A strain and method for producing notoginseng extract

Abstract

This invention discloses a bacterial strain and method for producing notoginseng extract, belonging to the field of bioengineering technology. This invention constructs recombinant cells or combinations of recombinant cells expressing alcohol dehydrogenase, amine dehydrogenase, ATP-dependent peptide synthase, polyphosphokinase 2-I, and polyphosphokinase 2-II, or replaces polyphosphokinase 2-I and polyphosphokinase 2-II with polyphosphokinase 2-III. The recombinant cells or combinations of recombinant cells are used to catalyze the synthesis of notoginseng extract from serine and oxalic acid. Using 100 g / L L-serine as a substrate, a maximum yield of 173 g / L L-notoginseng extract can be obtained. Therefore, this invention has good prospects for industrial application.

Description

Technical Field

[0001] This invention relates to a strain and method for producing notoginseng extract, belonging to the field of bioengineering technology. Background Technology

[0002] Decichine, also known as Panax notoginseng amino acid, chemically named β-oxalyl-L-diaminopropionic acid, is the main hemostatic active ingredient in Panax notoginseng, a precious traditional Chinese medicine. In 1981, Takuo Kosuge first extracted and isolated decichine from Panax notoginseng. Further research has demonstrated that decichine not only possesses hemostatic and platelet-increasing pharmacological activities, but also neuroprotective effects, reduces diabetic kidney damage, has anti-inflammatory properties, and lowers blood sugar levels. Decichine has one chiral carbon atom, thus having two isomers: the l-form and the d-form. As a special non-protein free amino acid, decichine is extremely rare in nature, and those isolated from natural plants are all in the l-configuration. With continuous research into the biological activity and in vivo metabolic characteristics of decichine, it has gained increasing attention as a high-value medicinal component and has become a research hotspot.

[0003] Currently, the industrial production of notoginseng is still immature, with most research remaining in the laboratory stage. There are two main methods for small-scale preparation of notoginseng: 1) Chemical synthesis; however, this method is costly, generates various byproducts, is difficult to purify and separate, and does not easily yield optically pure D / L notoginseng. 2) Extraction from plants such as ginseng and notoginseng (Araliaceae family). Extraction from plants is simple, easy to implement, and ensures quality. However, this method requires large amounts of organic solvents, is time-consuming, and has a low extraction rate. Therefore, developing an efficient method for synthesizing notoginseng, especially a green microbial synthesis method, would have greater application value. Summary of the Invention

[0004] The industrial production of Panax notoginseng is still immature. Both chemical synthesis and plant extraction methods have significant drawbacks, resulting in high costs and low yields. There is an urgent need for a method that is both high-yield and environmentally friendly.

[0005] This invention provides a method for synthesizing D / L-glucan using D / L-serine as a substrate. Serine is converted into 2,3-diaminopropionic acid by co-expression of NAD-dependent alcohol dehydrogenase (ADH) and NADH-dependent amine dehydrogenase (AmDH). An ATP-dependent peptide bond synthase condenses 2,3-diaminopropionic acid and oxalic acid to form glucan. ATP releases energy to be converted into AMP. Polyphosphate kinase PPK2-II is used to regenerate AMP into ADP, and polyphosphate kinase 2-I further generates ATP from ADP.

[0006] This invention provides the application of alcohol dehydrogenase, amine dehydrogenase, peptide bond synthase, polyphosphate kinase 2-I and polyphosphate kinase 2-II in the synthesis of Panax notoginseng.

[0007] In one embodiment, the application uses carboxylic acid as a substrate and microbial cells that overexpress alcohol dehydrogenase, amine dehydrogenase, peptide bond synthase, polyphosphate kinase 2-I and polyphosphate kinase 2-II or polyphosphate kinase 2-II as catalysts to catalyze the synthesis of Panax notoginseng.

[0008] In one embodiment, the carboxylic acid is D / L-serine and oxalic acid.

