CN115896194A - A kind of bacterial strain and method for producing notoginseng - Google Patents

Abstract

The invention discloses a bacterial strain and a method for producing notoginseng, belonging to the technical field of bioengineering. The present invention constructs a recombinant cell or a combination of recombinant cells expressing alcohol dehydrogenase, amine dehydrogenase, ATP-dependent peptide synthetase, polyphosphate kinase 2-I, polyphosphate kinase 2-II, or the polyphosphate kinase 2-II Phosphokinase 2-I and polyphosphate kinase 2-II are replaced by polyphosphate kinase 2-III, and recombinant cells or a combination of recombinant cells are used to catalyze the synthesis of notoginseng from serine and oxalic acid, using 100g/LL-serine As a substrate, a maximum of 173g/L L-Notoginseng can be obtained. Therefore, the present invention has good industrial application prospect.

Description

A strain and method for producing notoginseng

Technical Field

The invention relates to a strain and a method for producing notoginseng, and belongs to the technical field of bioengineering.

Background Art

Decichine, also known as decichine, has a chemical name of β-oxalyl-L-diaminopropionic acid. It is the main hemostatic active ingredient in the precious Chinese medicinal material Panax notoginseng of the Araliaceae family. In 1981, Takuo Kosuge first extracted and separated decichine from Panax notoginseng. Studies have further proved that decichine not only has hemostatic function and pharmacological activity of increasing platelet count, but also has neuroprotective effects, reduces diabetic kidney damage, anti-inflammatory and lowers blood sugar. Decichine has a chiral carbon atom, so it has two isomers, l-type and d-type. Decichine, as a special non-protein free amino acid, is extremely rare in nature, and all those isolated from natural plants are l-configuration. With the continuous research on the biological activity and metabolic characteristics of decichine, decichine as a high-value medicinal ingredient has been increasingly valued and has become a research hotspot in the future.

At present, the industrial production of notoginseng is still immature, and most of it is still in the laboratory research stage. There are two main methods for preparing notoginseng in small quantities: 1) Prepare notoginseng by chemical synthesis; the chemical synthesis method is prone to high costs, a variety of by-products are produced in the middle, the purification and separation operation is difficult, and it is not easy to obtain optically pure D/L notoginseng. 2) It is obtained by processing and extracting plants such as ginseng and Panax notoginseng in the Araliaceae family. Extracting notoginseng from the plant Chinese wool is simple, easy to implement, and has guaranteed quality. However, this method requires a large amount of organic solvents, is time-consuming, and has a low extraction rate. Therefore, developing a method for effectively synthesizing notoginseng, especially a green microbial synthesis method, has more efficient application value.

Summary of the invention

The industrial production of notoginseng is still immature. Both the chemical synthesis method and the plant extraction method have major defects, high cost and low yield. There is an urgent need for a high-yield and green and environmentally friendly method.

The invention provides a method for synthesizing D/L-notoginseng using D/L-serine as a substrate. Serine is converted into 2,3-diaminopropionic acid under the action of co-expressed NAD-dependent alcohol dehydrogenase (ADH) and NADH-dependent amine dehydrogenase (AmDH); ATP-dependent peptide bond synthetase condenses 2,3-diaminopropionic acid and oxalic acid to form notoginseng, ATP releases energy and is converted into AMP, polyphosphate kinase PPK2-II is used to regenerate AMP into ADP, and polyphosphate kinase 2-I further generates ADP into ATP.

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

In one embodiment, the application is to use carboxylic acid as a substrate and alcohol dehydrogenase, amine dehydrogenase, peptide bond synthetase, polyphosphate kinase 2-I and polyphosphate kinase 2-II or microbial cells overexpressing alcohol dehydrogenase, amine dehydrogenase, peptide bond synthetase, polyphosphate kinase 2-I and polyphosphate kinase 2-II as catalysts to catalyze the synthesis of Panax notoginseng.

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

In one embodiment, the overexpression is to express one or more genes encoding alcohol dehydrogenase, amine dehydrogenase, peptide synthetase, polyphosphate kinase 2-I, and polyphosphate kinase 2-II through a vector, or integrate them into the host genome for expression.

