CN115404192A - Engineering bacterium for synthesizing 5-amino-1-pentanol and 1,5-pentanediol and application - Google Patents

Engineering bacterium for synthesizing 5-amino-1-pentanol and 1,5-pentanediol and application Download PDF

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CN115404192A
CN115404192A CN202110578901.9A CN202110578901A CN115404192A CN 115404192 A CN115404192 A CN 115404192A CN 202110578901 A CN202110578901 A CN 202110578901A CN 115404192 A CN115404192 A CN 115404192A
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pdo
transaminase
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袁其朋
孙新晓
马琳
李文娜
申晓林
王佳
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Beijing University of Chemical Technology
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Abstract

The invention provides an engineering bacterium for synthesizing 5-AMP, which comprises a host bacterium and a plasmid vector transferred into the host bacterium, wherein a gene for coding alcohol dehydrogenase is introduced into the plasmid vector, so that 5-aminopentanal generates 5-AMP; wherein the alcohol dehydrogenase is Adh6, yqhD, yahK or YjgB. Genes coding for putrescine aminotransferase PatA and lysine decarboxylase CadA, and genes coding for enzymes DapA and LysC enhancing the diaminopimelate pathway are also introduced into the plasmid vector. The invention also provides an engineering bacterium for synthesizing 1,5-PDO, which is equivalent to that a gene coding transaminase is introduced into the engineering bacterium for synthesizing 5-AMP so as to catalyze 5-AMP to generate 5-hydroxypentanal, wherein the transaminase is CV2025 or WTA. The invention also provides an application of the engineering bacteria in the synthesis of 5-AMP or 1,5-PDO.

Description

Engineering bacterium for synthesizing 5-amino-1-pentanol and 1,5-pentanediol and application
Technical Field
The invention relates to the technical field of biological engineering, in particular to an engineering bacterium for synthesizing 5-amino-1-pentanol and 1,5-pentanediol and application thereof.
Background
5-Amino-1-pentanol (5-Amino-1-pentanol), abbreviated to 5-AMP, of formula C 5 H 14 NO, molecular weight 104.1702, is an important bifunctional compound, is a white soluble crystal, is a saccharification and ammoniation intermediate of many anticancer and anti-inflammatory drugs, and is widely applied to synthesis of medicines and pesticides. At present, 5-AMP is mainly used as a starting material for synthesizing an alkaloid Manzamine A with high medicinal value. The demand for 5-AMP is increasing as the demand for alkaloids in the pharmaceutical industry is increasing due to the anti-inflammatory, anti-HIV-1, anti-cervical, antifungal and antimalarial activity of Manzamine A.
There are many methods for chemically synthesizing aminoalcohols, and the methods generally used include a halohydrin amino substitution method, an amino acid reduction method, a grignard reagent addition method, and the like. However, the existing chemical synthesis method has the problems that the raw materials are expensive and not easy to obtain or the economic efficiency of generating a large amount of waste is not high, the sustainable development is not facilitated, and the like. For example, one existing method for synthesizing 5-AMP is mainly to synthesize 5-AMP from biomass-derived dihydropyrane by in situ generation of coupling 5-hydroxypentanal over a supported Ni catalyst and reductive amination thereof; the method can obtain 5-AMP with a yield of 82%. However, this method requires high temperature and high pressure, which is not suitable for sustainable development. No one currently produces 5-AMP by a biological synthesis method, so that the realization of green, high-efficiency and sustainable production of 5-AMP has important significance.
1,5-pentanediol (1, 5-pentandiol), abbreviated to 1,5-PDO, is a colorless viscous liquid with bitter taste, formula C 5 H 12 O 2 And the molecular weight is 104.15, the paint can be mixed and dissolved with water, low molecular alcohol and acetone, and is insoluble to benzene, dichloromethane and petroleum ether. Unlike even-numbered diols, 1,5-PDO has longer molecular chain and thus stronger chain flexibility, so that the polyester which participates in the synthesis has different properties in heat, force and crystallinity from the polyester synthesized by glycol, butanediol, hexanediol and the like. 1,5-PDO is a raw material for synthetic polyester, polyurethane, ink, paint, plastic, fiber, binder, plasticizer, and the like.
1,5-PDO is currently produced commercially mainly from petroleum feedstocks, mainly from the hydrogenation reduction of glutaric acid, the high reaction pressures, the complex by-products and the corrosion of the equipment by concentrated hydrochloric acid, and furthermore glutaric acid is not cheap and widely available. In recent years, 1,5-PDO with 86% yield can be obtained by three steps of dehydration-hydration-hydrogenation (DHH) reaction of biomass furfural raw material. Although furfural is cheap and easy to obtain, a noble metal catalyst is needed for reaction, the reaction conditions relate to high temperature and high pressure, and the consumption of energy is large, so that the long-term sustainable development is not facilitated. Therefore, the synthesis of 1,5-PDO by biological method using cheap Glucose as carbon source is an important direction of current research, and the pathway for synthesizing 1,5-PDO by biological method as shown in FIG. 1 (Wang J, li C, zou Y, et al. Bacterial synthesis of C3-C5 diode via extension amino acid catalysis [ J ]; cen X, liu Y, chen B, et al. Metabolism Engineering of Escherichia coli for De Novo Production of 1, 5-pentaneedfor Glucose [ J ]), and it can be seen from FIG. 1 that the pathway for synthesizing 1,5-PDO is relatively long and needs to consume a large amount of NADPH and ATP, etc. Since 1,5-PDO is a highly reducing compound, the synthetic process consumes a large amount of reducing power, and therefore, it is important to find a more energy-saving biosynthetic pathway.
