Efficient synthesis of L-methionine by engineering the one carbon module of Escherichia coli / 生物工程学报

L-methionine, also known as L-aminomethane, is one of the eight essential amino acids required by the human body and has important applications in the fields of feed, medicine, and food. In this study, an L-methionine high-yielding strain was constructed using a modular metabolic engineering strategy based on the M2 strain (Escherichia coli W3110 ΔIJAHFEBC/PAM) previously constructed in our laboratory. Firstly, the production of one-carbon module methyl donors was enhanced by overexpression of methylenetetrahydrofolate reductase (methylenetetrahydrofolate reductase, MetF) and screening of hydroxymethyltransferase (GlyA) from different sources, optimizing the one-carbon module. Subsequently, cysteamine lyase (hydroxymethyltransferase, MalY) and cysteine internal transporter gene (fliY) were overexpressed to improve the supply of L-homocysteine and L-cysteine, two precursors of the one-carbon module. The production of L-methionine in shake flask fermentation was increased from 2.8 g/L to 4.05 g/L, and up to 18.26 g/L in a 5 L fermenter. The results indicate that the one carbon module has a significant impact on the biosynthesis of L-methionine, and efficient biosynthesis of L-methionine can be achieved through optimizing the one carbon module. This study may facilitate further improvement of microbial fermentation production of L-methionine.

Construction of an l-cysteine hyper-producing strain of Escherichia coli based on a balanced carbon and sulfur module strategy / 生物工程学报

l-cysteine is an important sulfur-containing α-amino acid. It exhibits multiple physiological functions with diverse applications in pharmaceutical cosmetics and food industry. Here, a strategy of coordinated gene expression between carbon and sulfur modules in Escherichia coli was proposed and conducted for the production of l-cysteine. Initially, the titer of l-cysteine was improved to (0.38±0.02) g/L from zero by enhancing the biosynthesis of l-serine module (serAf, serB and serCCg) and overexpression of CysB. Then, promotion of l-cysteine transporter, increased assimilation of sulfur, reduction or deletion of l-cysteine and l-serine degradation pathway and enhanced expression of cysEf (encoding serine acetyltransferase) and cysBSt (encoding transcriptional dual regulator CysB) were achieved, resulting in an improved l-cysteine titer (3.82±0.01) g/L. Subsequently, expressions of cysM, nrdH, cysK and cysIJ genes that were involved in sulfur module were regulated synergistically with carbon module combined with utilization of sulfate and thiosulfate, resulting in a strain producing (4.17±0.07) g/L l-cysteine in flask shake and (11.94±0.1) g/L l-cysteine in 2 L bioreactor. Our results indicated that efficient biosynthesis of l-cysteine could be achieved by a proportional supply of sulfur and carbon in vivo. This study would facilitate the commercial bioproduction of l-cysteine.

Advances in the biosynthesis and application of bixin / 生物工程学报

Since synthetic pigments are potentially harmful to human health, natural ones such as bixin, one of the carotenoids, are favored. As the second widely used natural pigment in the world, there is significant interest in the biosynthetic pathway of bixin which has not been fully elucidated. This review summarizes the chemical properties, extraction methods, biosynthetic pathway and application of bixin. In addition, we compared the difference between traditional extraction methods and new extraction techniques. Moreover, we described the genes involved in the biosynthetic pathway of bixin and the effects of abiotic stress on the biosynthesis of bixin, and discussed the application of bixin in food, pharmaceutical and chemical industries. However, the researches on bixin biosynthesis pathway are mostly carried out at the transcriptome level and most of the gene functions have not been elucidated. Therefore, we propose to characterize the entire bixin biosynthetic pathway using techniques of genomics, bioinformatics, and phytochemistry. This will help facilitate the synthetic biology research of bixin and development of bixin into new drugs.