In one embodiment, the overexpression is to co-express the genes of the above five enzymes using one vector; or to co-express the genes of the above five enzymes using multiple vectors, each vector expressing at least one enzyme gene, and the genes expressed by each vector are different.

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

In one embodiment, when the genes encoding the five enzymes are connected to the vector, each gene contains a T7 promoter and an RBS binding site in front of it, and each gene is 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 cell uses Escherichia coli as a host, including but not limited to Escherichia coli BL21 (DE3).

In one embodiment, the alcohol dehydrogenase is derived from Saccharomyces cerevisiae S288C, Aeropyrum pernix K1, Thermus thermophilus HB8, Clostridium beijerinckii or Lactobacillus reuteri DSM20016.

In one embodiment, the NCBI accession numbers of the amino acid sequences of the alcohol dehydrogenase are NP_014555.1, BAA81251.2, WP_011228103.1, WP_012060249.1 and ABQ83742.1; the NCBI accession numbers of the corresponding nucleotide sequences are NC_001147.6, APE_2239.1, NC_006461.1, LZZG01000005.1, NC_009513.1.

In one embodiment, the amine dehydrogenase is from Burkholderia ambifariaAMMD, Vibriofurnissii, Rhodococcuspyridinivorans or Pseudomonasyamanorum.

In one embodiment, the NCBI accession numbers of the amino acid sequences of the amine dehydrogenase are WP_011659448.1, WP_004726087.1, WP_033097867.1 and WP_063029039.1; the NCBI accession numbers of the corresponding nucleotide sequences are NC_008391.1, NZ_CP040990.1, NZ_FNRX01000002.1, NZ_CP012400.2.

In one embodiment, the peptide synthetase is from Streptomyces rimosus, Acinetobacter lactuca, Trichoderma virens Gv29-8, Rhodococcus erythropolis PR4 or Bacillus safensis.

In one embodiment, the NCBI accession numbers of the amino acid sequences of the peptide synthetase are WP_050504588.1, WP_016145237.1, XP_013952649.1, WP_020907735.1 and WP_048238116.1; the NCBI accession numbers of the corresponding nucleotide sequences are NZ_CP023688.1, NZ_KB976991.1, NW_014013663.1, NC_012490.1, and NZ_LDUS01000007.1.

In one embodiment, the polyphosphate kinase 2-I is from Sinorhizobium_meliloti; the NCBI accession number of its amino acid sequence is NP_384613.1; the NCBI accession number of the corresponding nucleotide sequence is NC_003047REGION:complement(564142...565044).

In one embodiment, the polyphosphate kinase 2-II is from Acinetobacter johnsonii; the NCBI accession number of its amino acid sequence is BAC76403.1; and the NCBI accession number of the corresponding nucleotide sequence is AB092983REGION:339...1766.

In one embodiment, the notoginseng is D-notoginseng or L-notoginseng. When the notoginseng is L-notoginseng, the peptide synthetase from Rhodococcus erythropolis PR4, Bacillus safensis strains is selected.

The present invention also provides a combination of recombinant cells; the combination of recombinant cells consists of recombinant cells that overexpress one or more of alcohol dehydrogenase, amine dehydrogenase, peptide bond synthetase, polyphosphate kinase 2-I and polyphosphate kinase 2-II, and each recombinant cell does not repeat the expression with other recombinant cells.

In one embodiment, the combination of recombinant cells uses Escherichia coli as a host, such as Escherichia coli BL21 (DE3).

In one embodiment, the overexpression is to co-express genes of five enzymes using multiple vectors, each vector expresses a gene of at least one enzyme, and the genes expressed by each vector are different.

In one embodiment, the combined overexpression of the recombinant cell is the co-expression of 1 to 5 enzyme genes on one vector: one vector expresses 1 enzyme gene, with a total of 5 vectors; or one vector co-expresses 5 enzyme genes; or one vector co-expresses 2 to 3 enzyme genes, with a total of 2 vectors, wherein the gene encoding alcohol dehydrogenase and the gene encoding amine dehydrogenase are on one vector, and the gene encoding polyphosphate kinase 2-I and polyphosphate kinase 2-II are on another vector.