Disclosure of Invention
In view of the above, it is an object of the present invention to select an enzyme having catalytic efficiency in vitro from a large number of organisms or microorganisms capable of catalyzing 5-aminopentanal to 5-AMP, thereby achieving biosynthesis of 5-AMP.
Another objective of the present invention is to screen out enzymes having catalytic efficiency in vitro from enzymes catalyzing 5-AMP to produce 5-hydroxypentanal in a large number of organisms or microorganisms in vivo, to achieve efficient biosynthesis of 1,5-PDO, shorten 1,5-PDO biosynthesis pathway, achieve energy saving, and have high yield.
In order to achieve the aim, the invention provides an engineering bacterium for synthesizing 5-AMP, which comprises a host bacterium and a plasmid vector transferred into the host bacterium, wherein a gene for coding alcohol dehydrogenase is introduced into the plasmid vector, so that 5-aminopentanal generates 5-AMP; and the alcohol dehydrogenase is any one of Adh6, yqhD, yahK and YjgB or alcohol dehydrogenase with 60% or more of amino acid sequence similarity of any enzyme, wherein Adh6 is NADP-dependent alcohol dehydrogenase derived from saccharomyces cerevisiae, and YqhD, yahK and YjgB are NADPH-dependent aldehyde reductase derived from escherichia coli. Preferably, the amino acid sequence of YjgB is shown in SEQ ID NO. 1.
Wherein the host bacterium is a bacterium, a yeast or a fungus, wherein the bacterium or fungus is original or modified. Preferably, the host bacterium is escherichia coli, bacillus subtilis, corynebacterium glutamicum, saccharomyces cerevisiae or aspergillus niger.
Based on the engineering bacteria for synthesizing 5-AMP, a gene for coding putrescine aminotransferase PatA is also introduced into the plasmid vector to catalyze the pentanediamine to generate 5-aminopentanal. That is, pentanediamine synthesizes 5-AMP under the co-catalysis of putrescine transaminase PatA and the alcohol dehydrogenase.
Based on the engineering bacteria for synthesizing 5-AMP, a gene coding lysine decarboxylase CadA is also introduced into the plasmid vector to catalyze lysine to generate pentanediamine. That is, lysine synthesizes 5-AMP under the co-catalysis of lysine decarboxylase CadA, putrescine transaminase PatA and the alcohol dehydrogenase.
Among them, lysine decarboxylase CadA and putrescine transaminase PatA are derived from original or modified bacteria.
Based on the engineering bacteria for synthesizing 5-AMP, genes coding enzymes DapA and LysC for enhancing the diaminopimelate pathway are also introduced into the plasmid vector. Thus, 5-AMP was synthesized from a simple carbon source such as glucose under the co-catalysis of DapA, lysC, lysine decarboxylase CadA, putrescine transaminase PatA, and the alcohol dehydrogenase.
The invention also provides application of the engineering bacteria for synthesizing 5-AMP in synthesizing 5-AMP.
The invention provides an engineering bacterium for synthesizing 1,5-PDO, which comprises a host bacterium and a plasmid vector transferred into the host bacterium, wherein a gene for coding transaminase is transferred into the plasmid vector to catalyze 5-AMP to generate 5-hydroxypentanal, and the transaminase is transaminase CV2025 derived from violet bacillus or transaminase WTA derived from pseudomonas aeruginosa or the transaminase of which the amino acid sequence similarity of any enzyme is 60 percent or more. Wherein, the amino acid sequence of WTA is shown in SEQ ID NO. 2.
Based on the engineering bacteria for synthesizing 1,5-PDO, alcohol dehydrogenase ADH is also introduced into the plasmid vector, so that 5-hydroxypentanal is synthesized into 1,5-PDO. That is, 5-AMP is converted to 1,5-PDO under the combined action of the transaminase CV2025 or WTA, and the alcohol dehydrogenase ADH.
Wherein a gene encoding an enzyme that synthesizes 5-AMP is further introduced into the plasmid vector. The enzymes for synthesizing the 5-AMP comprise lysine decarboxylase CadA, putrescine transaminase PatA and alcohol dehydrogenase catalyzing 5-aminopentanal to generate the 5-AMP, wherein the alcohol dehydrogenase is any one of Adh6, yqhD, yahK and YjgB or alcohol dehydrogenase with the amino acid sequence similarity of any one of Adh6, yqhD, yahK and YjgB of 60% or more. The lysine decarboxylase CadA and putrescine transaminase PatA are mainly used for catalyzing lysine to generate 5-aminopentanal.