[0009] In one embodiment, the overexpression is achieved by expressing one or more genes encoding alcohol dehydrogenase, amine dehydrogenase, peptide bond synthase, polyphosphate kinase 2-I, or polyphosphate kinase 2-II via a vector or by integrating them into the host genome.

[0010] In one embodiment, the overexpression is achieved by using one vector to co-express the genes of the above five enzymes; or by using multiple vectors to co-express the genes of the above five enzymes, with each vector expressing at least one enzyme gene, and each vector expressing a different gene.

[0011] In one embodiment, the overexpression is the expression of genes for 1 to 5 enzymes on a vector: one vector expresses one enzyme gene; or one vector expresses 5 enzyme genes in total; or one vector expresses 2 to 3 enzyme genes in total, wherein the gene encoding alcohol dehydrogenase and the gene encoding amine dehydrogenase are on one vector, and the genes encoding polyphosphate kinase 2-I and polyphosphate kinase 2-II are on another vector.

[0012] In one implementation, when genes encoding five enzymes are ligated into a vector, each gene contains a T7 promoter and an RBS binding site before it, and a T7 terminator after it.

[0013] In one embodiment, the carrier includes, but is not limited to, pETDuet-1, pACYCDuet-1, pRSFDuet-1, pCDFduet-1, and pCOLDⅡ.

[0014] In one embodiment, the recombinant cells use Escherichia coli as a host, including but not limited to Escherichiacoli BL21(DE3).

[0015] In one embodiment, the alcohol dehydrogenase is derived from Saccharomyces cerevisiae S288C, Aeropyrum pernix K1, Thermus thermophilus HB8, Clostridium beijerinckii, or Lactobacillus reuteri DSM20016.

[0016] 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, respectively; and the NCBI accession numbers of the corresponding nucleotide sequences are NC_001147.6, APE_2239.1, NC_006461.1, LZZG01000005.1 and NC_009513.1.

[0017] In one embodiment, the amine dehydrogenase is derived from Burkholderia ambifaria AMMD, Vibriofurnissii, Rhodococcus pyridinivorans, or Pseudomonasyamanorum.

[0018] 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, respectively; and the corresponding NCBI accession numbers of the nucleotide sequences are NC_008391.1, NZ_CP040990.1, NZ_FNRX01000002.1 and NZ_CP012400.2.

[0019] In one embodiment, the peptide bond synthase is derived from Streptomyces rimosus, Acinetobacter lactuca, Trichoderma virens Gv29-8, Rhodococcus erythropolis PR4, or Bacillus safensis.

[0020] In one embodiment, the NCBI accession numbers of the amino acid sequences of the peptide bond synthase are WP_050504588.1, WP_016145237.1, XP_013952649.1, WP_020907735.1 and WP_048238116.1, respectively; and the corresponding NCBI accession numbers of the nucleotide sequences are NZ_CP023688.1, NZ_KB976991.1, NW_014013663.1, NC_012490.1 and NZ_LDUS01000007.1.

[0021] In one embodiment, the polyphosphate kinase 2-I is derived from Sinorhizobium meliloti; its amino acid sequence has an NCBI accession number of NP_384613.1; and the corresponding nucleotide sequence has an NCBI accession number of NC_003047REGION:complement(564142...565044).

[0022] In one embodiment, the polyphosphate kinase 2-II is derived from Acinetobacter johnsonii; its amino acid sequence has an NCBI accession number of BAC76403.1; and the corresponding nucleotide sequence has an NCBI accession number of AB092983REGION:339...1766.

[0023] In one embodiment, the notoginsenoside is D-notoginsenoside or L-notoginsenoside. When the notoginsenoside is L-notoginsenoside, a peptide bond synthase from the strains Rhodococcus erythropolis PR4 and Bacillus safensis is selected.

[0024] 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 synthase, polyphosphate kinase 2-I and polyphosphate kinase 2-II, and each recombinant cell does not repeat the expression of other recombinant cells.

[0025] In one embodiment, the combination of recombinant cells uses Escherichia coli as a host, such as Escherichiacoli BL21(DE3).

[0026] In one embodiment, the overexpression involves using multiple vectors to co-express the genes of five enzymes, with each vector expressing the gene of at least one enzyme, and each vector expressing a different gene.

[0027] In one embodiment, the overexpression of the recombinant cell combination is a vector that co-expresses 1 to 5 enzyme genes: one vector expresses 1 enzyme gene, for a total of 5 vectors; or one vector co-expresses 5 enzyme genes; or one vector co-expresses 2 to 3 enzyme genes, for a total of 2 vectors, wherein the gene encoding alcohol dehydrogenase and the gene encoding amine dehydrogenase are on one vector, and the genes encoding polyphosphate kinase 2-I and polyphosphate kinase 2-II are on another vector.

[0028] In one implementation, when genes encoding five enzymes are ligated into a vector, each gene contains a T7 promoter and an RBS binding site before it, and a T7 terminator after it.

[0029] In one embodiment, the carrier includes, but is not limited to, pETDuet-1, pACYCDuet-1, pRSFDuet-1, pCDFduet-1, and pCOLDⅡ.

[0030] Specifically, the present invention also provides a recombinant cell for synthesizing notoginseng extract, wherein the recombinant cell expresses genes encoding alcohol dehydrogenase, amine dehydrogenase, ATP-dependent peptide bond synthase, and polyphosphate kinase 2-I and polyphosphate kinase 2-II; the recombinant cell uses *Escherichia coli* as a host and pRSFDuet-1 as a vector to express genes encoding polyphosphate kinase 2-I and polyphosphate kinase 2-II, and pTDuet-1 as a vector to express genes encoding alcohol dehydrogenase and amine dehydrogenase. Each gene is preceded by a T7 promoter and an RBS binding site, and each gene is followed by a T7 terminator.

[0031] The present invention also provides a method for whole-cell catalytic production of notoginseng, which uses the recombinant cells or combinations of recombinant cells of the present invention as whole-cell catalysts to synthesize D-notoginseng (L-notoginseng) using oxalic acid and D-serine (L-serine) as substrates.

[0032] In one embodiment, the whole-cell catalyst is prepared by culturing and propagating recombinant cells or combinations of recombinant cells, enabling the recombinant cells or combinations of recombinant cells to express the five enzymes, and then collecting the recombinant cells. When using the whole-cell catalyst, in addition to providing the substrate, it is necessary to maintain appropriate temperature and pH, and if necessary, provide some coenzymes or nutrients to help the whole-cell catalyst better exert its catalytic effect.

[0033] In one embodiment, the whole-cell conversion production system includes cells with a 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 hexapolyphosphate 2-300 g / L, and pH 5.0-9.0; the reaction is carried out at 15-40°C for 1-48 h.

[0034] This invention also protects the application of the above-mentioned combination of recombinant cells or whole-cell catalytic production method of notoginseng in the production of notoginseng or products containing notoginseng or substances with notoginseng as a precursor.

[0035] Beneficial effects

[0036] (1) Through a reasonable expression strategy, this invention achieves the dual coenzyme regeneration of NAD and ATP on the basis of expressing five enzymes: alcohol dehydrogenase, amine dehydrogenase, polypeptide synthase, polyphosphate kinase 2-I, and polyphosphate kinase 2-II. This effectively ensures the continuous progress of enzyme-catalyzed reactions and increases the yield of Panax notoginseng.

[0037] (2) The common notoginseng extracts extracted from plants in nature are all of type D. This invention obtained alcohol dehydrogenase, amine dehydrogenase and peptide bond synthase that can use L-serine as a substrate. Based on this, L-ginseng was synthesized by biological method using L-serine and oxalic acid as raw materials. Detailed Implementation

[0038] 1. The strains and plasmids involved in this invention

[0039] pRSFDuet-1, pETDuet-1, pCDFDuet-1, pACYCDuet-1, pCOLDⅡ plasmids and Escherichia coli BL21(DE3) were purchased from Novagen.