In one embodiment, when the genes encoding the five enzymes are connected to the vector, each gene contains a T7 promoter and an RBS binding site in front of it, and each gene is 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 particular, the present invention also provides a recombinant cell for synthesizing notoginseng, wherein the recombinant cell expresses a gene encoding alcohol dehydrogenase, a gene encoding amine dehydrogenase, a gene encoding ATP-dependent peptide bond synthetase, and a gene encoding polyphosphate kinase 2-I and polyphosphate kinase 2-II; the recombinant cell uses Escherichia coli as a host, pRSFDuet-1 as a vector to express the genes encoding polyphosphate kinase 2-I and polyphosphate kinase 2-II, and pTDuet-1 as a vector to express the genes encoding alcohol dehydrogenase and amine dehydrogenase. Each gene contains a T7 promoter and an RBS binding site in front, and each gene is followed by a T7 terminator.

The present invention also provides a method for producing notoginseng by whole-cell catalysis, which utilizes the recombinant cell of the present invention or a combination of recombinant cells as a whole-cell catalyst, and uses oxalic acid and D-serine (L-serine) as substrates to synthesize D-notoginseng (L-notoginseng).

In one embodiment, the preparation of the whole cell catalyst is to culture and propagate recombinant cells or a combination of recombinant cells, and make the recombinant cells or the combination of recombinant cells express the five enzymes, and then collect the recombinant cells. When using the whole cell catalyst, in addition to providing a substrate, it is also necessary to maintain an appropriate temperature and pH, and if necessary, provide some coenzymes or nutrients to help the whole cell catalyst better exert its catalytic effect.

In one embodiment, the whole-cell conversion production system includes a cell wet weight of 1-200 g/L, D/L-serine 1-100 g/L, oxalic acid 1-100 g/L, ATP 0-1 g/L, NAD 0-1 g/L, sodium hexametaphosphate 2-300 g/L, pH 5.0-9.0; the reaction is carried out at 15-40° C. and the reaction time is 1-48 h.

The present invention also protects the use of the above-mentioned combination of recombinant cells or the method for producing notoginseng by whole-cell catalysis in the production of notoginseng or products containing notoginseng or substances with notoginseng as a precursor.

Beneficial Effects

(1) The present invention realizes the dual coenzyme regeneration of NAD and ATP based on the expression of five enzymes, namely alcohol dehydrogenase, amine dehydrogenase, polypeptide synthetase, polyphosphate kinase 2-I and polyphosphate kinase 2-II, through a reasonable expression strategy, effectively ensures the continuous progress of the enzyme catalytic reaction, and increases the yield of Panax notoginseng.

(2) Common notoginseng extracted from plants in nature are all D-type. The present invention obtains alcohol dehydrogenase, amine dehydrogenase and peptide synthetase that can use L-serine as a substrate. On this basis, L-notoginseng is synthesized by a biological method using L-serine and oxalic acid as raw materials.

DETAILED DESCRIPTION

1. Strains and plasmids involved in the present invention

pRSFDuet-1, pETDuet-1, pCDFDuet-1, pACYCDuet-1, pCOLDⅡ plasmids and Escherichia coli BL21 (DE3) were purchased from Novagen.

2. Construction of multi-gene co-expression system and cell culture

There are many methods for co-expression of multiple genes in Escherichia coli (for example, the method described in the article "Strategy for co-expression of multiple genes in Escherichia coli, Journal of Chinese Biotechnology, 2012, 32 (4): 117-122"). The present invention adopts the method described in Liu Xianglei's doctoral thesis (Synthetic biology technology to transform Escherichia coli to produce shikimic acid and resveratrol, 2016, Shanghai Institute of Pharmaceutical Industry) to construct recombinant Escherichia coli. In the following embodiment, when co-expressing multiple genes, each gene contains the T7 promoter and RBS binding site of Escherichia coli BL21 (DE3), and each gene is followed by a T7 terminator. In theory, because each gene has a T7 promoter and RBS binding site, the expression intensity of the gene is not greatly affected by the order of arrangement of the genes on the plasmid. The constructed plasmid is heat-transfected into Escherichia coli competent cells, and coated on a monoclonal antibody or mixed antibiotic solid plate, and positive transformants are screened to obtain recombinant Escherichia coli.