Based on the above engineered bacteria synthesizing 1,5-PDO, genes encoding enzymes DapA and LysC enhancing the diaminopimelate pathway were also introduced into the plasmid vector. Thus, a simple carbon source such as glucose is synthesized into 1,5-PDO under the co-catalysis of DapA, lysC, lysine decarboxylase CadA, putrescine transaminase PatA, the alcohol dehydrogenase, and the transaminase.
The invention also provides application of the engineering bacteria for synthesizing 1,5-PDO in synthesizing 1,5-PDO.
Referring to FIG. 2, the engineering bacteria for synthesizing 5-AMP provided by the present invention are mainly fermented and cultured in a medium containing simple carbon sources such as glucose, and the main reason why 5-AMP can be synthesized is that: the simple carbon source can be firstly converted into lysine through the metabolism of engineering bacteria for synthesizing 5-AMP, the lysine is catalyzed by lysine decarboxylase CadA to synthesize pentanediamine, the pentanediamine is converted into 5-hydroxypentanal under the action of putrescine transaminase PatA, and the 5-hydroxypentanal is converted into 5-AMP under the action of alcohol dehydrogenase.
5-AMP is converted into 5-hydroxypentanal under the action of the transaminase, and the 5-hydroxypentanal is converted into 1,5-PDO under the action of the metabolism of engineering bacteria.
Therefore, the engineering bacteria for synthesizing 5-AMP provided by the invention can be used for biologically synthesizing 5-AMP from pentanediamine, lysine, glucose and the like as sources, and the synthesis efficiency is high. The test proves that: 5-AMP is synthesized and cultured in a culture medium containing 1g/L of pentanediamine by fermentation, and 910 mg/L of 5-AMP can be detected by high performance liquid chromatography; the yield of 5-AMP synthesized by using lysine or simple carbon source as a source and using the engineering bacteria for synthesizing 5-AMP can reach 1448mg/L and 1900 mg/L respectively.
The engineering bacteria for synthesizing 1,5-PDO provided by the invention can be used for respectively biosynthesizing 1,5-PDO by taking 5-AMP, lysine or glucose and the like as sources, and has short synthesis path and high efficiency. The test proves that: the engineering bacteria for synthesizing the 1,5-PDO are subjected to fermentation culture in a culture medium containing 1g/L of 5-AMP, and the 1,5-PDO of 800 mg/L can be detected by high performance liquid chromatography; the yield of the 1,5-PDO synthesized by using the engineering bacteria for synthesizing the 1,5-PDO by taking lysine or a simple carbon source as a source can reach 790mg/L and 1019 mg/L respectively.
Therefore, the engineering bacteria provided by the invention can be used for realizing the high-efficiency biosynthesis of 5-AMP and 1,5-PDO respectively.
Drawings
FIG. 1 is a diagram showing the pathway for synthesizing 1,5-PDO by a conventional biological method.
FIG. 2 is a diagram of the biosynthetic pathways provided by the present invention for de novo synthesis and lysine addition for the production of 5-AMP and 1,5-PDO.
FIG. 3 is a graph showing the results of fermentation in the present invention, wherein 5-AMP is synthesized from the engineering bacteria ML1, ML2, ML3, ML4 and ML5 in combination with pentamethylenediamine.
FIG. 4 is a HPLC detection chart of 5-AMP standards.
FIG. 5 is a HPLC detection chart of a fermentation target product for synthesizing 5-AMP by using an engineering bacterium ML5 and combining pentanediamine in an embodiment of the invention.
FIG. 6 is a graph showing the results of fermentation in the present invention, which uses the combination of the engineering bacteria ML6 and ML7 and 5-AMP to produce 1,5-PDO.
FIG. 7 is an HPLC check of 1,5-PDO standard.
FIG. 8 is a HPLC chromatogram of a fermentation target for the production of 1,5-PDO using an engineered bacterium ML7 in combination with 5-AMP in an example of the present invention.
FIG. 9 is a graph showing the results of fermentation in the present example using engineered bacteria ML8 and ML9 to produce 5-AMP and 1,5-PDO by in vitro lysine addition.
FIG. 10 is a HPLC detection chart of a fermentation target product for producing 5-AMP by in vitro lysine addition using an engineered bacterium ML8 in an embodiment of the present invention.
FIG. 11 is a HPLC detection chart of fermentation target products for producing 1,5-PDO by in vitro lysine addition using the engineering bacteria ML9 in the embodiment of the present invention.
FIG. 12 is a graph showing the results of fermentation for the first synthesis of 5-AMP and 1,5-PDO from glucose using the engineered bacteria ML10 and ML10 in the example of the present invention.
FIG. 13 is a HPLC detection chart of a fermentation target product for first synthesizing 5-AMP from glucose using the engineering bacterium ML10 in the example of the present invention.
