CN115896194A - Bacterial strain and method for producing dencichine - Google Patents

Abstract

The invention discloses a bacterial strain and a method for producing dencichine, belonging to the technical field of biological engineering. The invention constructs recombinant cells or a combination of the recombinant cells for expressing alcohol dehydrogenase, amine dehydrogenase, ATP-dependent peptide synthetase, polyphosphate kinase 2-I and polyphosphate kinase 2-II, or replaces the polyphosphate kinase 2-I and the polyphosphate kinase 2-II with the polyphosphate kinase 2-III, utilizes the recombinant cells or the combination of the recombinant cells to catalyze serine and oxalic acid to synthesize dencichine, uses 100 g/LL-serine as a substrate, and can obtain 173g/L L-dencichine at most. Therefore, the invention has good industrial application prospect.

Description

Bacterial strain and method for producing dencichine

Technical Field

The invention relates to a bacterial strain and a method for producing dencichine, belonging to the technical field of biological engineering.

Background

Dencichine (dencichine) which is named as pseudo-ginseng acid and has a chemical name of beta-oxalyl-L-diaminopropionic acid is a main hemostatic active ingredient in pseudo-ginseng which is a precious Chinese medicinal material of Araliaceae and Panax, and Dianella canaliculata Zhuo Fu is extracted and separated from pseudo-ginseng for the first time in 1981. Dencichine has one chiral carbon atom and thus has two isomers, the l-form and the d-form. Dencichine as a special non-protein free amino acid has very low content in nature, and is separated from natural plants to obtain I-shaped dencichine. With the continuous research on the bioactivity and in vivo metabolic characteristics of dencichine, dencichine is more and more regarded as a high-value medicinal component and becomes a research hotspot in the future.

At present, the industrial production of dencichine is not mature, and most of the dencichine is still in a laboratory research stage. There are two main methods for preparing dencichine in small amount: 1) Preparing dencichine by chemical synthesis; the chemical synthesis method is easy to face high cost, various byproducts are generated in the middle, the purification and separation operations are difficult, and the optically pure D/L dencichine is not easy to obtain. 2) Is prepared from Panax ginseng C.A.Meyer and Panax notoginseng (Burk.) F.H.Chen of Araliaceae by extracting. The method for extracting the dencichine from the plant China wool is simple and convenient, is easy to realize and ensures the quality. However, the method needs a large amount of organic solvents, is time-consuming and has low extraction rate, so that the development of a method for effectively synthesizing the dencichine, particularly a green microbial synthesis method, has higher effective application value.

Disclosure of Invention

The industrial production of dencichine is still immature, the chemical synthesis method and the plant extraction method have great defects, the cost is high, the yield is low, and a method with high yield and environmental protection is urgently needed.

The invention provides a method for synthesizing D/L-dencichine by taking D/L-serine as a substrate, wherein serine is converted into 2,3-diaminopropionic acid under the action of coexpression of NAD-dependent Alcohol Dehydrogenase (ADH) and NADH-dependent amine dehydrogenase (AmDH); the ATP-dependent peptide bond synthetase condenses 2,3-diaminopropionic acid and oxalic acid to form dencichine, ATP releases energy which is converted to AMP, AMP is regenerated to ADP using polyphosphate kinase PPK2-II, and the ADP is further generated to ATP by polyphosphate kinase 2-I.

The invention provides application of alcohol dehydrogenase, amine dehydrogenase, peptide bond synthetase, polyphosphate kinase 2-I and polyphosphate kinase 2-II in synthesis of dencichine.

In one embodiment, the application is to catalytically synthesize dencichine by using carboxylic acid as a substrate and using alcohol dehydrogenase, amine dehydrogenase, peptide bond synthetase, polyphosphate kinase 2-I and polyphosphate kinase 2-II or microbial cells over expressing the alcohol dehydrogenase, the amine dehydrogenase, the peptide bond synthetase, the polyphosphate kinase 2-I and the polyphosphate kinase 2-II as catalysts.

In one embodiment, the carboxylic acids are D/L-serine and oxalic acid.

In one embodiment, the overexpression is expression of one or more of the genes encoding alcohol dehydrogenase, amine dehydrogenase, peptide bond synthase, polyphosphate kinase 2-I, polyphosphate kinase 2-II by a vector, or by integration into the genome of the host.