Cell culture: According to the classic recombinant E. coli culture and induced expression scheme, the recombinant E. coli was transferred to LB fermentation medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L) at a volume ratio of 2%. When the cell OD600 reached 0.6-0.8, IPTG with a final concentration of 0.4mM was added and induced expression was cultured at 20°C for 8h. After the induced expression was completed, the cells were collected by centrifugation at 4°C, 8000rpm, and 20min.

3. Selection of relevant enzymes

(1) Polyphosphate kinase 2-I

The gene smpkk encoding polyphosphate kinase 2-I from Sinorhizobium_meliloti was selected. The accession number of the gene smpkk on NCBI is NC_003047REGION:complement(564142..565044), and the corresponding amino acid sequence is NP_384613.1. The enzyme can catalyze AMP to generate ADP, and the phosphate group is provided by polyphosphate.

(2) Polyphosphate kinase 2-II

The gene ajpkk encoding polyphosphate kinase 2-II from Acinetobacter johnsonii was selected. The accession number of the gene ajpkk on NCBI is AB092983 REGION:339..1766, and the corresponding amino acid sequence is BAC76403.1. The enzyme can catalyze ADP to generate ATP, and the phosphate group is provided by polyphosphate.

(3) Polyphosphate kinase 2-III

The gene mhpkk encoding polyphosphate kinase 2-III from Meiothermus hypogaeus was selected. The accession number of the gene mhpkk on NCBI is NZ_BJXL01000029 REGION:complement(14116..14919), and the corresponding amino acid sequence is WP_119340583.1. The enzyme can catalyze AMP to directly generate ATP, with the phosphate group provided by polyphosphate.

4. Sample detection and analysis

The content of notoginseng was determined according to the literature (Qiao CF, Liu XM, Cui XM, et al. High-performance anion-exchange chromatography coupled with diode array detection for the determination of dencichine in Panax notoginseng and related species. Journal of Separation Science. 2013 Aug; 36(15): 2401-2406.)

The activity of ATP-dependent peptide synthetase was determined according to the literature: Petchey, Mark et al. "The Broad Aryl Acid Specificity of the Amide Bond Synthetase McbA Suggests Potential for the Biocatalytic Synthesis of Amides." Angewandte Chemie (International ed. in English) vol. 57, 36 (2018): 11584-11588.

The 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 dehydrogenases from Lactobacillus reuteri DSM20016, Process Biochemistry.

The activity of amine dehydrogenase was determined according to the literature: Abrahamson MJ, Vázquez-Figueroa E, Woodall NB, Moore JC, Bommarius AS. Development of an amine dehydrogenase for synthesis of chiral amines. Angew Chem Int Ed Engl. 2012; 51(16): 3969-3972.

Specific enzyme 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 generate 1 μmol of product in 1 min.

Example 1: Screening and expression of alcohol dehydrogenase

Alcohol dehydrogenase is widely present in various organisms. Alcohol dehydrogenase genes scadh, apadh, ttadh, cbadh, and lradh were synthesized based on 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 have access numbers of NP_014555.1, BAA81251.2, WP_011228103.1, WP_012060249.1, and ABQ83742.1 on NCBI. The synthesized genes were separately connected to the multiple cloning site of the pETDuet-1 vector, and induced to express in Escherichia coli BL21 (DE3), and 5 recombinant Escherichia coli were obtained.

Induced expression method: 5 kinds of recombinant E. coli were inoculated into LB fermentation medium at a volume ratio of 2%. When the cell OD ₆₀₀ reached 0.6-0.8, IPTG with a final concentration of 0.4mM was added and induced expression was cultured at 20°C for 8h. After the induction expression was completed, the cells were collected by centrifugation at 4°C, 8000rpm, and 20 minutes. After the cells were broken, the enzyme was purified by the His-tag labeling method, and the activity was measured after the pure enzyme was obtained.