FIG. 14 is a HPLC detection chart of a fermentation target product for first synthesizing 1,5-PDO from glucose by using an engineering bacterium ML11 in the embodiment of the invention.
In the sequence listing：
SEQ ID NO.1 is the amino acid sequence of a reductase YjgB derived from Escherichia coli.
SEQ ID No.2 is the amino acid sequence of the transaminase WTA derived from Pseudomonas aeruginosa.
Detailed Description
The technical solution of the present invention is further described in detail by the following embodiments.
In the present invention, there is no special requirement for the type of expression plasmid, and it is considered that various methods commonly used in the art can be adopted as the construction method for expressing the target gene in escherichia coli, for example, the target gene is linked to the vector after enzyme digestion treatment, and no further description is given. That is, unless otherwise specified, technical means used in the following examples are conventional means well known to those skilled in the art.
In the following examples, the E.coli strains Trans5 alpha and BW25113 are all common E.coli strains which are commercially available, wherein Trans5 alpha is used for vector construction, and BW25113 is used as a strain for fermentation; plasmid pZE12-luc is the same as that used by Lin et al (Lin Y, sun X, yuan Q, et al. Extension shikimate pathway for the production of pathological acid and its precursor cosmetic acid in Escherichia coli [ J ]. Metabolic Engineering, 2014, 23; plasmids pCS27 and pSA74 are identical to those employed by Shen et al (Shen X, mahajani M, wang J, et al. Elevating 4-hydroxyoumarin production through induced hydrolysis-mediated salicyl-CoA degradation [ J ]. Metabolic Engineering, 2017); the components of the LB culture medium are 10 g/L NaCl, 10 g/L peptone and 5 g/L yeast powder; 2% agar needs to be added into the solid LB culture medium; LB ampicillin-resistant Medium an additional ampicillin resistance was added to the LB medium.
Example 1 construction of 5-AMP-synthesizing engineering bacteria and Synthesis of 5-AMP
Construction of engineering bacteria for synthesizing 5-AMP
Respectively selecting alcohol dehydrogenases YqhD, yahK and YjgB from escherichia coli and alcohol dehydrogenase Adh6 from saccharomyces cerevisiae as enzymes for catalyzing 5-aminopentanal to synthesize 5-AMP. Wherein the amino acid sequence of YjgB is shown in SEQ ID NO. 1.
Putrescine transaminase PatA was chosen as the enzyme catalyzing the synthesis of 5-aminopentanal from pentanediamine.
The lysine decarboxylase CadA was chosen as the enzyme catalyzing lysine to pentanediamine.
Constructing engineering bacteria: the gene of the enzyme is coded, gene segments are obtained by PCR, then the segments and the carrier are subjected to double enzyme digestion by endonuclease, the fragments after enzyme digestion are subjected to gel recovery, and then the fragments are respectively connected with the medium copy plasmid pCS-patA. Plasmids pCS-patA-yqhD, pCS-patA-yahK, pCS-patA-yjgB, and pCS-patA-adh6 were obtained. Meanwhile, a recombinant plasmid pSA-potE containing the pentanediamine transporter PotE was constructed as shown in Table 1.
Competent cells were prepared by electroporation and dispensed 90 μ L into 1.5mL EP tubes for transformation. The constructed recombinant plasmids pCS-patA, pCS-patA-yqhD, pCS-patA-yahK, pCS-patA-yjgB or pCS-patA-adh6 were added to 1.5mL of EP tubes containing 90. Mu.L of competent cells, respectively, and mixed well. The plasmid is then electrotransferred into competent cells using an electrotransfer instrument. After the electroporation was completed, LB medium was added and the mixture was transferred to a 1.5mL EP tube and allowed to resuscitate for about 45 min. Then, the bacterial suspension was applied to a plate containing antibiotics and cultured overnight at 37 ℃. Preparation of 5-AMP-producing Strain ML1: BW (pSA-potE, pCS-patA). Electrotransformation was performed in the same way to obtain the strain ML2: BW (pSA-potE, pCS-patA-adh 6), ML3: BW (pSA-potE, pCS-patA-yqhD), ML4: BW (pSA-potE, pCS-patA-yahK), ML5: BW (pSA-potE, pCS-patA-yjgB).
Application of engineering bacteria for synthesizing 5-AMP: 5-AMP is produced by utilizing the engineering bacteria ML1, ML2, ML3, ML4 and ML5 to combine with pentanediamine for fermentation culture, single colonies are respectively picked on plates of the strains ML1, ML2, ML3, ML4 and ML5 for producing the 5-AMP, the single colonies are inoculated into 4mL of liquid LB culture medium with resistance, the liquid culture is cultured for 8 to 12 hours at 37 ℃, then the bacterial liquid is respectively transferred into 50mL of fermentation culture medium M9 with resistance, one thousandth of inducer IPTG is added for induction, and 1g/L of pentanediamine is added, wherein the fermentation culture medium M9 comprises: 2 g/L MOPS, 10 g/L glucose, 2 g/L yeast powder and 6.78 g/L Na 2 HPO 4 、0.5 g/L NaCl、3.0 g/L/ KH 2 PO 4 And 1.0g/L NH 4 Cl and adding corresponding antibiotics according to actual conditions. Then sampling at 12h, 24 h,48 h and 72h respectively and determining the intermediate product and the target product by high performance liquid chromatographyThe concentration, final yield are shown in FIG. 3. As can be seen in fig. 3: the ML5 strain produced a maximum of about 910 mg/L of 5-AMP.
