[Cloning and functional characterization of oxidosqualene cyclase gene for α-amyrin synthesis].

Ursolic acid has gradually attracted much attention due to its unique pharmacological activities and valuable market value in recent years. Currently, ursolic acid is mostly extracted from loquat leaves, but the plant extraction method has low yield and high cost, and chemical synthesis is not readily available, so the biosynthesis method provides a new source for ursolic acid. α-amyrin acts as the main precursor for the synthesis of ursolic acid, and its yield is positively correlated with ursolic acid yield. Oxidosqualene cyclase(OSC) belongs to a multigene family which can catalyze the common precursor 2,3-oxidosqualene to generate different types of triterpene backbones, and plays a decisive role in the synthesis of triterpenoids. However, there are fewer reported key genes catalyzing the synthesis of α-amyrin in medicinal plants, and the yield and proportion of α-amyrin in the catalyzed products have always been a focus of research. In this study, ItOSC2, MdOSC1, AaOSC2 and CrAS, four enzymes capable of catalyzing the production of α-amyrin from 2,3-oxidosqualene, were cloned from Iris tectorum, Malus domestica, Artemisia annua and Catharanthus roseus, subject to sequence alignment and phylogenetic tree analyses, and transformed into Saccharomyces cerevisiae as plasmids. After 7 days of fermentation, the yield and proportions of α-amyrin, ß-amyrin and ergosterol were measured. Finally, AaOSC2 with the best ability to catalyze the generation of α-amyrin was filtered out, providing a key gene element for the later construction of engineered yeast strains with high production of α-amyrin and ursolic acid.

The HHEX-ABI2/SLC17A9 axis induces cancer stem cell-like properties and tumorigenesis in HCC.

Accumulating evidence indicated that HHEX participated in the initiation and development of several cancers, but the potential roles and mechanisms of HHEX in hepatocellular carcinoma (HCC) were largely unclear. Cancer stem cells (CSCs) are responsible for cancer progression owing to their stemness characteristics. We reported that HHEX was a novel CSCs target for HCC. We found that HHEX was overexpressed in HCC tissues and high expression of HHEX was associated with poor survival. Subsequently, we found that HHEX promoted HCC cell proliferation, migration, and invasion. Moreover, bioinformatics analysis and experiments verified that HHEX promoted stem cell-like properties in HCC. Mechanistically, ABI2 serving as a co-activator of transcriptional factor HHEX upregulated SLC17A9 to promote HCC cancer stem cell-like properties and tumorigenesis. Collectively, the HHEX-mediated ABI2/SLC17A9 axis contributes to HCC growth and metastasis by maintaining the CSC population, suggesting that HHEX serves as a promising therapeutic target for HCC treatment.

Pathogenicity and drug resistance of the Eimeria tenella isolate from Yiwu, Zhejiang province, eastern China.

Chicken coccidiosis can cause severe enteritis with high mortality, which causes serious economic losses to the global breeding industry each year. The most virulent species is Eimeria tenella (E. tenella), but the infectivity of different E. tenella varies among geographic strains. At present, there are no reports related to the pathogenicity and drug resistance of E. tenella in Yiwu, Zhejiang province, China. A total of 600 fecal samples were collected from 10 farms in Zhejiang province, the overall oocyst prevalence was 54.2% (325/600). The prevalence was significantly higher (P < 0.01) in chickens under 40 d (97.5%) than that in chickens between 60 and 85-days-old (40.5%) and chickens over 90-days-old (24.5%). E. tenella stain was isolated from fecal samples of chickens in Yiwu and the pathogenicity of this isolate was determined, and then we recorded the survival rate, bloody stool score, lesion score, average weight gain. The results showed that all of the chickens infected with 5 × 105 sporulated oocysts of E. tenella died after the seventh day of infection, the bloody stool score and average lesion score of chickens from group 1 (5 × 105), group 2 (5 × 104), group 3 (5 × 103) and group 4 (5 × 102) decreased successively; the average weight gain (g) and relative weight gain (%) increased successively; the weight gain of the low-dose E. tenella infection groups (5 × 103 and 5 × 102) were higher than the other 2 groups (5 × 105 and 5 × 104) (P < 0.05). Finally, The E. tenella isolate was tested for sensitivity to 6 anticoccidial drugs (sulfachloropyrazine sodium, amproline, toltrazuril, clopidol, salinomycin, and nicarbazine) using 4 indexes including anticoccidial index(ACI), percent of optimum anticoccidial activity (POAA), reduction of lesion scores (RLS), and relative oocyst production (ROP). The results showed that this isolate has developed severe resistance to drugs of salinomycin and nicarbazine, moderate resistance to amproline and clopidol, slight resistance to toltrazuril, while the E. tenella isolate performed more sensitive to sulfachloropyrazine sodium.

Production of 11-Oxo-ß-Amyrin in Saccharomyces cerevisiae at High Efficiency by Fine-Tuning the Expression Ratio of CYP450:CPR.

The production of glycyrrhetinic acid (GA) and 11-oxo-ß-amyrin, the major bioactive components in liquorice, was typically inhibited by P450 oxidation in Saccharomyces cerevisiae. This study focused on optimizing CYP88D6 oxidation by balancing its expression with cytochrome P450 oxidoreductase (CPR) for the efficient production of 11-oxo-ß-amyrin in yeast. Results indicated that a high CPR:CYP88D6 expression ratio could decrease both 11-oxo-ß-amyrin concentration and turnover ratio of ß-amyrin to 11-oxo-ß-amyrin, whereas a high CYP88D6:CPR expression ratio is beneficial for improving the catalytic activity of CYP88D6 and 11-oxo-ß-amyrin production. Under such a scenario, 91.2% of ß-amyrin was converted into 11-oxo-ß-amyrin in the resulting S. cerevisiae Y321, and 11-oxo-ß-amyrin production was further improved to 810.6 mg/L in fed-batch fermentation. Our study provides new insights into the expression of cytochrome P450 and CPR in maximizing the catalytic activity of P450s, which could guide the construction of cell factories in producing natural products.

[Cloning and expression analysis of UDP-rhamnose synthase from Citrus sinensis].

Uridine diphosphate rhamnose(UDP-Rha), a glycoside donor synthesized with the catalysis of rhamnose synthase(RHM), is one of the important elements in the synthesis of rhamnosides. In this study, we cloned a RHM gene from Citrus sinensis(CsRHM) and analyzed its bioinformatic information and functions in vitro. The results showed the gene consisted of an open reading frame of 2 007 bp encoding 668 amino acid residues. The deduced protein had a presumed molecular weight of 75.27 kDa, a theoretical isoelectric point of 6.97, and the characteristic signal sequences(GxxxGxxG/A and YxxxK) of the RHM family. Multiple sequence alignments and the phylogenetic tree demonstrated that CsRHM shared homology with other RHMs. The results of enzymatic reactions in vitro showed that the recombinant protein CsRHM catalyzed the conversion of UDP-Glu to UDP-Rha, with the kinetic parameters V_(max), K_m, K_(cat), and K_(cat)/K_m of 0.373 7 µmol·L~(-1)·min~(-1), 21.29 µmol·L~(-1), 0.24 s~(-1), and 1.13×10~4 s~(-1)·L·mol~(-1), respectively. This study is the first report about CsRHM with validated catalytic function in vitro, which provides a foundation for further research on the biosynthesis of UDP-Rha.

Metabolic Engineering of Saccharomyces cerevisiae for de Novo Dihydroniloticin Production Using Novel CYP450 from Neem (Azadirachta indica).