When D-serine was used as substrate, the specific enzyme activities of the enzymes expressed by the genes of alcohol dehydrogenase scadh, apadh, ttadh, cbadh, and lradh were 124, 87, 56, 98, and 156 U/mg, respectively.

When L-serine was used as substrate, the specific enzyme activities of the enzymes expressed by the genes of alcohol dehydrogenase scadh, apadh, ttadh, cbadh, and lradh were 32, 46, 51, 39, and 78 U/mg, respectively.

Example 2: Screening and expression of amine dehydrogenase

Amine dehydrogenase is widely present in plants. Amine dehydrogenase genes baamdh, vfamdh, rpamdh, and pyamdh were synthesized based on the amine dehydrogenase gene information of Burkholderia ambifaria AMMD, Vibriofurnissii, Rhodococcus pyridinivorans, and Pseudomonas yamanorum on NCBI. The corresponding amino acid sequences have accession numbers of WP_011659448.1, WP_004726087.1, WP_033097867.1, and WP_063029039.1 on NCBI. The synthesized genes were separately connected to the pETDuet-1 vector and induced to express in Escherichia coli BL21 (DE3) to obtain 4 recombinant Escherichia coli. The induction expression method was the same as in Example 1.

When (R)-2-amino-3-oxopropionic acid was used as substrate, the specific enzyme activities of the enzymes expressed by the genes of amine dehydrogenases baamdh, vfamdh, rpamdh, and pyamdh were 156, 125, 107, and 114 U/mg, respectively.

When (S)-2-amino-3-oxopropionic acid was used as substrate, the specific enzyme activities of the enzymes expressed by the genes of amine dehydrogenases baamdh, vfamdh, rpamdh, and pyamdh were 138, 72, 89, and 102 U/mg, respectively.

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

ATP-dependent peptide synthetase is widely present in plants. The ATP-dependent peptide synthetase genes srabs, alabs, tvabs, reabs, and bsabs were synthesized based on the ATP-dependent peptide synthetase gene information of Streptomyces rimosus, Acinetobacter lactuca, Trichoderma virens Gv29-8, Rhodococcus erythropolis PR4, and Bacillus safensis on NCBI. The corresponding amino acid sequences have access numbers of WP_050504588.1, WP_016145237.1, XP_013952649.1, WP_020907735.1, and WP_048238116.1 on NCBI. The synthesized genes were separately connected to the pETDuet-1 vector and induced to express in Escherichia coli BL21 (DE3) to obtain 5 recombinant Escherichia coli. The induced expression method was the same as in Example 1.

When (R)-2,3-diaminopropionic acid and oxalic acid were 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 were 73, 121, 86, 45, and 49 U/mg, respectively.

When (S)-2,3-diaminopropionic acid and oxalic acid were 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 were 35, 36, 28, 45, and 68 U/mg, respectively.

Example 4: Construction of recombinant Escherichia coli expressing five enzymes simultaneously

As shown in Table 1, 4-5 enzyme genes were selected from the five plasmids of pETDuet-1, pACYCDuet-1, pRSFDuet-1, pCDFduet-1, and pCOLDⅡ, and the genes encoding 6 enzymes were connected to the same plasmid, or connected to two plasmids (2-3 genes were expressed on each plasmid), or connected to five plasmids (1 gene was expressed on each plasmid). The gene fragment was cloned into the multiple cloning site of the plasmid, and each gene contained the T7 promoter and RBS binding site of Escherichia coli BL21 (DE3) in front of each gene, and each gene was followed by a T7 terminator. The constructed recombinant plasmid was transformed into Escherichia coli BL21, and positive transformants were obtained by screening different mixed antibiotic plates according to the resistance genes on different plasmids, that is, recombinant Escherichia coli that can enhance the expression of 5 genes was obtained.

The recombinant E. coli was induced to express, and the cells were collected after the induction expression was completed. In a 100 mL reaction system, 200 g/L of cells, 100 g/L of L-serine, 100 g/L of oxalic acid, 1 g/L of ATP, 1 g/L of NAD, and 300 g/L of sodium hexamethaphosphate were added, and the reaction time was 24 h. The pH was 5.0-9.0 and the temperature was 15-40°C.

Table 1

Example 5: In vitro catalytic synthesis of D-notoginseng using five enzymes

The five genes of smpkk, ajpkk, lradh, baamdh and alabs were connected to the pEDT28a vector to obtain five recombinant vectors, and the five recombinant vectors were transformed into Escherichia coli BL21 to obtain recombinant Escherichia coli expressing five enzymes. Five pure enzymes were obtained after expression and purification in the same way as in Example 1. Then, 2 mg of each of the five pure enzymes, 100 g/L of D-serine, 100 g/L of oxalic acid, 1 g/L of ATP, 1 g/L of NAD, 60 g/L of sodium hexapolyphosphate, pH 7.0 were added to a 100 mL reaction system; the reaction was carried out at 35 ° C for 48 h. The yield of D-notoginseng was detected to be 62 g/L.

Example 6: In vitro catalytic synthesis of D-notoginseng using four enzymes

The four genes mhpkk, lradh, baamdh and alabs were connected to the pED28a vector to obtain four recombinant vectors, and the four recombinant vectors were transformed into Escherichia coli BL21 to obtain four recombinant Escherichia coli. Four pure enzymes were obtained after expression and purification using the same method as in Example 1. Then, 2 mg of each of the four pure enzymes, 100 g/L of D-serine, 100 g/L of oxalic acid, 1 g/L of ATP, 1 g/L of NAD, and 200 g/L of sodium hexapolyphosphate were added to a 100 mL reaction system, and the reaction was carried out at 35°C for 48 hours. The yield of D-notoginseng was detected to be 166 g/L.

Example 7: Synthesis of D-notoginseng using combined catalysis of recombinant cells

The five genes smpkk, ajpkk, lradh, baamdh and alabs were connected to the pEDTDuet-1 vector to obtain five recombinant vectors, which were transformed into Escherichia coli BL21 to obtain five recombinant Escherichia coli. The recombinant Escherichia coli was induced to express enzymes in the same manner as in Example 1. Then, 20 g/L of each of the five whole cells, 100 g/L of D-serine, 100 g/L of oxalic acid, 1 g/L of ATP, 100 g/L of sodium hexapolyphosphate, and 1 g/L of NAD were added to a 100 mL reaction system, and the reaction was carried out at 25 ° C for 24 h. The yield of D-notoginseng was detected to be 153 g/L.

Example 8: Synthesis of D-notoginseng and L-notoginseng using recombinant Escherichia coli whole cells

All the notoginseng in nature are of D type. The present invention further uses L-serine and oxalic acid as raw materials to synthesize L-notoginseng. Previously, L-notoginseng has not been synthesized by biological methods.

The genes reabs and bsabs encoding peptide synthetases from Rhodococcus erythropolis PR4 and Bacillus safensis were selected, together with genes encoding polyphosphate kinase 2-I, polyphosphate kinase 2-II, alcohol dehydrogenase and amine dehydrogenase, to construct recombinant bacteria Escherichia coli BL21 (DE3) / pRSFDuet-1-smpkk-ajpkk and pETDuet-1-lradh-baamdh-alabs or Escherichia coli BL21 (DE3) / pRSFDuet-1-smpkk-ajpkk and pETDuet-1-lradh-baamdh-bsabs according to the method described in Example 1, induced to express, and then the bacteria were collected.

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

Example 9: Synthesis of L-notoginseng using recombinant Escherichia coli whole cells

The following two recombinant bacteria were constructed: Escherichia coli BL21 (DE3)/pRSFDuet-1-mhpkk (named E1) and Escherichia coli BL21 (DE3)/pETDuet-1-lradh-baamdh-alabs (named E2).

According to the method described in Example 1, E1 and E2 were induced to express respectively, and then the cells were collected. In a 100 mL reaction system, the wet weight of E1 cells was 30 g/L, the wet weight of E2 cells was 50 g/L, L-serine was 100 g/L, oxalic acid was 100 g/L, sodium hexapolyphosphate was 300 g/L, NAD was 1 g/L, ATP was 1 g/L, pH was 7.0; the reaction was carried out at 40°C for 48 hours. After the conversion was completed, the content of L-notoginseng was determined by liquid chromatography to be 173 g/L.

Example 10: Synthesis of D-notoginseng using recombinant Escherichia coli whole cells

The following two recombinant bacteria were constructed: Escherichia coli BL21 (DE3)/pRSFDuet-1-smpkk-ajpkk-alabs (named E3) and Escherichia coli BL21 (DE3)/pACYCDuet-1-lradh-baamdh (named E4).

According to the method described in Example 1, E3 and E4 were induced to express respectively, and then the cells were collected. In a 100 mL reaction system, the wet weight of E3 cells was 100 g/L, the wet weight of E4 cells was 100 g/L, D-serine 50 g/L, oxalic acid 50 g/L, NAD 1 g/L, ATP 1 g/L, sodium hexapolyphosphate 30 g/L, pH 7.0; the reaction was carried out at 40°C for 12 hours. After the conversion was completed, the content of D-panax notoginseng was determined by liquid chromatography to be 65 g/L.

Example 11: Synthesis of L-notoginseng using recombinant Escherichia coli whole cells

The following two recombinant bacteria were constructed: Escherichia coli BL21 (DE3)/pRSFDuet-1-smpkk-ajpkk-bsabs (named E5) and Escherichia coli BL21 (DE3)/pACYCDuet-1-lradh-baamdh (E4).

According to the method described in Example 1, E5 and E4 were induced to express respectively, and then the cells were collected. In a 100 mL reaction system, the wet weight of E5 cells was 100 g/L, the wet weight of E4 cells was 100 g/L, L-serine was 10 g/L, oxalic acid was 10 g/L, NAD was 0.1 g/L, ATP was 0.1 g/L, sodium hexapolyphosphate was 20 g/L, pH was 7.0; the reaction was carried out at 30°C for 1 hour. After the conversion was completed, the content of L-notoginseng was determined by liquid chromatography to be 18 g/L.

Example 12: Synthesis of D-notoginseng using recombinant Escherichia coli whole cells

The following two recombinant bacteria were constructed: Escherichia coli BL21 (DE3)/pRSFDuet-1-lradh-baamdh (named E6) and Escherichia coli BL21 (DE3)/pACYCDuet-1-smpkk-ajpkk-alabs (named E7).

According to the method described in Example 1, E6 and E7 were induced to express respectively, and then the cells were collected. In a 100 mL reaction system, the wet weight of E6 cells was 100 g/L, the wet weight of E7 cells was 100 g/L, D-serine 50 g/L, oxalic acid 50 g/L, NAD 0.5 g/L, ATP 0.5 g/L, sodium hexapolyphosphate 60 g/L, pH 7.0; react at 40 ° C for 5 hours. After the conversion was completed, the content of D-panax notoginseng was determined by liquid chromatography to be 80 g/L.

Example 13: Synthesis of L-notoginseng using recombinant Escherichia coli whole cells

The following two recombinant bacteria were constructed: Escherichia coli BL21 (DE3)/pRSFDuet-1-lradh-baamdh (E6) and Escherichia coli BL21 (DE3)/pACYCDuet-1-mhpkk-bsabs (named E8).

According to the method described in Example 1, E6 and E8 were induced to express respectively, and then the cells were collected. In a 100 mL reaction system, the wet weight of E6 cells was 10 g/L, the wet weight of E8 cells was 20 g/L, L-serine was 1 g/L, oxalic acid was 1 g/L, NAD was 0 g/L, ATP was 0 g/L, sodium hexapolyphosphate was 2 g/L, pH was 7.0; the reaction was carried out at 30°C for 1 hour. After the conversion was completed, the content of L-notoginseng was determined by liquid chromatography to be 1.8 g/L.

Various changes and modifications can be made without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the 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.