Identifying the target product fermented and cultured by using the engineering bacteria ML5 and the pentamethylene diamine
Derivatization reagent: the first reagent was triethylamine-acetonitrile solution, 1.4 mL of triethylamine solution was pipetted into a 15 mL centrifuge tube (operating in a fume hood) using a pipette gun, 8.6 mL of acetonitrile was pipetted and added, and the mixture was vortexed uniformly and stored in a 4 ℃ refrigerator. The second reagent was phenylisothiocyanate solution, 135.18 mg of phenylisothiocyanate-acetonitrile solution was weighed into a 15 mL centrifuge tube (operating in a fume hood) using a fine balance, 10 mL of acetonitrile was aspirated by a pipette gun and added thereto, and the mixture was vortexed uniformly and stored in a 4 ℃ refrigerator.
The derivatization method comprises the following steps: 200 mu L of the supernatant of the sample to be detected is respectively taken. Adding 100 mu L of reagent 1 triethylamine-acetonitrile solution for derivatization catalysis, adding 100 mu L of reagent 2 phenyl isothiocyanate-acetonitrile solution for reaction, oscillating and mixing uniformly after adding, and standing for reaction for 1 h to fully perform derivatization reaction. And after the reaction is finished, adding 200 mu L of n-hexane to terminate the reaction, extracting and separating, fully shaking and uniformly mixing, and then centrifuging. And (3) placing a sample to be tested in the middle lower layer of the layered liquid, sucking the lower layer liquid by using a 1 mL disposable syringe, injecting the liquid into a small liquid-phase catheter after the liquid passes through a membrane, placing the small liquid-phase catheter in a small liquid-phase bottle, covering a small liquid-phase bottle cover, marking and performing HPLC analysis for later use.
Detecting the sample to be detected by adopting an HPLC analysis method, wherein the detection conditions are as follows:
and (3) chromatographic column: separating the column: diamonsil C18, ID 5 μm, 250X 4.6 mm, detection wavelength 254 nm;
mobile phase: a is methanol, B is 0.1 formic acid aqueous solution, the column temperature is 35 ℃;
the elution method comprises the following steps: the following were used at a flow rate of 1 mL/min: 20% to 40% solvent A for 10 min,40% to 80% solvent A for 10 min,80% solvent A for 5min, 80% to 20% solvent A for 2 min, and finally 20% solvent A for 3 min.
5-AMP standard solutions are prepared, the concentrations are respectively 0mg/L, 25 mg/L, 50 mg/L, 100 mg/L and 200 mg/L, and the 5-AMP standard solutions are subjected to derivatization treatment and HPLC detection, and figure 4 is an HPLC chart of the 100 mg/L5-AMP standard. FIG. 5 is an HPLC chart of fermentation products of respective fermentation cultures of recombinant Escherichia coli ML5 in combination with pentamethylenediamine. As can be seen from the figure: the peak-off time of the standard product shown in fig. 4 is about 18.24min, and the peak-off time of fig. 5 is also about 18.24min, so that the characteristic peaks are proved: the recombinant E.coli provided in this example can produce 5-AMP by fermentation with pentanediamine.
Example 2 construction of 5-hydroxy valeraldehyde and its application.
Selecting a transaminase CV2025 derived from the violet bacteria or a transaminase WTA derived from the pseudomonas aeruginosa as an enzyme catalyzing 5-AMP to generate 5-hydroxypentanal, wherein the amino acid sequence of the transaminase WTA is shown in SEQ ID NO. 2.
Construction of engineering bacteria: the gene coding the enzyme is subjected to PCR to obtain gene fragments, then the fragments and the vector are subjected to double digestion by endonuclease, the digested fragments are subjected to gel recovery, and then the target genes are respectively inserted into the plasmid pCS 27. Plasmids pCS-cv2025 and pCS-wtA were obtained, respectively.
Competent cells were prepared by electroporation and dispensed 90 μ L into 1.5mL EP tubes for transformation. The constructed recombinant plasmids pCS-cv2025, pCS-wtA were used. mu.L of each was added to a 1.5mL EP tube containing 90. Mu.L of competent cells, and mixed well. The plasmid is then electrotransferred into competent cells using an electrotransfer instrument. After the electroporation was completed, LB medium was added and the mixture was transferred to a 1.5mL EP tube and allowed to resuscitate for about 45 min. Then, the bacterial suspension was applied to a plate containing antibiotics and cultured overnight at 37 ℃. ML6: BW (pCS-cv 2025), ML7: BW (pCS-wtA).