Azadirachtin, a limonoid isolated from the neem tree, has attracted considerable interest due to its excellent performance in pest control. Studies have also reported pharmaceutical activities of dihydroniloticin, an intermediate in azadirachtin biosynthesis, but these pharmaceutical activities could not be validated due to the limited supply. In this study, AiCYP71CD2 was first identified as involved in azadirachtin biosynthesis in neem by expressing it in Nicotiana benthamiana and yeast (Saccharomyces cerevisiae). Homology modeling and molecular docking analysis revealed that AiCYP71CD2 may exhibit a higher ability in catalyzing tirucalla-7,24-dien-3ß-ol into dihydroniloticin compared with MaCYP71CD2 from Melia azedarach L. G310 was identified as the critical residue responsible for the higher catalytic ability of AiCYP71CD2. Condon-Optimized AiCYP71CD2 greatly improved the catalytic efficiency in yeast. De novo dihydroniloticin production using the novel AiCYP71CD2 was achieved by constructing the S. cerevisiae DI-3 strain, and the titer could reach up to 405 mg/L in a fermentor, which was an alternative source for dihydroniloticin.

[Construction of yeast cell factories for production of azadirachtin precursor tirucalla-7,24-dien-3ß-ol].

Azadirachtin, as a botanical insecticide, is a highly oxidized limonoid triterpenoid existing in the seeds of Azadirachta indica. However, due to the low content in the seeds, the production of azadirachtin by seed extraction has low yield. Chemical synthesis of azadirachtin is characterized by complex process and low yield. Synthetic biology provides an alternative for the supply of azadirach-tin. In this study, two oxidosqualene cyclases AiOSC1 and MaOSC1 respectively derived from A. indica and Melia azedarach were identified in yeast. A yeast strain producing tirucalla-7,24-dien-3ß-ol was constructed by integration of AiOSC1, Arabidopsis thaliana-derived squalene synthase gene(AtAQS2), and Saccharomyces cerevisiae-derived truncated 3-hydroxy-3-methyl-glutaryl coenzyme A reductase gene(PgtHMGR) into the delta site of yeast. Then, the function of MaCYP71BQ5 was successfully verified in yeast after this gene was introduced into the constructed yeast strain. This study not only laid a foundation for the biosynthesis of tirucalla-7,24-dien-3ß-ol, but also provided a chassis cell for the functional identification of cytochrome oxidases(CYP450 s) in azadirachtin biosynthesis pathway.

Co-production of polysaccharides, ginsenosides and succinic acid from Panax ginseng residue: A typical industrial herbal waste.

Co-production of polysaccharides, ginsenosides and succinic acid was achieved from Panax ginseng residue (PGR) in this study. Physico-chemical separation was first applied to recover the released polysaccharides and ginsenoside. Enzymatic hydrolysis was then conducted to covert the left PGR into mono-sugars which was following transformed into succinic acid by constructing a succinic acid-producing strain of Escherichia coli-ZW333. Results indicated that the yields of polysaccharides and ginsenosides increased according to the increase of deconstruction content of PGR. A total sugar yield reached 52 g/L at 10% PGR loading and increased to 94.33 g/L following fed-batch enzymatic hydrolysis. Finally, 56.28 g/L succinic acid was produced. In total, 18 g ginseng polysaccharides, 230 mg ginsenosides and 39 g succinic acid were produced from 100 g PGR. Accordingly, the total economic output could reach RMB 80,149 from 1 t PGR, illustrating the great value increasement of PGR by this industrially possible process.

[Regulation of ß-mercuryl alcohol metabolic flow in Saccharomyces cerevisiae cells].

In this study, citrate synthase gene(CIT2), and malate synthase gene(MLS1) were successfully knocked out in ß-amyrin-producing yeast cells by using CRISPR/CAS9. The promoter of phosphoglucose isomerase gene(PGI1) was replaced by that of cytochrome c oxidase subunit Ⅶa(Cox9)to weaken its expression, aiming to channel more carbon flux into the NADPH-producing pathway. The fermentation results showed that CIT2 deletion had no effect on the ß-amyrin production. Compared with the control strain, the production of ß-amyrin was increased by 1.85 times after deleting MLS1, reaching into 3.3 mg·L~(-1). By replacing the promoter of PGI1, the ß-amyrin yield was 3.75 times higher than that of the control strain, reaching up to 6.7 mg·L~(-1). This study successfully knocked out the CITT2 and MLS1 genes and weakened the PGI1 gene by using CRISPR/CAS9, which directly influenced the production of ß-amyrin and provided some reference for the the metabolic engineering of triterpernoid producing strain.

[Study of heterologous efficient synthesis of ß-amyrin and high-density fermentation].

In this study, the synthetic pathway of ß-amyrin was constructed in the pre-constructed Saccharomyces cerevisiae chassis strain Y0 by introducing ß-amyrin synthase from Glycyrrhiza uralensis, resulting strain Y1-C20-6, which successfully produced ß-amyrin up to 5.97 mg·L~(-1). Then, the mevalonate pyrophosphate decarboxylase gene(ERG19), mevalonate kinase gene(ERG12), 3-hydroxy-3-methylglutaryl-CoA synthase gene(ERG13), phosphomevalonate kinase gene(ERG8) and IPP isomerase gene(IDI1)were overexpressed to promoted the metabolic fluxto the direction of ß-amyrin synthesis for further improving ß-amyrin production, resulting the strain Y2-C2-4 which produced ß-amyrin of 10.3 mg·L~(-1)under the shake flask fermentation condition. This is 100% higher than that of strain Y1-C20-6, illustrating the positive effect of the metabolic engineering strategy applied in this study. The titer of ß-amyrin was further improved up to 157.4 mg·L~(-1) in the fed-batch fermentation, which was almost 26 fold of that produced by strain Y1-C20-6. This study not only laid the foundation for the biosynthesis of ß-amyrin but also provided a favorable chassis strain for elucidation of cytochrome oxidases and glycosyltransferases of ß-amyrin-based triterpenoids.

Efficient production of glycyrrhetinic acid in metabolically engineered Saccharomyces cerevisiae via an integrated strategy.

BACKGROUND: Glycyrrhetinic acid (GA) is the most important ingredient in licorice due to its outstanding anti-inflammatory activity and wide application in the medicine and cosmetics industries. Contemporary industrial production of GA by acid hydrolysis of glycyrrhizin which was extracted from Glycyrrhiza plants, is not environment-friendly and devastates farmland since the Glycyrrhiza rhizomes grow up to 10 m underground. RESULTS: In this study, GA was produced through metabolically engineering Saccharomyces cerevisiae by introducing the entire heterogeneous biosynthetic pathway of GA. Codon optimized CYP88D6 and CYP72A154, combined with ß-AS (ß-amyrin synthase encoding gene) and the NADPH-cytochrome P450 reductase gene of Arabidopsis thaliana were introduced into S. cerevisiae. The resulting strain (Y1) produced 2.5 mg/L of ß-amyrin and 14 µg/L of GA. The cytochrome b5 from G. uralensis (GuCYB5) was identified and the introduction of this novel GuCYB5 increased the efficiency of GA production by eightfold. The joint utilization of the GuCYB5 gene along with 10 known MVA pathway genes from S. cerevisiae were overexpressed in a stable chromosome integration to achieve higher GA production. Using the combined strategy, GA concentration improved by 40-fold during batch fermentation. The production was further improved to 8.78 mg/L in fed-batch fermentation, which was increased by a factor of nearly 630. CONCLUSIONS: This study first investigated the influence of carbon flux in the upstream module and the introduction of a newly identified GuCYB5 on GA production. The newly identified GuCYB5 was highly effective in improving GA production. An integrated strategy including enzyme discovery, pathway optimization, and fusion protein construction was provided in improving GA production, achieving a 630 fold increase in GA production. The metabolically engineered yeast cell factories provide an alternative approach to glycyrrhetinic acid production, replacing the traditional method of plant extraction.

Efficient production of succinic acid from herbal extraction residue hydrolysate.