In one embodiment, the overexpression is co-expression of genes of the 5 enzymes by using one vector; or co-expressing the 5 enzyme genes by using a plurality of vectors, wherein each vector expresses at least 1 enzyme gene, and the genes expressed by each vector are different.

In one embodiment, the overexpression is the expression of 1 to 5 genes for enzymes on one vector: one vector expresses 1 enzyme gene; or a vector co-expressing 5 enzyme genes; or one vector co-expresses 2 to 3 enzyme genes, wherein the gene coding for alcohol dehydrogenase and the gene coding for amine dehydrogenase are on one vector, and the genes coding for polyphosphate kinase 2-I and polyphosphate kinase 2-II are on the other vector.

In one embodiment, the genes encoding the 5 enzymes are ligated into a vector, each gene preceded by a T7 promoter and an RBS binding site and followed by a T7 terminator.

In one embodiment, the vector includes, but is not limited to, pETDuet-1, pACYCDuet-1, pRSFDuet-1, pCDFduet-1, and pCOLDII.

In one embodiment, the recombinant cells are based on E.coli, including but not limited to Escherichia coli BL21 (DE 3).

In one embodiment, the alcohol dehydrogenase is from Saccharomyces cerevisiae S288C, aeropyrum pernix K1, thermus thermophilus HB8, clostridium beijerinckii or Lactobacillus reuteri DSM20016.

In one embodiment, the NCBI of the amino acid sequence of the alcohol dehydrogenase has access numbers NP _014555.1, baa81251.2, wp _011228103.1, wp _012060249.1, and ABQ83742.1, respectively; the access number of the corresponding nucleotide sequence on NCBI is NC-001147.6, APE _2239.1, NC _006461.1, LZG01000005.1 and NC _009513.1.

In one embodiment, the amine dehydrogenase is from Burkholderia ambifaria AMMD, vibriofurnissi, rhodococcus pyrinidivorans or Pseudomonas anyanorum.

In one embodiment, the amino acid sequence of the amine dehydrogenase has an access number at the NCBI of WP _011659448.1, WP _004726087.1, WP _033097867.1 and WP _063029039.1, respectively; the access number of the corresponding nucleotide sequence on NCBI is NC-008391.1, NZ _CP040990.1, NZ _FNRX01000002.1 and NZ _CP012400.2.

In one embodiment, the peptide bond synthetase is derived from Streptomyces rimosus, acinetobacter lactuca, trichoderma virens Gv29-8, rhodococcus erythropolis PR4 or Bacillus safensis.

In one embodiment, the amino acid sequence of the peptide bond synthetase has access numbers on NCBI of WP _050504588.1, WP _016145237.1, XP _013952649.1, WP _020907735.1 and WP _048238116.1, respectively; the access number of the corresponding nucleotide sequence on NCBI is NZ _ CP023688.1, NZ _ KB976991.1, NW _014013663.1, NC _012490.1, NZ _LDUS01000007.1.

In one embodiment, the polyphosphate kinase 2-I is from Sinorhizobium meliloti; the access number on NCBI of the amino acid sequence is NP-384613.1; the access number on NCBI of the corresponding nucleotide sequence is NC _003047region (564142.. 565044).

In one embodiment, the polyphosphate kinase 2-II is derived from acinetobacter johnsonii; the access number on NCBI of the amino acid sequence is BAC76403.1; the access number on the NCBI of the corresponding nucleotide sequence is AB092983REGION:339.. 1766.

In one embodiment, the dencichine is D-dencichine or L-dencichine. When the dencichine is L-dencichine, a peptide bond synthetase derived from Rhodococcus erythropolis PR4, bacillus safensis strain was selected.

The invention also provides a combination of recombinant cells; the combination of recombinant cells consists of recombinant cells overexpressing one or more of alcohol dehydrogenase, amine dehydrogenase, peptide bond synthase, polyphosphate kinase 2-I, and polyphosphate kinase 2-II, respectively, each recombinant cell not being repeatedly expressed with other recombinant cells.

In one embodiment, the combination of recombinant cells is hosted in E.coli, such as Escherichia coli BL21 (DE 3).