The application comprises the following steps: respectively using the engineering bacteria ML6 and ML7 to combine with 5-AMP to carry out microbial fermentation to produce 1,5-PDO
Picking single colonies from plates of strains ML6 and ML7 for producing 5-hydroxypentanal, inoculating to a liquid LB culture medium with resistance of 4mL, culturing at 37 ℃ for 8-12h, transferring the bacterial liquid to a fermentation medium M9 with resistance of 50mL, adding one thousandth of IPTG for induction, and adding 1g/L of 5-AMP. Then samples were taken at 12h, 24 h,48 h and 72h and the concentration of the target product was determined by HPLC, and the final yields are shown in FIG. 6.
The detection result shows that 5-hydroxypentanal is not accumulated, but 1,5-PDO is detected instead. Namely, the product is directly reduced into the final product 1,5-PDO by the endogenous reducing power of the large intestine. The ML7 strain produces 1,5-PDO at a maximum yield of about 800 mg/L.
Identifying target products obtained by microbial fermentation by respectively using the engineering bacteria ML7 and 5-AMP
The detection method of the fermentation target product 1,5-PDO in the embodiment is similar to the detection method of the fermentation target product 5-AMP provided in the embodiment 1, and the main difference is that:
the 1,5-PDO was analyzed by an organic acid analytical Column, model Amine HPX-87H Ion Exclusion Column, 300 mm. Times.7.8 mm. Refractive index detector of Shimadzu high performance liquid chromatography (HITACHI). The mobile phase was 5 mM H 2 SO 4 Aqueous solution, column box temperature was set at 58 ℃. The flow rate was 0.55 mL/min.
FIG. 7 is HPLC chart of 1,5-PDO standard product, and FIG. 8 is HPLC chart of target product of recombinant Escherichia coli ML7 combined with 5-AMP respectively fermentation culture. As can be seen from the figure: the peak-off time of the standard product shown in fig. 7 was about 31.03 min, and the peak of fig. 8 was also characteristic at about 31.03 min, and the characteristic peak of 5-hydroxypentanal was not shown, which can be explained by: the recombinant E.coli provided in this example in combination with 5-AMP can directly produce 1,5-PDO by fermentation culture.
Example 3 engineering bacteria for synthesizing 5-AMP and 1,5-PDO by combining lysine added in vitro and application thereof
Lysine directly added into a culture medium in the fermentation process is screened from bacteria, and the modified lysine decarboxylase CadA, putrescine transaminase PatA and alcohol dehydrogenase YjgB are used for catalyzing and generating 5-AMP. On the basis, transaminase WTA of pseudomonas aeruginosa is introduced to catalyze and generate 1,5-PDO.
Construction of engineering bacteria: coding the genes of the enzymes, obtaining gene fragments of lysine decarboxylase CadA, putrescine transaminase PatA, alcohol dehydrogenase YjgB and transaminase WTA by PCR respectively, then carrying out enzyme digestion on the gene fragments and a plasmid vector by using restriction endonuclease, carrying out gel recovery or column recovery on the fragments after enzyme digestion, and then inserting a target gene into a plasmid to obtain a recombinant plasmid: pSA-cadA-potE and pCS-patA-yjgB-wtA, as shown in Table 1.
Competent cells were prepared by electroporation and dispensed 90. Mu.L of EP tubing in 1.5mL for transformation. The mixed plasmid of the constructed plasmid pSA-cadA-potE 4. Mu.L and pCS-patA-yjgB-wtA 2. Mu.L was added to a 1.5mL centrifuge tube containing 90. Mu.L of competent cells, and mixed well. The plasmid is then electrotransferred into competent cells using an electrotransfer instrument. After the completion of the electrotransfer, 600. Mu.L of LB medium was added, and the mixture was transferred to a 1.5mL centrifuge tube and revived at 37 ℃ for 30 min. Then, 200. Mu.L of the suspension was applied to the plate and cultured overnight at 37 ℃. 5-AMP-producing strain ML8: BW (pSA-cadA-potE, pCS-patA-yjgB), 1, 5-PDO-producing strain ML9: BW (pSA-cadA-potE, pCS-patA-yjgB-wtA).
The application comprises the following steps: fermentation of engineered strains ML8 and ML9 in combination with in vitro addition of lysine to produce 5-AMP and 1,5-PDO microorganisms, respectively
Single colonies were picked from plates of 5-AMP-producing ML8 strain and 1, 5-PDO-producing ML9 strain, and inoculated into 4mL of a chloramphenicol-and kanamycin-resistant LB solution, and cultured at 37 ℃ for 8 to 12 hours, after which the solutions were transferred to 50mL of a resistant fermentation medium M9, and one in a thousand IPTG was added for induction, and 5 g/L of lysine was added. Then samples were taken at 12h, 24 h,48 h and 72h and the concentration of the target product was determined by HPLC, and the final yields are shown in FIG. 9.
As can be seen in fig. 9: the final yield of 5-AMP synthesized by in vitro lysine addition reaches 1448mg/L, and the yield of 1,5-PDO synthesized by in vitro lysine addition reaches 790mg/L.
