phosphate are added into the reaction system, 6-allose phosphate can be generated, and the reaction conversion rate is high; after RpiB and AlsE are added simultaneously, the generated 6-allose phosphate is converted into acetyl phosphate under the action of RpiA, Rpe and Fpk, and is further converted into acetyl coenzyme A by phosphate acetyltransferase, and the reaction conversion rate is very high as can be seen from the detection results of Au5P and acetyl coenzyme A.
TABLE 3 substance addition and metabolite formation tables of the system (concentration unit of reactant or product: mM; amount unit of enzyme: μ g)
Figure BDA0003394526780000081
Remarking: the buffer solution contains Tris, NaCl and MgCl2Three major components, pH 7.5, 37 ℃.
In addition, in the same reaction system containing reactants such as glycolaldehyde GALD, phosphate ion Phos, erythrose 4-phosphate, thiamine pyrophosphate ThDP and the like, respectively: a: no aldolase added (black line); b: adding DeoC (red line); c: addition of the aldolase DeoC, the allose-6-phosphate isomerase RpiB and the psicose-6-phosphate 3-epimerase AlsE (blue line); d: DeoC, RpiB, AlsE and Fpk (green line) were added. The GC-MS test shows the content of allose 6-phosphate, and the results are shown in FIG. 2 (peak time of allose 6-phosphate is 26.06 min). As can be seen from the graphs in FIG. 2 and the corresponding reaction conditions, allose 6-phosphate was produced only after addition of the aldolase DeoC; the generated 6-phosphoric acid allose is decomposed more completely only after adding DeoC, RpiB, AlsE and Fpk at the same time.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and the like that are within the spirit and principle of the present invention are included in the present invention.

Claims (9)

1. A preparation method of 6-allose phosphate is characterized in that glycolaldehyde and 4-erythrose phosphate are reacted to generate the 6-allose phosphate under the catalysis of aldolase.
2. The method of claim 1, wherein the aldolase is Escherichia coliEscherichia coliA source of deoxyribose aldolase; preferably, the catalytic reaction condition is 35-40 ℃ for 20-60 min; preferably, the glycolaldehyde is prepared from formaldehyde.
3. The preparation method of the acetyl phosphate is characterized by comprising the following steps:
a step of obtaining allose 6-phosphate according to the method of any of claims 1 to 2;
a step of converting the psicose-6-phosphate into psicose-6-phosphate, converting the psicose-6-phosphate into fructose-6-phosphate, and finally converting the fructose-6-phosphate into acetyl phosphate.
4. The method of claim 3, wherein the step of converting the allose 6-phosphate to fructose 6-phosphate comprises the step of converting the allose 6-phosphate to psicose 6-phosphate and further to fructose 6-phosphate; the step of converting fructose-6-phosphate into acetyl phosphate is realized by converting fructose-6-phosphate and phosphate into acetyl phosphate under the catalysis of phosphoketolase.
5. The method of claim 4, wherein the step of converting the allose 6-phosphate to fructose 6-phosphate is catalyzed by a combination of allose-6-phosphate isomerase and psicose-6-phosphate 3-epimerase.
6. The method of claim 5, wherein the phosphoketolase is fructose-6-phosphate phosphoketolase or phosphoketolase, preferably of microbial origin such as more preferably of microbial originBifidobacterium adolescentisFructose-6-phosphate phosphoketolase; more preferably, a coenzyme of phosphoketolase, such as thiamine pyrophosphate, is further added to the reaction system.
7. The preparation method of acetyl coenzyme A is characterized by comprising the following steps:
a step of preparing acetyl phosphate by the method according to any one of claims 3 to 6;
a step of converting acetyl phosphate into acetyl-CoA by a phosphate acetyltransferase; preferably, the enzyme and the starting material are added simultaneously in the reaction;
optionally, said acetyl-coa is further used in the preparation of acetyl-coa derived compounds.
8. The method according to any one of claims 1 to 7, wherein it is carried out in a whole-cell manner; preferably, it is carried out by whole cells of Escherichia coli; more specifically, it is prepared by introducing aldolase gene, and further, a gene for synthesizing poly-3-hydroxybutyrate, more specifically PhaA, PhaB and PhaC, into E.coli to prepare whole cells, and preferably these genes are controlled by, for example, IPTG inducible promoter.
9. A process for utilizing glycolaldehyde by a cyclic reaction pathway, characterized by comprising the steps of:
1) reacting glycolaldehyde with 4-erythrose phosphate under aldolase catalysis to generate 6-allose phosphate; preferably, the aldolase is Escherichia coliEscherichia coliA source of deoxyribose aldolase; preferably, the catalytic reaction condition is 35-40 ℃ for 20-60 min; preferably, the glycolaldehyde is prepared from formaldehyde;
2) the allose-6-phosphate isomerase catalyzes the conversion of the allose-6-phosphate into the psicose-6-phosphate;
3) then the psicose-6-phosphate 3-epimerase converts the psicose-6-phosphate into fructose-6-phosphate; preferably, the method comprises the steps of converting the 6-psicose phosphate into the 6-psicose phosphate and further converting the 6-psicose phosphate into the 6-fructose phosphate; more preferably, by a combination of allose-6-phosphate isomerase and psicose-6-phosphate 3-epimerase catalysis;
4) finally, converting fructose-6-phosphate into acetyl phosphate and erythrose-4-phosphate by fructose-6-phosphate phosphoketolase;
5) repeating the reaction in the step 1) to realize circulation.
CN202111478556.8A 2021-12-06 2021-12-06 Preparation method of 6-allose phosphate, acetyl phosphate and acetyl coenzyme A Pending CN114369633A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111478556.8A CN114369633A (en) 2021-12-06 2021-12-06 Preparation method of 6-allose phosphate, acetyl phosphate and acetyl coenzyme A