In one embodiment, the overexpression is co-expression of 5 enzyme genes using multiple vectors, each vector expressing at least 1 enzyme gene, and each vector expressing a different gene.

In one embodiment, the combined overexpression of the recombinant cell is the co-expression of 1 to 5 genes for enzymes on one vector: one vector expresses 1 enzyme gene, and 5 vectors in total; or a vector co-expressing 5 enzyme genes; or 2 vectors which co-express 2 to 3 enzyme genes, wherein the gene coding alcohol dehydrogenase and the gene coding amine dehydrogenase are on one vector, and the genes coding polyphosphate kinase 2-I and polyphosphate kinase 2-II are on the other vector.

In one embodiment, the genes encoding the 5 enzymes are ligated into a vector, each gene preceded by a T7 promoter and an RBS binding site and followed by a T7 terminator.

In one embodiment, the vector includes, but is not limited to, pETDuet-1, pACYCDuet-1, pRSFDuet-1, pCDFduet-1, and pCOLD II.

In particular, the present invention also provides a recombinant cell for synthesizing dencichine, which expresses a gene encoding alcohol dehydrogenase, a gene encoding amine dehydrogenase, a gene encoding ATP-dependent peptide bond synthase, and genes encoding polyphosphate kinase 2-I, polyphosphate kinase 2-ii; the recombinant cell takes escherichia coli as a host, pRSFDuet-1 as a vector to express genes coding polyphosphate kinase 2-I and polyphosphate kinase 2-II, and pTDuet-1 as a vector to express genes coding alcohol dehydrogenase and amine dehydrogenase. Each gene contained a T7 promoter and RBS binding site in front of it and a T7 terminator behind it.

The invention also provides a method for producing dencichine by whole-cell catalysis, which is characterized in that the recombinant cell or the combination of the recombinant cell is used as a whole-cell catalyst, and oxalic acid and D-serine (L-serine) are used as substrates to synthesize the D-dencichine (L-dencichine).

In one embodiment, the whole cell catalyst is prepared by culturing, propagating, and allowing the recombinant cells or combination of recombinant cells to express the 5 enzymes, and then harvesting the recombinant cells. When using the whole-cell catalyst, in addition to providing the substrate, it is necessary to maintain the appropriate temperature and pH, and if necessary, to provide some co-enzymes or nutrients to help the whole-cell catalyst better perform the catalytic function.

In one embodiment, the whole cell transformation production system comprises 1-200g/L of cell wet weight, 1-100g/L of D/L-serine, 1-100g/L of oxalic acid, 0-1g/L of ATP, 0-1g/L of NAD, 2-300g/L of sodium hexametaphosphate, and pH5.0-9.0; reacting at 15-40 ℃ for 1-48h.

The invention also protects the application of the combination of the recombinant cells or the method for producing the dencichine by the whole cell catalysis in the production of the dencichine or the products containing the dencichine or the substances taking the dencichine as the precursor.

Advantageous effects

(1) The method realizes double coenzyme regeneration of NAD and ATP on the basis of expressing five enzymes of alcohol dehydrogenase, amine dehydrogenase, polypeptide synthetase, polyphosphate kinase 2-I and polyphosphate kinase 2-II through a reasonable expression strategy, effectively ensures the continuous proceeding of enzyme catalytic reaction and improves the yield of dencichine.

(2) The D-type dencichine extracted from plants in nature is common. The invention obtains alcohol dehydrogenase, amine dehydrogenase and peptide bond synthetase which can take L-serine as a substrate, and on the basis, L-serine and oxalic acid are taken as raw materials to synthesize and obtain the L-dencichine by a biological method.

Detailed Description

1. The invention relates to a strain and a plasmid

pRSFDuet-1, pETDuet-1, pCDFDuet-1, pACYCDuet-1, pCOLD II plasmid and E.coli Escherichia coli BL21 (DE 3) from Novagen.