Identifying target products of microbial fermentation by respectively using the engineering bacteria ML8 and ML9 in combination with 5-AMP
The target products of the fermentation were detected using the methods provided in example 1 and example 2, respectively. The detection results are shown in fig. 10 and 11, respectively.
Wherein, comparing FIG. 4 and FIG. 10, FIG. 10 shows a characteristic peak at about 18.24min, which is substantially the same as the peak-off time of the standard 5-AMP shown in FIG. 4, so that 5-AMP can be produced by adding lysine to the recombinant Escherichia coli ML8 in vitro provided in this example through microbial fermentation.
Comparing FIG. 7 with FIG. 11, FIG. 11 shows a characteristic peak at about 31.03 min, which is substantially consistent with the peak-off time of the 1,5-PDO standard shown in FIG. 7, therefore, the recombinant Escherichia coli ML9 provided in this example can be used to produce 1,5-PDO by adding lysine in vitro through microbial fermentation.
Example 4 Synthesis of 5-AMP and 1,5-PDO from the beginning and use thereof
After achieving the synthesis of 1,5-PDO by biotransformation of lysine, the synthesis of 1,5-PDO is followed de novo. By enhancing the expression of the diaminopimelate pathway enzymes DapA and LysC.
Constructing engineering bacteria: the gene encoding the above enzyme was digested with endonuclease, the fragment and the vector were digested with PCR to obtain a gene fragment, and the digested fragment was recovered by gel extraction, after which the desired gene was inserted into a plasmid to obtain plasmid pZE-dapA-lysC shown in Table 1.
Competent cells were prepared by electroporation and dispensed 90 μ L into 1.5mL EP tubes for transformation. The constructed recombinant plasmid was added to a 1.5mL EP tube containing 90. Mu.L of competent cells, and mixed well. The plasmid is then electrotransferred into competent cells using an electrotransfer instrument. After the electroporation was completed, LB medium was added and the mixture was transferred to a 1.5mL EP tube and allowed to resuscitate for about 45 min. Then, the bacterial suspension was applied to a plate containing antibiotics and cultured overnight at 37 ℃. Respectively preparing a strain ML10 of the engineering bacteria for synthesizing the 5-AMP: BW (pSA-cadA-potE, pCS-patA-yjgB pZE-dapA-lysC) and the engineered strain ML11BW for the synthesis of 1, 5-PDO: (pSA-cadA-potE, pCS-patA-yjgB-wtA pZE-dapA-lysC).
The application comprises the following steps: production of 5-AMP and 1,5-PDO by microbial fermentation using ML10 and ML11 strains respectively and using glucose as source
Respectively picking single colonies from plates of strains ML10 and ML11, inoculating the single colonies into a liquid LB culture medium with resistance of 4mL, culturing at 37 ℃ for 8-12h, then respectively transferring the bacterial liquid into a fermentation culture medium M10 with resistance of 50mL, and adding one thousandth of IPTG for induction, wherein the fermentation culture medium M10 comprises: 25 glucose monohydrate in g/L, anhydrous magnesium sulfate in 1g/L, ammonium sulfate in 16 g/L, potassium dihydrogen phosphate in 1g/L, high-quality calcium carbonate in 10 g/L and yeast powder in 2 g/L. Then samples were taken at 12h, 24 h,48 h and 72h and the concentrations of the intermediate product and the target product were determined by high performance liquid chromatography, and the final yields are shown in FIG. 12.
As can be seen in fig. 12: the yield of synthesized 5-AMP reaches 1900 mg/L and the yield of synthesized 1,5-PDO reaches 1019 mg/L by taking glucose as a source.
Identifying target products of microbial fermentation by respectively using the engineering bacteria ML8 and ML9 in combination with 5-AMP
The target products of the fermentation were detected using the methods provided in example 1 and example 2, respectively. The detection results are shown in fig. 13 and 14, respectively.
Wherein, comparing FIG. 4 and FIG. 13, the time of peak appearance of FIG. 13 has a characteristic peak around 18.24min, which is substantially the same as the time of peak appearance of the standard 5-AMP product shown in FIG. 4, so that the recombinant Escherichia coli ML10 provided by this embodiment can be used to produce 5-AMP from simple carbon sources such as glucose through microbial fermentation.
Comparing FIG. 7 with FIG. 14, FIG. 14 shows a characteristic peak at about 31.03 min, which is substantially consistent with the peak-out time of the 1,5-PDO standard shown in FIG. 7, therefore, the production of 1,5-PDO from simple carbon sources such as glucose can be realized by the microbial fermentation culture using the recombinant Escherichia coli ML10 provided in this embodiment.
TABLE 1 strains and plasmids
Figure DEST_PATH_IMAGE001
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention and not to limit it; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art will understand that: modifications to the specific embodiments of the invention or equivalent substitutions for parts of the technical features may be made; without departing from the spirit of the invention, it is intended to cover all modifications within the scope of the invention as claimed.