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111478556.8A CN114369633A (en) 2021-12-06 2021-12-06 Preparation method of 6-allose phosphate, acetyl phosphate and acetyl coenzyme A

Publications (1)

Publication Number Publication Date
CN114369633A true CN114369633A (en) 2022-04-19

Family

ID=81140058

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111478556.8A Pending CN114369633A (en) 2021-12-06 2021-12-06 Preparation method of 6-allose phosphate, acetyl phosphate and acetyl coenzyme A

Country Status (1)

Country Link
CN (1) CN114369633A (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106755172A (en) * 2016-12-16 2017-05-31 中国科学院天津工业生物技术研究所 Using glycolaldehyde synthesis of acetyl coacetylase and its new way of derived product

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106755172A (en) * 2016-12-16 2017-05-31 中国科学院天津工业生物技术研究所 Using glycolaldehyde synthesis of acetyl coacetylase and its new way of derived product

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YUFENG MAO ET AL: "Non-natural Aldol Reactions Enable the Design and Construction of Novel One-Carbon Assimilation Pathways in vitro", 《FRONTIERS IN MICROBIOLOGY》, vol. 12, 2 June 2021 (2021-06-02), pages 8 - 9 *

Similar Documents

Publication Publication Date Title
KR101814648B1 (en) Microorganisms and methods for the biosynthsis of butadiene
US20210332393A1 (en) Microorganisms and methods for improving product yields on methanol using acetyl-coa synthesis
Derrington et al. Carboxylic acid reductases (CARs): An industrial perspective
Tang et al. Metabolic engineering of Cupriavidus necator H16 for improved chemoautotrophic growth and PHB production under oxygen-limiting conditions
US11371063B2 (en) Microorganisms and methods for the production of butadiene using acetyl-coA
CN106755172B (en) New way for synthesizing acetyl coenzyme A and derivative products thereof by using glycolaldehyde
US20230136909A1 (en) Microorganisms and methods for enhancing the availability of reducing equivalents in the presence of methanol, and for producing 3-hydroxyisobutyrate or methacrylic acid related thereto
KR20140145935A (en) Microorganisms and methods for the biosynthsis of butadiene
CN112877272B (en) Escherichia coli engineering bacteria of N-acetylglucosamine and fermentation production method
KR20120123742A (en) Microorganisms and methods for the biosynthesis of p-toluate and terephthalate
Zhang et al. Bioconversion of methanol into value-added chemicals in native and synthetic methylotrophs
EP2971021A2 (en) Microorganisms and methods for producing butadiene and related compounds by formate assimilation
US20190017078A1 (en) Microorganisms and methods for producing butadiene and related compounds by formate assimilation
Bonk et al. Rational design of thiolase substrate specificity for metabolic engineering applications
Li et al. Enhancing the glucose flux of an engineered EP-Bifido pathway for high poly (hydroxybutyrate) yield production
US20210079334A1 (en) Fermentation systems and methods with substantially uniform volumetric uptake rate of a reactive gaseous component
EP3901256A1 (en) Optimized production of cbga from olivetol acid and geranyl pyrophosphate via synnphb
CN114901815A (en) Microorganisms and methods for increasing cofactors
CN114369633A (en) Preparation method of 6-allose phosphate, acetyl phosphate and acetyl coenzyme A
US20230407350A1 (en) Microorganisms capable of producing poly(hiba) from feedstock
EP3601547A1 (en) 3-hydroxybutyryl-coa dehydrogenase variants and methods of use
Keasling et al. Engineering controllable alteration of malonyl-CoA levels to enhance polyketide production and versatility in E. coli
CN116376947A (en) Method suitable for screening bacterial strain with high yield of non-protein amino acid
NZ621318A (en) Microorganisms and methods for producing 1,3-butanediol and related alcohols
NZ621318B2 (en) Microorganisms and methods for producing 1,3-butanediol and related alcohols

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination