Heterologous biomimetic synthesis of active ingredients in traditional Chinese medicine:a new mode for protection and development of traditional Chinese medicine resources / 中国中药杂志

Heterologous biomimetic synthesis of the active ingredients of traditional Chinese medicine(TCM) is a new mode of resource acquisition and has shown great potential in the protection and development of TCM resources. According to synthetic biology and by constructing biomimetic microbial cells and imitating the synthesis of active ingredients in medicinal plants and animals, the key enzymes obtained from medicinal plants and animals are scientifically designed and systematically reconstructed and optimized to realize the heterologous synthesis of the active ingredients in microorganisms. This method ensures an efficient and green acquisition of target products, and also achieves large-scale industrial production, which is conducive to the production of scarce TCM resources. Additiona-lly, the method playes a role in agricultural industrialization, and provides a new option for promoting the green and sustainable deve-lopment of TCM resources. This review systematically summarized the important progress in the heterologous biomimetic synthesis of TCM active ingredients from three research areas: biosynthesis of terpenoids, flavonoids, phenylpropanoids, alkaloids and other active ingredients, key points and difficulties in heterologous biomimetic synthesis, and biomimetic cells with complex TCM ingredients. This study facilitated the application of new generation of biotechnology and theory to the development of TCM.

Construction of cell factories for production of patchoulol in Saccharomyces cerevisiae / 中国中药杂志

Patchoulol is an important sesquiterpenoid in the volatile oil of Pogostemon cablin, and is also considered to be the main contributing component to the pharmacological efficacy and fragrance of P. cablin oil, which has antibacterial, antitumor, antioxidant, and other biological activities. Currently, patchoulol and its essential oil blends are in high demand worldwide, but the traditional plant extraction method has many problems such as wasting land and polluting the environment. Therefore, there is an urgent need for a new method to produce patchoulol efficiently and at low cost. To broaden the production method of patchouli and achieve the heterologous production of patchoulol in Saccharomyces cerevisiae, the patchoulol synthase(PS) gene from P. cablin was codon optimized and placed under the inducible strong promoter GAL1 to transfer into the yeast platform strain YTT-T5, thereby obtaining strain PS00 with the production of(4.0±0.3) mg·L~(-1) patchoulol. To improve the conversion rate, this study used protein fusion method to fuse SmFPS gene from Salvia miltiorrhiza with PS gene, leading to increase the yield of patchoulol to(100.9±7.4) mg·L~(-1) by 25-folds. By further optimizing the copy number of the fusion gene, the yield of patchoulol was increased by 90% to(191.1±32.7) mg·L~(-1). By optimizing the fermentation process, the strain was able to achieve a patchouli yield of 2.1 g·L~(-1) in a high-density fermentation system, which was the highest yield so far. This study provides an important basis for the green production of patchoulol.

Construction of cell factories for production of valencene in Saccharomyces cerevisiae / 药学学报

Valencene, a kind of sesquiterpenoid with a citrus flavor, is mainly found in Valencia orange and is commonly used in cosmetics and food additives, as well as industrial synthetic nootkatone. In this study, synthetic biology was used to create a Saccharomyces cerevisiae cell factory to produce valencene. Fistly, valencene synthase gene (CnVS) from Callitropsis nootkatensis was inserted into the chromosome of the chassis strain YTT-T5. The resulting strain VAL-01 could produce 1.1 mg·L-1 valencene. Protein fusion technique was used, different valencene synthases were compared and the copy number of key genes was adjusted, yielding valencene to 436.4 mg·L-1. Then, knocking-out the transcription factor ROX1 resulted in valencene improvement by 17.4%. Moreover, the induction system of galactose was regulated, transcription factor PDR3 and INO2 were overexpressed. The engineered strain VAL-10 could produce 2 798.6 mg·L-1 valencene by high cell density fermentation method (nearly 2 500 times higher than VAL-01). This study provides a basis for green production of valencene.

Construction of cell factories for high production of ginsenoside Rh_2 in Saccharomyces cerevisiae / 中国中药杂志

Ginsenoside Rh_2 is a rare active ingredient in precious Chinese medicinal materials such as Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma, and Panacis Quinquefolii Radix. It has important pharmacological activities such as anti-cancer and improving human immunity. However, due to the extremely low content of ginsenoside Rh_2 in the source plants, the traditional way of obtaining it has limitations. This study intended to apply synthetic biological technology to develop a cell factory of Saccharomyces cerevisiae to produce Rh_2 by low-cost fermentation. First, we used the high protopanaxadiol(PPD)-yielding strain LPTA as the chassis strain, and inserted the Panax notoginseng enzyme gene Pn1-31, together with yeast UDP-glucose supply module genes[phosphoglucose mutase 1(PGM1), α-phosphoglucose mutase(PGM2), and uridine diphosphate glucose pyrophosphorylase(UGP1)], into the EGH1 locus of yeast chromosome. The engineered strain LPTA-RH2 produced 17.10 mg·g~(-1) ginsenoside Rh_2. This strain had low yield of Rh_2 while accumulated much precursor PPD, which severely restricted the application of this strain. In order to further improve the production of ginsenoside Rh_2, we strengthened the UDP glucose supply module and ginsenoside Rh_2 synthesis module by engineered strain LPTA-RH2-T. The shaking flask yield of ginsenoside Rh_2 was increased to 36.26 mg·g~(-1), which accounted for 3.63% of the dry weight of yeast cells. Compared with those of the original strain LPTA-RH2, the final production and the conversion efficiency of Rh_2 increased by 112.11% and 65.14%, respectively. This study provides an important basis for further obtaining the industrial-grade cell factory for the production of ginsenoside Rh_2.

Dynamic control of ERG20 expression to improve production of monoterpenes by engineering Saccharomyces cerevisiae / 中国中药杂志

Monoterpenes are widely used in cosmetics, food, medicine, agriculture and other fields. With the development of synthetic biology, it is considered as a potential way to create microbial cell factories to produce monoterpenes. Engineering Saccharomyces cerevisiae to produce monoterpenes has been a research hotspot in synthetic biology. In S. cerevisiae, the production of geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) is catalyzed by a bifunctional enzyme farnesyl pyrophosphate synthetase(encoded by ERG20 gene) which is inclined to synthesize FPP essential for yeast growth. Therefore, reasonable control of FPP synthesis is the basis for efficient monoterpene synthesis in yeast cell factories. In order to achieve dynamic control from GPP to FPP biosynthesis in S. cerevisiae, we obtained a novel chassis strain HP001-pERG1-ERG20 by replacing the ERG20 promoter of the chassis strain HP001 with the promoter of cyclosqualene cyclase(ERG1) gene. Further, we reconstructed the metabolic pathway by using GPP and neryl diphosphate(NPP), cis-GPP as substrates in HP001-pERG1-ERG20. The yield of GPP-derived linalool increased by 42.5% to 7.6 mg·L~(-1), and that of NPP-derived nerol increased by 1 436.4% to 8.3 mg·L~(-1). This study provides a basis for the production of monoterpenes by microbial fermentation.

Engineering CrtW and CrtZ for improving biosynthesis of astaxanthin in Escherichia coli / 中国天然药物

This study engineered β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ to improve biosynthesis of astaxanthin in Escherichia coli. Firstly, crtW was randomly mutated to increase CrtW activities on conversion from β-carotene to astaxanthin. A crtW* mutant with A6T, T105A and L239M mutations has improved 5.35-fold astaxanthin production compared with the wild-type control. Secondly, the expression levels of crtW* and crtZ on chromosomal were balanced by simultaneous modulation RBS regions of their genes using RBS library. The strain RBS54 selected from RBS library, directed the pathway exclusively towards the desired product astaxanthin as predominant carotenoid (99%). Lastly, the number of chromosomal copies of the balanced crtW-crtZ cassette from RBS54 was increased using a Cre-loxP based technique, and a strain with 30 copies of the crtW*-crtZ cassette was selected. This final strain DL-A008 had a 9.8-fold increase of astaxanthin production compared with the wild-type control. Fed-batch fermentation showed that DL-A008 produced astaxanthin as predominant carotenoid (99%) with a specific titer of 0.88 g·L without addition of inducer. In conclusion, through constructing crtW mutation, balancing the expression levels between crtW* and crtZ, and increasing the copy number of the balanced crtW*-crtZ cassette, the activities of β-carotene ketolase and β-carotene hydroxylase were improved for conversion of β-carotene to astaxanthin with higher efficiency. The series of conventional and novel metabolic engineering strategies were designed and applied to construct the astaxanthin hetero-producer strain of E. coli, possibly offering a general approach for the construction of stable hetero-producer strains for other natural products.

Optimization of UDP-glucose supply module and production of ginsenoside F1 in Saccharomyces cerevisiae / 中国中药杂志

Ginsenoside F1 is a rare ginsenoside in medicinal plants such as Panax ginseng,P. notogingseng and P. quinquefolius. It has strong pharmacological activities of anti-tumor,anti-oxidation and anti-aging. In order to directly produce ginsenoside F1 by using inexpensive raw materials such as glucose,we integrated the codon-optimized P.ginseng dammarenediol-Ⅱ synthase(Syn Pg DDS),P.ginseng protopanaxadiol synthase(Syn Pg PPDS),P. ginseng protopanaxatriol synthase(Syn Pg PPTS) genes and Arabidopsis thaliana cytochrome P450 reductase(At CPR1) gene into triterpene chassis strain BY-T3. The transformant BY-PPT can produce protopanaxatriol. Then we integrated the Sacchromyces cerevisiae phosphoglucomutase 1(PGM1),phosphoglucomutase 2(PGM2) and UDP-glucose pyrophosphorylase 1(UGP1) genes into chassis strain BY-PPT. The UDP-glucose supply module increased UDP-glucose production by 8. 65 times and eventually reached to 44. 30 mg·L-1 while confirmed in the transformant BY-PPT-GM. Next,we integrated the UDPglucosyltransferase Pg3-29 gene which can catalyze protopanaxatriol to produce ginsenoside F1 into chassis strain BY-PPT-GM. The transformant BY-F1 produced a small amount of ginsenoside F1 which was measured as 0. 5 mg·L-1. After the fermentation process was optimized,the titer of ginsenoside F1 could be increased by 900 times to 450. 5 mg·L-1. The high-efficiency UDP-glucose supply module in this study can provide reference for the construction of cell factories for production of saponin,and provide an important basis for further obtaining high-yield ginsenoside yeast cells.

Biosynthesis analysis of hederagenin pathway and its construction in yeast cells / 中国中药杂志

Hederagenin is an effective constituent of many medical plants, such as Clematidis Radix, and has a wide range of applications in anti-tumor, anti-inflammatory, antidepressant, hepatoprotective antibacterial, et al. In order to obtain the efficient production of yeast cells for hederagenin,we successfully cloned and screened out a P450 gene MdMA02 from Malus×domestica which can catalyze oleanolic acid C-23 oxidation with our developed plug and play platform. Its amino acid homology is only 32% as compared to characterized CYP72A68v2. By transforming MdMA02 to the oleanolic acid-producing strain BY-OA, a hederagenin-producing strain was constructed and hederagenin's titer could achieve 101 mg·L⁻¹ using high cell density fermentation, which was 337 times higher than in shake flasks culturing. This study provides a basis for further research on promoting the creation of oleanane-type pentacyclic triterpenoids biosynthetic pathway analysis and relative cell factories construction.

Construction of efficient yeast cell factories for production of ginsenosides precursor dammarenediol-II / 药学学报

Dammarenediol-Ⅱ is an important precursor in the biosynthesis pathway of ginsenosides which are the main active components of Panax quinquefolius and Panax ginseng. For constructing a dammarenediol- Ⅱ-producing cell factory, the triterpenoid precursors of yeast are improved significantly by the modular pathway engineering strategy on the basis of an MVA optimized strain. The strain overexpressing Salvia miltiorrhiza SmFPS and Arabidopsis thaliana AtSQS2 could yield 67.4 mg·g−1 squalene, accounting for about 6.74% of cell dry weight. In our further work, an Arabidopsis thaliana 2,3-oxidosqualene synthase AtSQE2 was found to be able to increase the downstream lanosterol yield by 22-fold, reaching 47.9 mg·g−1. Then, regulating dammarenediol-Ⅱ synthase gene expression, using anti-sense RNA technology for regulation of ERG7 in the ergosterol pathway, and optimizing fermentation process were successively performed. Finally, the synthesis flux of triterpenes was increased to 10 g·L−1 for the first time, and we constructed an efficient cell factory that can produce 15 g·L−1 dammarenediol-Ⅱ, which lays a solid foundation of industrial synthesis of dammarane-type ginsenosides.