In this study, six different herbal-extraction residues were evaluated for succinic acid production in terms of chemical composition before and after dilute acid pretreatment (DAP) and sugar release performance. Chemical composition showed that pretreated residues of Glycyrrhiza uralensis Fisch (GUR) and Morus alba L. (MAR) had the highest cellulose content, 50% and 52%, respectively. Higher concentrations of free sugars (71.6 g/L total sugar) and higher hydrolysis yield (92%) were both obtained under 40 FPU/g DM at 10% solid loading for GUR. Using scanning electron microscopy (SEM), GUR was found to show a less compact structure due to process of extraction. Specifically, the fibers in pretreated GUR were coarse and disordered compared with that of GUR indicated by SEM. Finally, 65 g/L succinic acid was produced with a higher yield of 0.89 g/g total sugar or 0.49 g/g GUR. Our results illustrate the potential of GUR for succinic acid production.

[Investigation and optimization on ability of enzymatic hydrolysis of Mori Cortex residue].

Residue of Mori Cortex was studied to optimize its enzymatic hydrolysis process, and explore its potential as a carbon source for biochemistry and biofuel production. The cellulose content of diluted acid pretreated (DAP) and non-pretreated from Mori Cortex were measured in this study, and the results showed that the cellulose content of DAP and non-pretreated from Mori Cortex were 52.5% and 47%, respectively. This higher cellulose content indicated that residue of Mori Cortex had the potential to act as a carbon source for biochemistry and biofuel production. Enzymatic hydrolysis of pretreated and non-pretreated from Mori Cortex was conducted under different enzyme loading amount. 40 FPU·(g DW）⁻¹ enzyme loading was determined as the optimal amount by comparing the yield of sugar and the rate of enzymolysis. Under this condition, the concentrations of glucose, xylose, arabinose sugar were 23.82, 4.84, 3.6 g·L⁻¹, and the corresponding enzymatic hydrolysis rate was 45.33% which was 2.3 times higher than that of non-pretreated from Morus alba residues. Fed-batch enzymatic hydrolysis was conducted finally to get higher sugar yield, and the final glucose concentration reached up to 38 g·L⁻¹ with the enzymatic hydrolysis rate of 36.19%. The results indicated that Mori Cortex residue had higher cellulose and hemicellulose contents, so it had the potential to become a carbon source to produce the bio-chemicals and biofuels. Through enzymatic hydrolysis, it can be converted into microbial available monosaccharides; and through fermentation, it can be converted into high value-added chemicals, biofuels, etc., to solve the problem of residue pollution, and achieve the sustainable development and greening of Chinese pharmaceutical production process.

[Influence of light on gene expression of key synthesis enzyme genes FtANR and FtLAR about proanthocyanidin in seeds of homologous plant of food and medicine Fagopyrum tataricum].

Tartary buckwheat Fagopyrum tataricum is an important medicinal and functional herb due to its rich content of flavonoids in the seeds. F.tataricum exhibited good functions for free radicals scavenging， anti-oxidation， anti-aging activities. Although much genetic knowledge of the synthesis， regulation， accumulation of rutin， the genetic basis of proanthocyanidins(PAs) in tartary buckwheat and their related gene expression changes under different lights(blue， red， far red， ultraviolet light) remain largely unexplored. In this study， we cloned one anthocyanidin reductase gene(ANR) and two leucocyanidin reductase gene(LAR) named FtANR，FtLAR1，FtLAR3 involved in formation of(+)-catechin and(-)-epicatechin precusor proanthocyanidin by digging out F. tataricum seed transcriptome data. The expression data showed that the opposite influence of red light on these gene transcript level compared to others lights. The expression levels of FtANR and FtLAR1 decreased and FtLAR3 appeared increment after exposed in the red light， while the expression levels of those genes appeared opposite result after exposed in the blue and far red light.

[Recent advances of synthetic biology for production of functional ingredients in Chinese materia medica].

The functional ingredients in Chinese materia medica are the main active substance for traditional Chinese medicine and most of them are secondary metabolites derivatives. Until now,the main method to obtain those functional ingredients is through direct extraction from the Chinese materia medica. However, the income is very low because of the high extraction costs and the decreased medicinal plants. Synthetic biology technology, as a new and microbial approach, can be able to carry out large-scale production of functional ingredients and greatly ease the shortage of traditional Chinese medicine ingredients. This review mainly focused on the recent advances in synthetic biology for the functional ingredients production.