[0040] 2. Construction of a multi-gene co-expression system and cell culture

[0041] There are various methods for co-expressing multiple genes in *E. coli* (e.g., the method described in the article "Multi-gene Co-expression Strategy of *E. coli*, *Chinese Journal of Biotechnology*, 2012, 32(4):117-122"). This invention uses the method described in Liu Xianglei's doctoral dissertation (Synthetic Biology Technology to Modify *E. coli* to Produce Shikimic Acid and Resveratrol, 2016, Shanghai Institute of Pharmaceutical Industry) to construct recombinant *E. coli*. In the following examples, when co-expressing multiple genes, each gene is preceded by the T7 promoter and RBS binding site of *E. coli* BL21(DE3), and each gene is followed by a T7 terminator. Theoretically, because each gene is preceded by a T7 promoter and RBS binding site, the gene expression intensity is not significantly affected by the gene arrangement order on the plasmid. The constructed plasmid is heat-transformed into competent *E. coli* cells and plated on monoclonal antibody or mixed antibiotic solid plates. Positive transformants are screened to obtain recombinant *E. coli*.

[0042] Cell culture: Following the classic recombinant E. coli culture and induction expression protocol, recombinant E. coli were transferred to LB fermentation medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl) at a volume ratio of 2%. When the cell OD600 reached 0.6-0.8, IPTG was added to a final concentration of 0.4 mM, and expression was induced at 20°C for 8 h. After induction, cells were collected by centrifugation at 4°C, 8000 rpm, and 20 min.

[0043] 3. Selection of relevant enzymes

[0044] (1) Polyphosphate kinase 2-I

[0045] The gene smpkk, encoding polyphosphate kinase 2-I, from *Sinorhizobium meliloti*, was selected. The smpkk gene has the NCBI accession number NC_003047REGION:complement(564142..565044) and the corresponding amino acid sequence NP_384613.1. This enzyme catalyzes the conversion of AMP to ADP, with polyphosphate groups provided by the polyphosphate groups.

[0046] (2) Polyphosphate kinase 2-II

[0047] The gene ajpkk encoding polyphosphate kinase 2-II from *Acinetobacter johnsonii* was selected. The sequence of ajpkk, with accession number AB092983 REGION:339..1766 on NCBI, corresponds to the amino acid sequence BAC76403.1. This enzyme catalyzes the conversion of ADP to ATP, with polyphosphate groups provided by polyphosphate.

[0048] (3) Polyphosphate kinase 2-III

[0049] The gene *mhpkk* encoding polyphosphate kinase 2-III from *Meiothermus hypogaeus* was selected. The sequence of *mhpkk*, with NCBI accession number NZ_BJXL01000029, REGION:complement(14116..14919), corresponds to the amino acid sequence WP_119340583.1. This enzyme catalyzes the direct conversion of AMP to ATP, with polyphosphate groups provided by polyphosphate.

[0050] 4. Sample detection and analysis

[0051] The method for determining the content of dencichine was based on 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.)

[0052] The activity of ATP-dependent peptide synthase was determined according to the literature: Petchey, Mark et al. "The Broad ArylAcid 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.

[0053] Alcohol dehydrogenase activity was determined according to the literature: Zhenghong Hu, Pu Jia, Yajun Bai, Tai-ping Fan, Xiaohui Zheng, Yujie Cai, Characterization of five alcohol dehydrogenases from Lactobacillus reuteri DSM20016, Process Biochemistry.

[0054] The amine dehydrogenase activity 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.

[0055] Enzyme activity (U mg) -1 Enzyme activity (U) is defined as the amount of enzyme required to produce 1 μmol of product per mg of enzyme.

[0056] Example 1: Screening and expression of alcohol dehydrogenase

[0057] Alcohol dehydrogenases are widely found in various organisms. 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 alcohol dehydrogenase genes scadh, apadh, ttadh, cbadh, and lradh were synthesized. Their corresponding amino acid sequences have NCBI accession numbers of NP_014555.1, BAA81251.2, WP_011228103.1, WP_012060249.1, and ABQ83742.1, respectively. The synthesized genes were individually ligated into the multiple cloning site of the pETDuet-1 vector and induced to express in *E. coli* BL21(DE3), resulting in five recombinant *E. coli* strains.

[0058] Induction method: Five recombinant Escherichia coli strains were inoculated into LB fermentation medium at a volume ratio of 2%. When the cell OD... 600 After reaching a concentration of 0.6-0.8, IPTG was added to a final concentration of 0.4 mM, and expression was induced at 20°C for 8 h. After induction, cells were collected by centrifugation at 4°C, 8000 rpm for 20 minutes. After cell lysis, the enzyme was purified using the His-tag method, and its activity was measured after obtaining the purified enzyme.

[0059] When D-serine is used as a substrate, the specific enzyme activities of the enzymes expressed by the genes of alcohol dehydrogenases scadh, apadh, ttadh, cbadh, and lradh are 124, 87, 56, 98, and 156 U / mg, respectively.

[0060] When L-serine is used as a substrate, the specific enzyme activities of the enzymes expressed by the genes of alcohol dehydrogenases scadh, apadh, ttadh, cbadh, and lradh are 32, 46, 51, 39, and 78 U / mg, respectively.

[0061] Example 2: Screening and Expression of Amine Dehydrogenases

[0062] Amine dehydrogenases are widely found in plants. Based on the amine dehydrogenase gene information of *Burkholderia ambifaria* AMMD, *Vibriofurnissii*, *Rhodococcus pyridinivorans*, and *Pseudomonas yamanorum* on NCBI, the amine dehydrogenase genes baamdh, vfamdh, rpamdh, and pyamdh were synthesized. Their corresponding amino acid sequences have NCBI accession numbers of WP_011659448.1, WP_004726087.1, WP_033097867.1, and WP_063029039.1, respectively. The synthesized genes were individually ligated into the pETDuet-1 vector and induced to express in *Escherichia coli* BL21(DE3), resulting in four recombinant *E. coli* strains. The induction method was the same as in Example 1.

[0063] When (R)-2-amino-3-oxopropionic acid is used as a substrate, the specific enzyme activities of the amine dehydrogenases baamdh, vfamdh, rpamdh, and pyamdh expressed by their respective genes are 156, 125, 107, and 114 U / mg, respectively.

[0064] When (S)-2-amino-3-oxopropionic acid is used as a substrate, the specific enzyme activities of the amine dehydrogenases baamdh, vfamdh, rpamdh, and pyamdh expressed by their respective genes are 138, 72, 89, and 102 U / mg.

[0065] Example 3: Screening and expression of ATP-dependent peptide synthases

[0066] ATP-dependent peptide synthases are widely found in plants. Based on the ATP-dependent peptide synthase gene information of *Streptomyces rimosus*, *Acinetobacter lactuca*, *Trichoderma virens* Gv29-8, *Rhodococcus erythropolis* PR4, and *Bacillus safensis* from NCBI, the ATP-dependent peptide synthase genes *srabs*, *alabs*, *tvabs*, *reabs*, and *bsabs* were synthesized. The corresponding amino acid sequences have the following NCBI accession numbers: WP_050504588.1, WP_016145237.1, XP_013952649.1, WP_020907735.1, and WP_048238116.1. The synthesized genes were individually ligated into the pETDuet-1 vector and induced to express in *E. coli* BL21(DE3), resulting in five recombinant *E. coli* strains. The induction method was the same as in Example 1.

[0067] When (R)-2,3-diaminopropionic acid and oxalic acid are used as substrates, the specific enzyme activities of the ATP-dependent peptide synthases rsrabs, alabs, tvabs, reabs, and bsabs expressed by their respective genes are 73, 121, 86, 45, and 49 U / mg.

[0068] When (S)-2,3-diaminopropionic acid and oxalic acid are used as substrates, the specific enzyme activities of the ATP-dependent peptide synthases srabs, alabs, tvabs, reabs, and bsabs expressed by their respective genes are 35, 36, 28, 45, and 68 U / mg.

[0069] Example 4: Construction of recombinant Escherichia coli expressing five enzymes simultaneously

[0070] As shown in Table 1, from five plasmids—pETDuet-1, pACYCDuet-1, pRSFDuet-1, pCDFduet-1, and pCOLDⅡ—genes encoding six enzymes were selected and ligated to the same plasmid, or to two plasmids (each expressing 2-3 genes), or to five plasmids (each expressing one gene). The gene fragments were cloned into the multiple cloning site of the plasmids. Each gene was preceded by the T7 promoter and RBS binding site of *Escherichia coli* BL21(DE3), and followed by a T7 terminator. The constructed recombinant plasmids were transformed into *E. coli* BL21. Positive transformants were obtained by screening with different mixed antibiotic plates based on the resistance genes on different plasmids, thus obtaining recombinant *E. coli* strains capable of enhanced expression of five genes.

[0071] Recombinant Escherichia coli was induced to express the compound. After induction, the bacterial cells were collected and placed in a 100 mL reaction system containing 200 g / L bacterial cells, 100 g / L L-serine, 100 g / L oxalic acid, 1 g / L ATP, 1 g / L NAD, and 300 g / L sodium hexametaphosphate. The reaction time was 24 h. The pH range was 5.0-9.0, and the temperature was 15-40℃.

[0072] Table 1

[0073]

[0074]

[0075] Example 5: In vitro synthesis of D-glucosinolate using five enzymes

[0076] Five genes, smpkk, ajpkk, lradh, baamdh, and alabs, were ligated into the pEDT28a vector to obtain five recombinant vectors. These five recombinant vectors were then transformed into *Escherichia coli* BL21, yielding recombinant *E. coli* strains expressing the five enzymes, respectively. After expression and purification using the same method as in Example 1, five pure enzymes were obtained. Then, 2 mg of each of the five pure enzymes, 100 g / L D-serine, 100 g / L oxalic acid, 1 g / L ATP, 1 g / L NAD, and 60 g / L sodium hexametaphosphate were added to a 100 mL reaction system at pH 7.0. The reaction was carried out at 35°C for 48 h. The yield of D-glucosinolate was measured to be 62 g / L.

[0077] Example 6: In vitro synthesis of D-glucosinolate using four enzymes

[0078] The genes mhpkk, lradh, baamdh, and alabs were ligated into the pED28a vector to obtain four recombinant vectors. These four recombinant vectors were then transformed into *Escherichia coli* BL21 to obtain four recombinant *E. coli* strains. After expression and purification using the same method as in Example 1, four pure enzymes were obtained. Then, 2 mg of each of the four pure enzymes, 100 g / L D-serine, 100 g / L oxalic acid, 1 g / L ATP, 1 g / L NAD, and 200 g / L sodium hexametaphosphate were added to a 100 mL reaction system at pH 7.0. The reaction was carried out at 35°C for 48 h. The yield of D-glucosinolate was measured to be 166 g / L.

[0079] Example 7: Synthesis of D-Gynostemin using Combinatorial Catalysis of Recombinant Cells

[0080] Five genes, smpkk, ajpkk, lradh, baamdh, and alabs, were ligated into the pEDTDuet-1 vector to obtain five recombinant vectors. These five recombinant vectors were then transformed into *Escherichia coli* BL21 to obtain five recombinant *E. coli* strains. The recombinant *E. coli* strains were induced to express enzymes using the same method as in Example 1. Then, 20 g / L each of the five whole-cell enzymes, 100 g / L D-serine, 100 g / L oxalic acid, 1 g / L ATP, 100 g / L sodium hexametaphosphate, and 1 g / L NAD were added to a 100 mL reaction system, pH 8.0; the reaction was carried out at 25 °C for 24 h. The yield of D-glucosinolate was measured to be 153 g / L.

[0081] Example 8: Synthesis of D-glucosinolates and L-glucosinolates using whole-cell catalysis by recombinant Escherichia coli

[0082] In nature, all notoginsenosides are in the D form. This invention further synthesizes L-glucosinolate using L-serine and oxalic acid as raw materials. Previously, L-glucosinolate could not be synthesized by biological methods.