2. Construction of polygene co-expression system and culture of cell

Coli polygene co-expression has various methods (for example, the method described in the article "coli polygene co-expression strategy, journal of Chinese bioengineering, 2012, 32 (4): 117-122"), and the invention adopts the method described in the doctor paper of Liu Xianglei (synthetic biology technology for transforming escherichia coli to produce shikimic acid and resveratrol, 2016, shanghai pharmaceutical industry research institute) to construct recombinant escherichia coli. In the following examples, when multiple genes are co-expressed, each gene is preceded by the T7 promoter and RBS binding site of E.coli BL21 (DE 3), and each gene is followed by a T7 terminator. Theoretically, since each gene is preceded by a T7 promoter and an RBS binding site, the expression intensity of the gene is not greatly affected by the order of arrangement of the genes on the plasmid. And (3) thermally transducing the constructed plasmid into an escherichia coli competent cell, coating the escherichia coli competent cell on a monoclonal antibody or mixed antibiotic solid plate, and screening to obtain a positive transformant, namely the recombinant escherichia coli.

And (3) culturing the cells: according to the classical recombinant Escherichia coli culture and induction expression scheme, the recombinant Escherichia coli is transferred into an LB fermentation medium (peptone 10g/L, yeast powder 5g/L, naCl 10 g/L) according to the volume ratio of 2%, when the OD600 of the cells reaches 0.6-0.8, IPTG with the final concentration of 0.4mM is added, and the cells are subjected to induction expression culture at 20 ℃ for 8h. After the induction expression was completed, the cells were collected by centrifugation at 8000rpm for 20min at 4 ℃.

3. Selection of the relevant enzymes

(1) Polyphosphate kinase 2-I

Selecting the gene smpkk encoding polyphosphate kinase 2-I from Sinorhizobium meliloti, which gene smpkk has an access number NC _003047region at NCBI (564142.. 565044) and the corresponding amino acid sequence is NP _384613.1. The enzyme may catalyze the production of ADP from AMP, providing phosphate groups from polyphosphate.

(2) Polyphosphate kinase 2-II

The gene ajpkk from Acinetobacter johnsonii encoding polyphosphate kinase 2-II was selected, the gene ajpkk having a sequence with an accession number AB092983REGION:339.. 1766 at NCBI and the corresponding amino acid sequence BAC76403.1. The enzyme can catalyze ADP to generate ATP, and phosphate groups are provided by polyphosphoric acid.

(3) Polyphosphate kinase 2-III

The gene mhpkk encoding polyphosphate kinase 2-III from Meiothermus hypogaeus was selected, the sequence of the access number NZ _ BJXL01000029 REGION: completion (14116.. 14919) on gene mhpkk NCBI corresponding to the amino acid sequence WP _119340583.1. This enzyme catalyzes the direct production of ATP from AMP, which provides phosphate groups from polyphosphate.

4. Detection analysis of samples

Method for measuring the content of dencichine by measurement according to the literature (Qiao CF, liu XM, cui XM, et al, high-performance and exchange chromatography coupled with diode array detection for the determination of concentration of dencichine in Panax nanoparticles and related species, journal of Separation science 2013 Aug;36 (24015): 1-2406.)

ATP-dependent peptide synthetase enzyme activity was determined according to literature: petchey, mark et al, "The Broad Aryl Acid Specificity of The Amide Bond Synthesis McbA Suggests potentials for The biological Synthesis of The amides," Angewandte Chemie (International in English) vol.57, 36 (2018): 11584-11588.

Alcohol dehydrogenase activity was determined according to the literature: ZHENGHONG Hu, pu Jia, yajun Bai, tai-ping Fan, xiaohui Zheng, yujie Cai, characterisation of five alcohol products from Lactobacillus reuteri DSM20016, process Biochemistry.

Amine dehydrogenase activity was determined according to the literature: abrahamson MJ, V a zquez-Figueroa E, woodall NB, moore JC, bommarius AS.development of an amine dehydrogenase for synthesis of a chiral amines.Angew Chem Int Ed Engl.2012;51 (16):3969-3972.

Specific activity (U mg) ^-1 ) Is defined as the unit of enzyme activity per mg of enzyme. One unit of enzyme activity (U) is defined as the amount of enzyme required to produce 1. Mu. Mol of product in 1 min.