Construction of cell factories for high production of nerolidol in Saccharomyces cerevisiae / 中国中药杂志

Nerolidol is an important constituent of terpenoid essential oil and has excellent anti-tumor, anti-bacterial, and anti-oxidative properties. For realizing heterogenous production of nerolidol, our research firstly integrated the codon-optimized Actinidia sinensis nerolidol synthase gene (NES) into the terpenoid chassis strain FPP-001, and obtained NES-001 that could produce 2.71 mg•L⁻¹ nerolidol. Then, the N-terminal of the NES was fused with FPS by linker peptide GGGS. With this strategy, nerolidol production improved by 59.80-fold, reaching 162.07 mg•L⁻¹. Finally, by introduction of auxotrophic marker TRP1 in NES-002, the resulting strain NES-003 could produce 1 711.53 mg•L⁻¹ by high cell density fermentation method. This study provides the basis for the fermentative production of nerolidol and other sesquiterpenoids.

Study of heterologous efficient synthesis of cucurbitadienol / 中国中药杂志

Cucurbitadienol has anti-inflammation, anti-cancer activities, and acts as a precursor of traditional Chinese medicine active ingredients mogroside and cucurbitacine. For construction of a Sacchromyces cerevisiae cell factory for production of cucurbitadienol, we firstly cloned a cucurbitadienol synthase (CBS) gene from Siraitia grosvenorii. Then, through heterologous expression of CBS in the triterpenoid chassis strain WD-2091, the engineered strain could produced 27.44 mg•L ⁻¹ cucurbitadienol, which was determined by GC-MS. Further regulation of CBS expression led to cucurbitadienol's titer increasing by 202.07% and reaching 82.89 mg•L ⁻¹ in the shake flask fermentation and 1 724.10 mg•L ⁻¹ in the high cell density fermentation. Our research promotes the cucurbitane-type tetracyclic triterpenoids synthesis pathway analysis progress and provides the basis for further obtaining cell factories for production of cucurbitadienol tetracyclic triterpenoids.

Construction of cell factories for production of lupeol in Saccharomyces cerevisiae / 中国中药杂志

Lupane-type triterpenoids, such as betulinic acid, are derived from lupeol and have excellent properties in anti-HIV, anti-cancer activities and so on. For realizing heterogenous production of lupane-type triterpenoids, our research firstly integrated all the seven genes in the MVA pathway in Saccharomyces cerevisiae to increase the supply of squalene (triterpenoids universal precursor) in a single step using the DNA assembler method. Next, cell factories for production of lupeol was constructed by integrating Arabidopsis thaliana lupeol synthetic gene (AtLUP) into chromosome of triterpenoid chassis strain. Results showed that the MVA pathway, about 20 kb nucleotide length, could be assembled in one-pot process and the doubled MVA pathway could significantly improve squalene by 500-fold, reaching 354.00 mg•L⁻¹. NK2-LUP was obtained by introducing AtLUP gene on chromosome, and could produce 8.23 mg•L⁻¹ lupeol. This study supports the possibility of large-scale biosynthetic pathway assembly in S.cerevisiae and lays the foundation of obtaining cell factories for production of lupan-type triterpenoids at the same time.

Research progress of synthetic biology for tanshinones / 中国中药杂志

Synthetic biology research methods which design and build a new artificial biological systems (medicinal plants or microorganisms system) with specific physiological functions through clarifying and simulating the basic law of the biosynthesis of active components of traditional Chinese medicine, is considered to be a potential method to produce an abundant resources of bioactive components. Tanshinones is a kind of diterpene quinone compounds with important pharmacological activities from traditional Chinese medicine Salvia miltiorrhiza. This article systematically introduced the research progress of the synthetic biology of S. miltiorrhiza, in order to provide references for studies on other terpenoid bioactive components of traditional Chinese medicines, and give new research strategies for the sustainable development of traditional Chinese medicine resources.

Status and future of natural resource for Chinese materia medica / 中国中药杂志

For thousands of years, the natural resource for Chinese materiamedica has been the foundation of the traditional Chinese medicine industry, which provides abundant medicine for human. In recent years, increasing demands and irrational exploitation led to a lot of problems such as rapid decrease of traditional Chinese herbs reserves, low quality of medicine and dismishing traditional cultures. These restricted the development of the traditional Chinese medicine. To solve these problems, scientists have done much work on investigating traditional Chinese medicine resources, exploring the metabolic pathway of bioactive ingredients, cultivating new varieties, and carrying out synthetic biology. These studies provided a theoretical basis for sustainable utilizationand future developmentof traditional Chinese medicine resources.