Advances in biosynthesis of cordycepin from Cordyceps militaris / 生物工程学报

Cordycepin as the main active ingredient of Cordyceps militaris, a traditional medicinal fungus in China, has many physiological functions such as anti-cancer, anti-tumor and anti-virus activity. The most potential route for effective cordycepin production has been considered as liquid fermentation of C. militaris though with low productivity at present. Thus, it is urgent to apply both process engineering strategy and metabolic engineering strategy to enhance the productivity of cordycepin. In this review, the effects of medium components (i.e. the carbon/nitrogen source, precursor substances and metal ions) and operation factors (i.e. pH, dissolved oxygen and light) on cordycepin biosynthesis in liquid fermentation system are summarized. Besides, separation of cordycepin, the gene cluster involved and predicted biosynthesis pathways of cordycepin are also discussed, providing possible solutions of finally realizing efficient production of cordycepin.

Biosynthesis and transcriptional regulation of tanshinones and salvianolic acids / 药学学报

Tanshinones and salvianolic acids are important materials in the treatment of coronary heart disease, myocardial infarction, hypertension, hyperlipidemia, stroke and others illnesses. In recent years, with the development of genomics, transcriptome, metabolomics and bioinformatics, many advances have been made in the biosynthesis and transcriptional regulation of tanshinones and salvianolic acids. This paper summarizes these advances and suggests further study on the downstream synthesis pathways and transcriptional regulatory mechanisms to reveal new molecular mechanism of synthesis, transport, regulation and modification. Additionally, we discuss the design and construction of new biological pathways to increase the expression of biosynthesis genes and the production of secondary components, is a newly developing research field.

Expression of saponin biosynthesis related genes in different tissues of / 中国中药杂志

The relationship between saponin content of in different parts of the organization and expression of ginsenoside biosynthesis related gene was obtained by the correlation analysis between saponin content and gene expression. The 14 tissue parts of were studied, six saponins in Samples (ginsenoside Rg₁, Re, Rb₁, Rc, Rb₂ and Rd), group saponins and total saponins were determined by high performance liquid chromatography and vanillin-sulfuric acid colorimetric method. Simultaneously, the expression levels of 7 ginsenoside biosynthesis related genes ( and ) in different tissues of were determined by Real-time fluorescence quantitative PCR. Although 7 kinds of ginsenoside biosynthesis related enzyme gene in the involved in ginsenoside synthesis, the expression of and P450 genes had no significant effect on the content of monosodium saponins, grouping saponins and total saponins, and had significant or extremely significant on the contents of single saponins Re, Rg1, Rb1, Rd, group saponin PPD and PPT, total saponin TMS and total saponin TS (<0.05 or <0.01). The biosynthesis of partial saponins, grouping saponins and total saponins in was affected by the interaction of multiple enzyme genes in the saponin synthesis pathway, the content of saponins in different tissues of was determined by the differences in the expression of key enzymes in the biosynthetic pathway. Therefore, this study further clarified that and was the key enzyme to control the synthesis of saponins in by correlation analysis, the biosynthesis of ginsenosides in was regulated by these five kind of enzymes in cluster co-expression of interaction mode.

Differentially expressed genes in heads and tails of Angelica sinensis diels: Focusing on ferulic acid metabolism / 中国结合医学杂志

<p><b>OBJECTIVE</b>To explore the scientific connotation of the discrepant pharmaceutical activities between the head and tail of Angelica sinensis diels (AS), an important herb extensively utilized in Chinese medicine, by the approach of transcriptome sequencing.</p><p><b>METHODS</b>Ten samples of AS were randomly collected in Min County, Gansu Province of China. Transcriptome sequencing of AS was accomplished in a commercial ILLumina HiSeq-2000 platform. The transcriptome of each head and tail of AS were fixed in a gene chip, and detected under the procedure of Illumina HiSeq-2000. Differentially expressed unigenes between the heads and tails of AS were selected by Shanghai Biotechnology Corporation (SBC) online analysis system, based on Gene Ontology, Kyoto Encyclopedia of Genes and Genomes and relevant bioinformatic database.</p><p><b>RESULTS</b>Totally 63,585 unigenes were obtained from AS by high-throughput sequencing platform. Among which 3359 unigenes were identified as differentially expressed unigenes between the heads and tails of AS by SBC analysis system scanning. Of which 15 differentially expressed unigenes participate in the metabolic regulation of phenylpropanoid biosynthesis (PB) pathway and ferulic acid metabolites, in response to the distinguished pharmaceutical actions of the heads and tails of AS.</p><p><b>CONCLUSION</b>Different content of ferulic acid in the heads and tails of AS is related to the differentially expressed genes, particularly involved in the PB pathway.</p>

HPPR encodes the hydroxyphenylpyruvate reductase required for the biosynthesis of hydrophilic phenolic acids in Salvia miltiorrhiza / 中国天然药物

Salvia miltiorrhiza is a medicinal plant widely used in the treatment of cardiovascular and cerebrovascular diseases. Hydrophilic phenolic acids, including rosmarinic acid (RA) and lithospermic acid B (LAB), are its primary medicinal ingredients. However, the biosynthetic pathway of RA and LAB in S. miltiorrhiza is still poorly understood. In the present study, we accomplished the isolation and characterization of a novel S. miltiorrhiza Hydroxyphenylpyruvate reductase (HPPR) gene, SmHPPR, which plays an important role in the biosynthesis of RA. SmHPPR contained a putative catalytic domain and a NAD(P)H-binding motif. The recombinant SmHPPR enzyme exhibited high HPPR activity, converting 4-hydroxyphenylpyruvic acid (pHPP) to 4-hydroxyphenyllactic acid (pHPL), and exhibited the highest affinity for substrate 4-hydroxyphenylpyruvate. SmHPPR expression could be induced by various treatments, including SA, GA, MeJA and Ag, and the changes in SmHPPR activity were correlated well with hydrophilic phenolic acid accumulation. SmHPPR was localized in cytoplasm, most likely close to the cytosolic NADPH-dependent hydroxypyruvate reductase active in photorespiration. In addition, the transgenic S. miltiorrhiza hairy roots overexpressing SmHPPR exhibited up to 10-fold increases in the products of hydrophilic phenolic acid pathway. In conclusion, our findings provide a new insight into the synthesis of active pharmaceutical compounds at molecular level.

Transcriptome analysis reveals candidate genes involved in esculentoside A biosynthesis in Phytolacca americana / 药学学报

In order to study the biosynthesis pathway of esculentoside A, the Illumina HiSeq 4000 highthroughput sequencing method was used to analyze the transcriptome of Phytolacca americana seedlings. The 9.60 Gb clean data were obtained after the transcriptome of P. americana assembled by Trinity software. The total 63 957 unigenes were obtained after assembly and the average length was 988.82 bp, among them 24 517 unigenes (38.33%) were annotated in the public databases Nr, Swiss-Prot, COG, KOG, Pfam, GO and KEGG. According to the assignment of KEGG pathway, 53 unigenes were involved in terpenoid backbone biosynthesis and 8 unigenes involved in triterpenoid biosynthesis. Additionally, there were 417 unigenes assigned to other secondary metabolic pathways in P. americana. The post-modification enzyme genes involved in the esculentoside A biosynthesis were also analyzed in the transcriptome of P. americana. The results indicated that 130 unigenes may have the function of CYP450 which was involved in oxidation/hydroxylation modification of P. americana secondary metabolites. Furthermore, 46 unigenes had the function of glycosyltransferase UGT. The transcriptome data of P. americana laid a foundation for studying the biosynthesis pathway of esculentoside A and other secondary metabolites, and also provided theoretical basis for formation of medicinal materials quality.

Research progress on triterpenoid saponins in Panax ginseng and its ecological effects / 中草药

Panax ginseng is well-known as an important medicinal resource in China. Triterpenoid saponins are the main secondary metabolite of P. ginseng with important ecological effects and its accumulation is influenced by many factors as well. In order to provide the valuable reference for researching and developing triterpenoid saponins in P. ginseng, the research progress in the type of triterpenoid saponins, biosynthetic pathway of triterpenoid saponins, influence factors of triterpenoid saponins accumulation, and ecological effects of ginsenosides were reviewed in this paper.

Identification of genes related to biosynthesis of methyleugenol in Asarum sieboldii by transcriptome sequence analysis / 中草药

Objective: To obtain the transcriptome sequence database and differentially expressed genes of Asarum sieboldii, and to identify the genes related to the biosynthesis of methyleugenol, the main chemical compound in this species. Methods: Roots and leaves of the plants were chosen as experiment materials. The transcriptome sequence database was constructed by applying an Illumina Hiseq 4000 Sequencing Platform. Unigenes were assembled by BLAST similarity searches and annotated with GO and KEGG orthologs identifiers. Moreover, differentially expressed genes were analyzed. Results: 12.25 Gb database was obtained, among which 129 003 unigenes were annotated to be involved in 52 GO-terms and 363 metabolic pathways. After analysis, 439 differentially expressed genes were observed, the up-regulated genes account for 38.3% and the down-regulated genes account for 61.7%. In addition, 136 unigenes involved in phenylpropanoid biosynthesis in A. sieboldii, and 44 unigenes that were associated with biosynthesis of methyleugenol were identified. Conclusion: Unigenes explored in this study will significantly contribute to genome-wide research and analysis of this species.

The Study and Regulation of Active Ingredients Has Broad Application Prospect in the Production of Chinese Herbal Medicine / 世界科学技术-中医药现代化

The resources and quality of Chinese herbal medicine is the foundation and guarantee for the sustainable development of the whole industry chain of traditional Chinese medicine.The effective component,having clinical curative effect,is the material base of Chinese herbal medicine,and is the core for the quality of medicinal material factors (quality).The accumulation of efficacy material is closely related to the genetic background of the original plant.By clarifying active ingredients biosynthesis,revealing medicinal material quality formation mechanism,regulating the content of effective components,the quality of medicine could be guarantee and ascend.The quality control technology based on active ingredients of Traditional Chinese medicine quality includes integrated analysis of high-throughput sequencing and systems biology data,revealing synthetic genetic mechanisms of active ingredients,excavating molecular markers of the quality,creating excellent germplasm,forming cultivation techniques,establishing classification standard of medicinal materials,and finally realizes the full industrial chain control of Chinese herbal medicine production.Taken all of these together could ensure the quality of medicinal materials.

Research process in biosynthesis pathway and regulation mechanism of phenolic acids in roots and rhizomes of Salvia miltiorrhiza / 中草药

Phenolic acids with significant biological activity and pharmacological effects are a class of active ingredients in the roots and rhizomes of Salvia miltiorrhiza used for the treatment of cardiovascular and cerebrovascular diseases. With increasing the demand of clinical and market, enhancing the contents and utilization efficiency of phenolic acids in the roots and rhizomes of S. miltiorrhiza has the important practical significance for sustainable development. In this paper, the research progress in study on genomics, proteomics, and metabonomics in recent years was carried out to summarize the biosynthesis pathway, key enzymes genes, regulation mechanism, and bioconversion and utilization of phenolic acids in the roots and rhizomes of S. miltiorrhiza, which lays the scientific foundation for the efficient production and comprehensive utilization of phenolic acids resources in S. miltiorrhiza.

Study progress on triterpenoids in Ganoderma lucidum / 中草药

The paper introduced the progress in study on triterpenoids in Ganoderma lucidum in recent years. The kinds of ganoderma triterpene, the pharmacological action, the biosynthesis pathway of ganoderma triterpene, and the influence factors of ganoderma triterpene accumulation were reviewed to provide the reference for the future development and utilization of ganoderma triterpene.

Comparison on chemical components and hypoglycemic activities between two closely-related Chinese materia medica / 中草药

Potentillae Discoloris cum Radice Herba and Agrimoniae Herba are closely-related medicinal plants in Rosaceae with specific hypoglycemic activities and applied as Chinese materia medica in clinical practice for the treatment of diabetes. Based upon pharmaphylogeny theories, the biosynthetic pathways of the principal active components of the above two Chinese herbs were summarized, and the commonness and differences of the active components were compared further and the correlation between material basis and curative effects was summarized as well. The results indicated that the common biosynthetic pathways of the two Chinese herbs accounted for 60%; The common components of tiliroside and quercetin, etc. as well as the differentiated components of potengriffioside A and rosolic acid, etc. are also the active components; The differences of the characteristic components and their contents may lead to the differences of functions and modes of action between the two Chinese herbs.