Example 1: screening and expression of alcohol dehydrogenase

Alcohol dehydrogenase is widely present in various organisms, and alcohol dehydrogenase genes scadh, apadh, ttadh, cbadh and lrah are obtained by total synthesis according to the alcohol dehydrogenase gene information of Saccharomyces cerevisiae S288C, aeropyrum pernix K1, thermus thermophilus HB8, clostridium beijerinckii and Lactobacillus reuteri DSM20016 on NCBI. The corresponding amino acid sequences are respectively the access numbers on NCBI: NP-014555.1, BAA81251.2, WP _011228103.1, WP _012060249.1, ABQ83742.1. The synthesized genes are respectively and independently connected to the multiple cloning sites of pETDuet-1 vector, and induced to express in Escherichia coli BL21 (DE 3), and 5 recombinant Escherichia coli are obtained.

The induction expression method comprises the following steps: respectively transferring the 5 recombinant escherichia coli into an LB fermentation culture medium according to the inoculation amount of 2% in volume ratio, and when the cells OD ₆₀₀ After reaching 0.6-0.8, IPTG was added to a final concentration of 0.4mM, and expression-induced culture was carried out at 20 ℃ for 8 hours. After the induction expression was completed, the cells were collected by centrifugation at 8000rpm for 20 minutes at 4 ℃. After the cells are broken, the enzyme is purified by a His-tag label method, and the activity is measured after the pure enzyme is obtained.

When D-serine is taken as a substrate, the specific enzyme activities of the enzymes expressed by the genes of alcohol dehydrogenase scadh, apadh, ttadh, cbadh and lradh are respectively as follows: 124. 87, 56, 98 and 156U/mg.

When L-serine is taken as a substrate, the specific enzyme activities of the enzymes expressed by the genes of alcohol dehydrogenase scadh, apadh, ttadh, cbadh and lradh are respectively as follows: 32. 46, 51, 39 and 78U/mg.

Example 2: screening and expression of amine dehydrogenases

Amine dehydrogenase is widely present in plants, and amine dehydrogenase genes baamdh, vfamdh, rpamdh and pyamdh are obtained by total synthesis according to the amine dehydrogenase gene information of Burkholderia ambifaria AMMD, vibrio furnissii, rhodococcus pyrinidivorans and Pseudomonas aeruginosa on NCBI. The corresponding amino acid sequences are respectively as follows on NCBI: WP _011659448.1, WP _004726087.1, WP _033097867.1, WP _063029039.1. The genes obtained by synthesis are respectively and independently connected to pETDuet-1 vectors, and induced and expressed in Escherichia coli BL21 (DE 3), so as to obtain 4 kinds of recombinant Escherichia coli. The method of inducible expression is the same as in example 1.

When (R) -2-amino-3-oxo propionic acid is used as a substrate, the specific enzyme activities of the enzymes expressed by the genes of the amine dehydrogenases baamdh, vfamdh, rpamdh and pyamdh are respectively as follows: 156. 125, 107 and 114U/mg.

When (S) -2-amino-3-oxo propionic acid is used as a substrate, the specific enzyme activities of the enzymes expressed by the genes of the amine dehydrogenases baamdh, vfamdh, rpamdh and pyamdh are respectively as follows: 138. 72, 89, 102U/mg.

Example 3: screening and expression of ATP-dependent peptide synthetases

ATP-dependent peptide synthetase is widely present in plants, and ATP-dependent peptide synthetase genes srabs, ababs, tvabs, reabs and bsabs are obtained by total synthesis based on the gene information of Streptomyces rimosus, acinetobacter lactuca, trichoderma virens Gv29-8, rhodococcus erythropolis PR4 and ATP-dependent peptide synthetase on NCBI of Bacillus safensis, respectively. The access numbers of the corresponding amino acid sequences on NCBI are respectively as follows: WP _050504588.1, WP _016145237.1, XP _013952649.1, WP _020907735.1 and WP _048238116.1. The genes obtained by synthesis are respectively and independently connected to pETDuet-1 vectors, and are induced and expressed in Escherichia coli BL21 (DE 3), and 5 recombinant Escherichia coli are obtained. The method of inducible expression is the same as in example 1.

When (R) -2,3-diaminopropionic acid and oxalic acid are used as substrates, the specific enzyme activities of the enzymes expressed by the genes of ATP-dependent peptide synthetases rsrabs, alabs, tvabs, reabs and bsabs are respectively as follows: 73. 121, 86, 45 and 49U/mg.