[0083] Genes encoding peptide synthases, reabs and bsabs, derived from Rhodococcus erythropolis PR4 and Bacillus safensis, were selected and, together with genes encoding polyphosphate kinase 2-I, polyphosphate kinase 2-II, alcohol dehydrogenase, and amine dehydrogenase, were used 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. These were induced to express the recombinant bacteria according to the method described in Example 1, and the bacterial cells were then collected.

[0084] In a 100 mL reaction system, the wet weight of cells was 100 g / L, D-serine or L-serine was 100 g / L, oxalic acid was 100 g / L, ATP was 1 g / L, NAD was 1 g / L, sodium hexapolyphosphate was 150 g / L, and pH was 6.0. The reaction was carried out at 30 °C for 48 h. When D-serine was used as the substrate, the yield of D-glucosinolate was 133 g / L. When L-serine was used as the substrate, the yield of L-glucosinolate after transformation was 58 g / L and 161 g / L, respectively.

[0085] Example 9: Synthesis of L-Gynostemin using whole-cell catalysis by recombinant Escherichia coli

[0086] 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).

[0087] According to the method described in Example 1, E1 and E2 cells were induced to express the cells, 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, and pH was 7.0. The reaction was carried out at 40 °C for 48 h. After transformation, the L-glucan content was determined by liquid chromatography to be 173 g / L.

[0088] Example 10: Synthesis of D-Gynostemin using whole-cell catalysis by recombinant Escherichia coli

[0089] 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).

[0090] According to the method described in Example 1, E3 and E4 cells were induced to express the cells, 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 was 50 g / L, oxalic acid was 50 g / L, NAD was 1 g / L, ATP was 1 g / L, sodium hexapolyphosphate was 30 g / L, and the pH was 7.0. The reaction was carried out at 40 °C for 12 h. After transformation, the D-glucosinolate content was determined by liquid chromatography to be 65 g / L.

[0091] Example 11: Synthesis of L-Ginsenoside using whole-cell catalysis by recombinant Escherichia coli

[0092] 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).

[0093] According to the method described in Example 1, E5 and E4 cells were induced to express the cells, 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, and the pH was 7.0. The reaction was carried out at 30 °C for 1 h. After transformation, the L-glucan content was determined by liquid chromatography to be 18 g / L.

[0094] Example 12: Synthesis of D-Gynostemin using whole-cell catalysis by recombinant Escherichia coli

[0095] 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).

[0096] According to the method described in Example 1, E6 and E7 cells were induced to express the cells, 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 was 50 g / L, oxalic acid was 50 g / L, NAD was 0.5 g / L, ATP was 0.5 g / L, sodium hexapolyphosphate was 60 g / L, and the pH was 7.0. The reaction was carried out at 40 °C for 5 h. After transformation, the D-glucosinolate content was determined by liquid chromatography to be 80 g / L.

[0097] Example 13: Synthesis of L-Gynostemin using whole-cell catalysis by recombinant Escherichia coli

[0098] 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).

[0099] According to the method described in Example 1, E6 and E8 cells were induced to express the cells, 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, and the pH was 7.0. The reaction was carried out at 30°C for 1 h. After transformation, the L-glucosinolate content was determined by liquid chromatography to be 1.8 g / L.

[0100] Various modifications and alterations may be made without departing from the spirit and scope of this invention. Therefore, the scope of protection of this invention should be determined by the claims.