SEQUENCE LISTING
<110> Beijing university of chemical industry
<120> engineering bacteria for synthesizing 5-amino-1-pentanol and 1,5-pentanediol and application
<130> 2021
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 339
<212> PRT
<213> NADPH-dependent aldehyde reductase YjgB
<400> 1
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<210> 2
<211> 458
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<213> transaminase WTA of pseudomonas aeruginosa
<400> 2
MNSQRTTSQWRELDAAHHLHPFTDTASLNQAGARVMTRGEGVYLWDSEGNKIIDGMAGLWCVNVGYGRKDFAEAARRQMEELPFYNTFFKTTHPAVVELSSLLAEVTPAGFDRVFYTNSGSESVDTMIRMVRRYWDVQGKPEKKTLIGRWNGYHGSTIGGASLGGMKYMHEQGDLPIPGMAHIEQPWWYKHGKDMTPDEFGVVAARWLEEKILEIGADKVAAFVGEPIQGAGGVIVPPATYWPEIERICRKYDVLLVADEVICGFGRTGEWFGHQHFGFQPDLFTAAKGLSSGYLPIGAVFVGKRVAEGLIAGGDFNHGFTYSGHPVCAAVAHANVAALRDEGIVQRVKDDIGPYMQKRWRETFSRFEHVDDVRGVGMVQAFTLVKNKAKRELFPDFGEIGTLCRDIFFRNNLIMRACGDHIVSAPPLVMTRAEVDEMLAVAERCLEEFEQTLKARGL*

Claims (10)

1. An engineering bacterium for synthesizing 5-AMP, which comprises a host bacterium and a plasmid vector transferred into the host bacterium, and is characterized in that: introducing a gene encoding an alcohol dehydrogenase into the plasmid vector to produce 5-AMP from 5-aminopentanal; wherein the alcohol dehydrogenase is any one of Adh6, yqhD, yahK and YjgB or alcohol dehydrogenase with the similarity of any one amino acid sequence thereof being 60% or more, adh6 is NADP-dependent alcohol dehydrogenase derived from Saccharomyces cerevisiae, yqhD, yahK and YjgB are NADPH-dependent aldehyde reductase derived from Escherichia coli, and the amino acid sequence of YjgB is shown in SEQ ID NO. 1.
2. The engineered bacterium for synthesizing 5-AMP according to claim 1, wherein: the plasmid vector also has a gene encoding putrescine transaminase PatA introduced therein to catalyze the formation of 5-aminopentanal from pentanediamine.
3. The engineered bacterium for synthesizing 5-AMP according to claim 2, wherein: the plasmid vector also has introduced into it a gene encoding lysine decarboxylase CadA to catalyze the formation of pentanediamine from lysine.
4. The engineered bacterium for synthesizing 5-AMP according to claim 3, wherein: the plasmid vector also introduced genes encoding diaminopimelate pathway enhancing enzymes DapA and LysC.
5. The use of the engineered bacteria of any one of claims 1 to 4 for the synthesis of 5-AMP.
6. An engineering bacterium for synthesizing 1,5-PDO, comprising a host bacterium and a plasmid vector transferred into the host bacterium, wherein a gene coding transaminase is introduced into the plasmid vector to catalyze 5-AMP to generate 5-hydroxypentanal, the transaminase is transaminase CV2025 derived from rhodobacter violaceum or transaminase with 60% or more similarity of amino acid sequence thereof, or transaminase WTA derived from pseudomonas aeruginosa or transaminase with 60% or more similarity of amino acid sequence thereof, and the amino acid sequence of the transaminase WTA derived from pseudomonas aeruginosa is shown as SEQ ID No. 2.
7. The engineered bacterium for synthesizing 1,5-PDO according to claim 6, wherein: the plasmid vector also has introduced into it an alcohol dehydrogenase ADH to synthesize 1,5-PDO from 5-hydroxypentanal.
8. The engineered bacterium for synthesizing 1,5-PDO according to claim 7, wherein: the enzyme for synthesizing the 5-AMP comprises lysine decarboxylase CadA, putrescine transaminase PatA and alcohol dehydrogenase catalyzing 5-aminopentanal to generate the 5-AMP, wherein the alcohol dehydrogenase is any one of Adh6, yqhD, yahK and YjgB or alcohol dehydrogenase with the similarity of 60% to any one of the Adh6, yqhD, yahK and YjgB, the Adh6 is NADP-dependent alcohol dehydrogenase derived from saccharomyces cerevisiae, the YqhD, yahK and YjgB are all NADPH-dependent aldehyde reductases derived from escherichia coli, and the amino acid sequence of the YjgB is shown in SEQ ID NO. 1.
9. The engineered bacterium of claim 8, wherein said engineered bacterium comprises: the plasmid vector also introduced genes encoding diaminopimelate pathway enhancing enzymes DapA and LysC.
10. Use of the engineered bacterium for synthesizing 1,5-PDO according to any one of claims 6 to 9 for synthesizing 1,5-PDO.
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