When (S) -2,3-diaminopropionic acid and oxalic acid are used as substrates, the specific enzyme activities of the enzymes expressed by the genes of ATP-dependent peptide synthetases srabs, alabs, tvabs, reabs and bsabs are: 35. 36, 28, 45 and 68U/mg.

Example 4 construction of recombinant E.coli simultaneously expressing 5 enzymes

As shown in Table 1, selection was made from five plasmids, pETDuet-1, pACYCDuet-1, pRSFDuet-1, pCDFduet-1, pCOLD II, and genes for 4 to 5 enzymes selected from genes encoding 6 enzymes were ligated to the same plasmid, or to two plasmids (2 to 3 genes expressed on each plasmid), or to five plasmids (1 gene expressed on each plasmid). Cloning gene fragments into the multiple cloning sites of plasmids, wherein each gene contains a T7 promoter and an RBS binding site of Escherichia coli BL21 (DE 3) in front of the gene, and a T7 terminator behind the gene. And (3) transforming the constructed recombinant plasmid into escherichia coli BL21, and screening by using different mixed antibiotic plates according to resistance genes on different plasmids to obtain a positive transformant, namely the recombinant escherichia coli capable of enhancing expression of 5 genes is obtained.

Carrying out inducible expression on the recombinant escherichia coli, collecting thalli after the inducible expression is finished, and reacting for 24 hours in a 100mL reaction system, wherein the thalli are 200g/L, the L-serine is 100g/L, the oxalic acid is 100g/L, the ATP is 1g/L, the NAD is 1g/L and the sodium hexametaphosphate is 300 g/L. pH5.0-9.0; the temperature is 15-40 ℃.

TABLE 1

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Example 5: in vitro catalytic synthesis of D-dencichine by using five enzymes

The five genes smpkk, ajpkk, lrah, baamdh and alabs are respectively connected to a pEDT28a vector to obtain 5 recombinant vectors, and the 5 recombinant vectors are respectively transformed into Escherichia coli BL21 to obtain recombinant Escherichia coli for respectively expressing 5 enzymes. Five pure enzymes were obtained after expression and purification in the same manner as in example 1. Then, adding each of the five pure enzymes of 2mg, 100g/L of D-serine, 100g/L of oxalic acid, 1g/L of ATP, 1g/L of NAD, 60g/L of sodium hexametaphosphate and pH 7.0 into a 100mL reaction system; the reaction is carried out at 35 ℃ for 48h. The yield of the D-dencichine is detected to be 62g/L.

Example 6: in-vitro catalytic synthesis of D-dencichine by using four enzymes

The four genes of mhpkk, lrah, baamdh and alabs are respectively connected to pED28a vector to obtain 4 recombinant vectors, and the 4 recombinant vectors are respectively transformed into Escherichia coli BL21 to obtain 4 recombinant Escherichia coli. Four kinds of pure enzymes were obtained after expression and purification in the same manner as in example 1. Then adding 2mg of each of the four pure enzymes, 100g/L of D-serine, 100g/L of oxalic acid, 1g/L of ATP, 1g/L of NAD, 200g/L of sodium hexametaphosphate and pH 7.0 into a 100mL reaction system; the reaction is carried out at 35 ℃ for 48h. The yield of the D-dencichine is 166g/L.

Example 7: combined catalytic synthesis of D-dencichine by using recombinant cells

The five genes smpkk, ajpkk, lrah, baamdh and alabs are respectively connected to pEDTDuet-1 vector to obtain 5 recombinant vectors, and the 5 recombinant vectors are respectively transformed into Escherichia coli BL21 to obtain 5 recombinant Escherichia coli. The recombinant E.coli was induced to express the enzyme in the same manner as in example 1. Then, 20g/L of each of the five whole cells, 100g/L of D-serine, 100g/L of oxalic acid, 1g/L of ATP, 100g/L of sodium hexametaphosphate, 1g/L of NAD and 8.0 of pH are added into a 100mL reaction system; the reaction is carried out at 25 ℃ for 24h. The yield of the D-dencichine is detected to be 153g/L.