Optimization of synthetic pathway and fermentation process of yeast cell factories for production of oleanoic acid / 中国中药杂志

<p><b>OBJECTIVE</b>To optimize the synthetic pathway and fermentation process of yeast cell factories for production of oleanoic acid.</p><p><b>METHOD</b>Using the DNA assembler method, one copy of Glycyrrhiza glabra beta-amyrin synthase (GgbAS), Medicago truncatula oleanolic acid synthase (MtOAS) and Arabidopsis thaliana cytochrome P450 reductase 1 (AtCPR1) genes were introduced into Saccharomyces cerevisiae strain BY-OA, resulting in strain BY-20A. YPD medium with different glucose concentration were then used to cultivate strain BY-2OA.</p><p><b>RESULT</b>Increasing gene copies of GgbAS, MtOAS and AtCPR1 resulted in increased beta-amyrin and oleanolic acid production. The strain BY-2OA produced 136.5 mg x L(-1) beta-amyrin and 92.5 mg x L(-1) oleanolic acid, which were 54% and 30% higher than the parent strain BY-OA. Finally, the titer of oleanolic acid increased to 165.7 mg x L(-1) when cultivated in YPD medium with 40 mg x L(-1) glucose.</p><p><b>CONCLUSION</b>Production of oleanoic acid increased significantly in the yeast strain BY-2OA, which can provide the basis for creating an alternative way for production of oleanoic acid in place of extraction from plant sources.</p>

Construction of Saccharomyces cerevisiae cell factories for lycopene production / 中国中药杂志

For microbial production of lycopene, the lycopene synthetic genes from Pantoea agglomerans were integrated into Saccharomyces cerevisiae strain BY4742, to obtain strain ZD-L-000 for production of 0.17 mg · L(-1) lycopene. Improving supplies of isoprenoid precursors was then investigated for increasing lycopene production. Four key genes were chosen to be overexpressed, inclu- ding truncated 3-hydroxy-3-methylglutaryl-CoA reductase gene (tHMG1), which is the major rate-limiting enzyme in the mevalonate (MVA) pathway, a mutated global regulatory factor gene (upc2.1), a fusion gene of FPP synthase (ERG20) and endogenous GGPP synthase (BTS1), which is a key enzyme in the diterpenoid synthetic pathway, and GGPP synthase gene (SaGGPS) from Sulfolobus acidocaldarius. Over-expression of upc2.1 could not improve lycopene production, while over-expression of tHMGI , BTS1-ERG20 and SaGGPS genes led to 2-, 16. 9- and20. 5-fold increase of lycopene production, respectively. In addition, three effective genes, tHMG1, BTS1-ERG20 and SaGGPS, were integrated into rDNA sites of ZD-L-000, resulting in strain ZD-L-201 for production of 13.23 mg · L(-1) lycopene, which was 77-fold higher than that of the parent strain. Finally, two-phase extractive fermentation was performed. The titer of lycopene increased 10-fold to 135.21 mg · L(-1). The engineered yeast strains obtained in this work provided the basis for fermentative production of lycopene.

Cloning and characterization of cDNA encoding Psammosilene tunicoides squalene synthase / 药学学报

The total triterpene saponins of Psammosilene tunicoides have significant pharmacologic activity. Psammosilene tunicoides squalene synthase (PSS) is a gateway enzyme to regulate the biosynthesis of total triterpene saponins extracted from the root of Psammosilene tunicoides which is an endangered species. In this paper, cDNA encoding of PSS was cloned by the degenerate primer PCR and rapid-amplification of cDNA ends (RACE). The full-length of cDNA of PSS is 1663 bp, with an open reading frame (ORF) of 1 245 bp, encoding 414 amino acid polypeptide (calculated molecular mass, 47.69 kDa), 5'UTR (untranslated region) and 3'UTR are 260 bp and 158 bp, respectively. The deduced amino acid sequence of PSS has higher homology with the known squalene synthases of several species such as Panax notoginseng (83%), Panax ginseng (82%) and Glycyrrhiza glabra (82%) than that with Schizosacharomyces pombe (35%), Candida albicans (39%) and Homo sapiens (47%). The characterization of PSS was done by a series of methods, such as prokaryotic expression, the activity of enzyme in vitro, capillary gas chromatography (GC) and capillary gas chromatography mass spectrometry (GC-MS). The results showed that the cell-free extract of E. coli transformed with the recombinant plasmid can effectively convert farnesyl diphosphate into squalene in vitro. GenBank accession number is EF585250. Our research provided important base for the study of Psammosilene tunicoides secondary metabolism and metabolic engineering.