Claims

1. The application of alcohol dehydrogenase, amine dehydrogenase, ATP-dependent peptide synthase, polyphosphate kinase 2-I, and polyphosphate kinase 2-II in the synthesis of notoginseng extract, characterized in that, Using D / L-serine and oxalic acid as substrates, Lactobacillus reuteri alcohol dehydrogenase derived from DSM20016, Burkholderia ambifaria AMMD-derived amine dehydrogenases, Acinetobacter lactuca or Bacillus safensis ATP-dependent peptide synthase from which it originates Sinorhizobium meliloti Polyphosphate kinase 2-I and its source Meiothermus hypogaeus Polyphosphate kinase 2-II source or overexpression Lactobacillus reuteri alcohol dehydrogenase derived from DSM20016, Burkholderia ambifaria AMMD-derived amine dehydrogenases, Acinetobacter lactuca or Bacillus safensis ATP-dependent peptide synthase from which it originates Sinorhizobium meliloti Polyphosphate kinase 2-I from source Meiothermus hypogaeus Microbial cells derived from polyphosphate kinase 2-II act as catalysts to catalyze the synthesis of notoginseng. The NCBI accession number for the amino acid sequence of the alcohol dehydrogenase is ABQ83742.1, and the NCBI accession number for the amino acid sequence of the amine dehydrogenase is WP_011659448.

1. Acinetobacter lactuca , Bacillus safensis The NCBI accession numbers for the amino acid sequences of the ATP-dependent peptide synthases are WP_016145237.1 and WP_048238116.1, respectively. The NCBI accession number for the amino acid sequence of polyphosphate kinase 2-I is NP_384613.

1. The amino acid sequence of polyphosphate kinase 2-II is shown in GenBank:BAC76403.

1.

2. The application according to claim 1, characterized in that, The overexpression refers to the expression of one or more genes encoding alcohol dehydrogenase, amine dehydrogenase, ATP-dependent peptide synthase, polyphosphate kinase 2-I, and polyphosphate kinase 2-II via a vector or by integration into the host genome.

3. The application according to claim 2, characterized in that, The overexpression is achieved by using one vector to co-express the genes of five enzymes; or by using multiple vectors to co-express the genes of five enzymes, with each vector expressing at least one enzyme gene, and each vector expressing a different gene.

4. The application according to claim 2 or 3, characterized in that, The carriers include, but are not limited to, pETDuet-1, pACYCDuet-1, pRSFDuet-1, pCDFduet-1, and pCOLDⅡ.

5. A combination of recombinant cells, characterized in that, By separate overexpression Lactobacillus reuteri alcohol dehydrogenase derived from DSM20016, Burkholderia ambifaria AMMD-derived amine dehydrogenases, Acinetobacter lactuca or Bacillus safensis ATP-dependent peptide synthase from which it originates Sinorhizobium meliloti Polyphosphate kinase 2-I and its source Meiothermus hypogaeus The cells consist of one or more recombinant polyphosphate kinase 2-II sources, and each recombinant cell does not express the same polyphosphate kinase 2-II as other recombinant cells. The NCBI accession number for the amino acid sequence of the alcohol dehydrogenase is ABQ83742.1, and the NCBI accession number for the amino acid sequence of the amine dehydrogenase is WP_011659448.

1. Acinetobacter lactuca , Bacillus safensis The NCBI accession numbers for the amino acid sequences of the ATP-dependent peptide synthases are WP_016145237.1 and WP_048238116.1, respectively. The NCBI accession number for the amino acid sequence of polyphosphate kinase 2-I is NP_384613.

1. The amino acid sequence of polyphosphate kinase 2-II is shown in GenBank:BAC76403.

1.

6. The combination of recombinant cells according to claim 5, characterized in that, The recombinant cells use Escherichia coli as the host bacterium, including Escherichia coli BL21 (DE3).

7. A method for whole-cell catalytic production of notoginseng extract, characterized in that, It utilizes the combination of recombinant cells as described in claim 5 or 6 as a whole-cell catalyst to synthesize notoginseng using serine and oxalic acid as substrates.

8. The method according to claim 7, characterized in that, In the whole-cell transformation production system, the cell wet weight is 1-200 g / L, D / L-serine is 1-100 g / L, oxalic acid is 1-100 g / L, ATP is 0-1 g / L, NAD is 0-1 g / L, sodium hexapolyphosphate is 2-300 g / L, and pH is 5.0-9.0; the reaction is carried out at 15-40℃ for 1-48 h.

9. The use of the combination of recombinant cells as described in claim 5 or 6 or the method as described in claim 7 or 8 in the production of notoginseng extract or products containing notoginseng extract or substances with notoginseng extract as a precursor.

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