Example 8: catalytic synthesis of D-dencichine and L-dencichine by recombinant escherichia coli whole cell

In the nature, the dencichine is D-type, and the invention further synthesizes the L-dencichine by taking L-serine and oxalic acid as raw materials. Heretofore, L-dencichine has not been synthesized biologically.

Rhodococcus erythropolis PR4, genes reabs and bsabs encoding peptide synthetases from Bacillus safensis, together with genes encoding polyphosphate kinase 2-I, polyphosphate kinase 2-II, alcohol dehydrogenase and amine dehydrogenase, were selected to construct recombinant bacteria Escherichia coli BL21 (DE 3)/pRSFDuet-1-smpkk-ajpkk and pETDuet-1-lrah-baamdh-abs or Escherichia coli BL21 (DE 3)/pRSFDuet-1-smpkk-ajpkk and pETDuet-1-lrah-baamdh-bsabs, which were induced to express according to the method described in example 1, and then the bacteria were collected.

In a 100mL reaction system, the wet weight of cells is 100g/L, D-serine or L-serine is 100g/L, oxalic acid is 100g/L, ATP is 1g/L, NAD is 1g/L, sodium hexametaphosphate is 150g/L, and the pH value is 6.0; the reaction is carried out at 30 ℃ for 48h. When D-serine is taken as a substrate, the yield of the D-dencichine is 133g/L. When the L-serine is taken as a substrate, the yield of the L-dencichine is 58g/L and 161g/L after the conversion is finished.

Example 9: synthesis of L-notoginseng essence by recombinant colibacillus whole cell catalysis

The following 2 recombinant bacteria Escherichia coli BL21 (DE 3)/pRSFDuet-1-mhpkk (named E1), escherichia coli BL21 (DE 3)/pETDuet-1-lrah-baamdh-abs (named E2) were constructed.

Expression of E1 and E2 was induced according to the method described in example 1, and the cells were collected. In a 100mL reaction system, the wet weight of E1 cells is 30g/L, the wet weight of E2 cells is 50g/L, L-serine 100g/L, oxalic acid 100g/L, sodium hexametaphosphate 300g/L, NAD 1g/L, ATP 1g/L and pH 7.0; the reaction was carried out at 40 ℃ for 48h. After the conversion is finished, the content of the L-dencichine is 173g/L by liquid chromatography.

Example 10: catalytic synthesis of D-dencichine by recombinant escherichia coli whole cell

The following 2 recombinant bacteria Escherichia coli BL21 (DE 3)/pRSFDuet-1-smpkk-ajpkk-alabs (named E3), escherichia coli BL21 (DE 3)/pACYCDuet-1-lrah-baamdh (named E4) were constructed.

The E3 and E4 were separately induced to express according to the method described in example 1, and the cells were collected. In a 100mL reaction system, the wet weight of an E3 cell is 100g/L, the wet weight of an E4 cell is 100g/L, 50g/L of D-serine, 50g/L of oxalic acid, 1g/L of NAD, 1g/L of ATP, 30g/L of sodium hexametaphosphate and pH 7.0; the reaction is carried out at 40 ℃ for 12h. And after the conversion is finished, the content of the D-dencichine is 65g/L by liquid chromatography.

Example 11: synthesis of L-dencichine by recombinant escherichia coli whole cell catalysis

The following 2 recombinant bacteria Escherichia coli BL21 (DE 3)/pRSFDuet-1-smpkk-ajpkk-bsabs (designated E5), escherichia coli BL21 (DE 3)/pACYCDuet-1-lrah-baamdh (E4) were constructed.

According to the method described in example 1, E5 and E4 were separately induced to express, and then the cells were collected. In a 100mL reaction system, the wet weight of an E5 cell is 100g/L, the wet weight of an E4 cell is 100g/L, 10g/L of L-serine, 10g/L of oxalic acid, 0.1g/L of NAD, 0.1g/L of ATP, 20g/L of sodium hexametaphosphate and pH is 7.0; the reaction was carried out at 30 ℃ for 1h. And after the conversion is finished, the content of the L-dencichine is 18g/L by liquid chromatography.

Example 12: catalytic synthesis of D-dencichine by recombinant escherichia coli whole cell

The following 2 recombinant bacteria Escherichia coli BL21 (DE 3)/pRSFDuet-1-lrah-bamdh (designated E6), escherichia coli BL21 (DE 3)/pACYCDuet-1-smpkk-ajpkk-abs (designated E7) were constructed.

Expression of E6 and E7 was induced according to the method described in example 1, and the cells were collected. In a 100mL reaction system, the wet weight of E6 cells is 100g/L, the wet weight of E7 cells is 100g/L, 50g/L of D-serine, 50g/L of oxalic acid, 0.5g/L of NAD, 0.5g/L of ATP, 60g/L of sodium hexametaphosphate and pH 7.0; the reaction is carried out at 40 ℃ for 5h. And after the conversion is finished, the content of the D-dencichine is 80g/L by liquid chromatography.

Example 13: synthesis of L-dencichine by recombinant escherichia coli whole cell catalysis

The following 2 recombinant bacteria Escherichia coli BL21 (DE 3)/pRSFDuet-1-lrah-baamdh (E6), escherichia coli BL21 (DE 3)/pACYCDuet-1-mhpkk-bsabs (designated E8) were constructed.

Expression of E6 and E8 was induced according to the method described in example 1, and the cells were collected. In a 100mL reaction system, the wet weight of an E6 cell is 10g/L, the wet weight of an E8 cell is 20g/L, 1g/L of L-serine, 1g/L of oxalic acid, 0g/L of NAD, 0g/L of ATP, 2g/L of sodium hexametaphosphate and pH 7.0; the reaction was carried out at 30 ℃ for 1h. After the conversion is finished, the content of the L-dencichine is 1.8g/L by liquid chromatography.

Various changes and modifications can be made without departing from the spirit and scope of the invention, and the scope of the invention should be determined from the appended claims.

Claims (10)

1. Alcohol dehydrogenase, amine dehydrogenase, peptide bond synthetase, polyphosphate kinase 2-I and polyphosphate kinase 2-II are used in synthesizing dencichine.

2. The use of claim 1, wherein the synthesis of dencichine is catalyzed by using carboxylic acid as a substrate and using microbial cells overexpressing alcohol dehydrogenase, amine dehydrogenase, peptide bond synthase, polyphosphate kinase 2-I, and polyphosphate kinase 2-II as a catalyst; the carboxylic acid is D/L-serine and oxalic acid.

3. Use according to claim 2, wherein the overexpression is the expression of one or more of the genes encoding alcohol dehydrogenase, amine dehydrogenase, peptide bond synthase, polyphosphate kinase 2-I, polyphosphate kinase 2-II, by means of a vector, or by integration into the genome of the host.

4. The use of claim 3, wherein said overexpression is the co-expression of 5 enzyme genes using one vector; or co-expressing genes of 5 enzymes by using a plurality of vectors, wherein each vector expresses genes of at least 1 enzyme, and the genes expressed by each vector are different.

5. Use according to claim 3 or 4, wherein the vector includes, but is not limited to, pETDuet-1, pACYCDuet-1, pRSFDuet-1, pCDFduet-1, pCOLD II.

6. A combination of recombinant cells consisting of recombinant cells overexpressing one or more of alcohol dehydrogenase, amine dehydrogenase, peptide bond synthase, polyphosphate kinase 2-I and polyphosphate kinase 2-II, respectively, each recombinant cell not being overexpressed by other recombinant cells.

7. The combination of recombinant cells according to claim 6, wherein the recombinant cells are host bacteria of Escherichia coli, including Escherichia coli BL21 (DE 3).

8. A method for producing dencichine by whole-cell catalysis is characterized in that the combination of the recombinant cells of claim 6 or 7 is used as a whole-cell catalyst, and serine and oxalic acid are used as substrates to synthesize the dencichine.

9. The method as claimed in claim 8, wherein in the whole cell transformation production system, the wet weight of the cells is 1-200g/L, the D/L-serine is 1-100g/L, the oxalic acid is 1-100g/L, the ATP is 0-1g/L, the NAD is 0-1g/L, the sodium hexametaphosphate is 2-300g/L, and the pH is 5.0-9.0; reacting at 15-40 ℃ for 1-48h.

10. Use of a combination of recombinant cells according to claim 6 or 7 or a method according to claim 8 or 9 for the production of dencichine or a product containing dencichine or a substance which uses dencichine as a precursor.