Artemisia herba alba & Artemisia monosperma: The Discovery of the first potential Egyptian plant sources for the Pharmaceutical Commercial Production of Artemisinin and Some of Its Related Analogues.

Artemisinin has received considerable attention in the last few decades as the current drug of choice for treatment of malaria and a number of other diseases. Because of the development of resistance against other malarial drugs, the demand for artemisinin has rapidly increased during the past decade. However, the supply of artemisinin is troublesome as neither total nor semi-synthesis is economically feasible and the only plant species known to produce artemisinin, Artemisia annua L., contains only low amounts of this compound ranging from (0.01-0.6%) of dry weight. The wish to improve the overall supply of artemisinin at a reduced market price has encouraged interest to hit upon novel plant sources for artemisinin production as alternative to A. annua. In our current study a fingerprint profile method was developed for the detection and quantification of artemisinin and its related analogues in the methanolic extract of Artemisia monosperma and Artemisia herba alba using a high-performance liquid chromatography (HPLC) with diode-array detector (DAD) and ion trap mass spectrometry. Artemisinin, dihydroartemisinin (α and β isomers), artemisitene, dihydroartemisinic aldehyde, dihydroartemisinic acid, dihydroartemisinic alcohol were detected and quantified in the methanolic extract of Artemisia monosperma using LC-MS peak at concentrations of 3.6, 1.9, 0.27, 0.06, 0.08 and 1.95 % of dry plant weight respectively in addition to the detection of arteannuin B and artemisinic acid at a trace levels. While artemisinin, artemisitene, dihydroartemisinic acid, and artemisinic acid were detected and quantified in the methanolic extract of Artemisia herba alba at concentrations of 4.9, 0.35, 0.08 and 0.04 % of dry plant weight respectively . The high unexpected concentration of artemisinin and some of its related analogues detected in this study reported Artemisia herba alba and Artemisia monosperma for the first time as a novel potential plant sources for artemisinin and some of its related analogues that may be helpful for its commercial pharmaceutical production and could lead to the improvement of the overall supply of artemisinin at a reduced market price offering an acceptable price for most patients.especially that these Artemisia species are abundant in distribution in Egyptian desert.

Changes in the flux of hexosamine biosynthesis pathway in skeletal muscle of rats with insusulin resistance induced by high fat diet / 中华内分泌代谢杂志

Objective To investigate the effect of hexosamine biosynthesis pathway on the development of insulin resistance induced by high fat diet.Methods Normal male SD rats were randomly divided into three groups:control(fed with normal chow),high fat(fed with high fat diet for 13 weeks),and rosiglitazone (intragastric administration with rosiglitazone for 5 weeks)groups.After 13 weeks,all the rats were sacrificed,serum and muscle triglycerides(TG),serum total cholesterol(TC),and serum and muscle free fatty acids(FFA) were measured.Insulin sensitivity wss evaluated by insulin sensitivity index(ISI)and glucose infused rat(GIR) with the hyperinsulinemic englycemic clamp technique.The flux of HBP in skeletal muscle was detected with the expression level of glutamine-fructose-6-phosphate transaminase(GFAT)mRNA(RT-PCR),the content of UDPGlcNAc(HPLC)and the level of O-GlcNAc glycosylation in skeletal muscle proteins(Western blot). Results Compared with control group,senlm TG,TC,FFA and muscle TG,FFA levels of high fat group increased(aII P<0.01).both ISI and GIR decreased(both P<0.01),and the leveIs of GFAT mRNA(0.51±0.05 vs 0.18±0.02),UDP-GlcNAc[(6.18±0.86 vs 2.42±0.36)nmol/g],and O-GIcNAc glycosylation of skeletal muscle proteins in high fat group were raised(all P<0.01).In rosiglitazone group,serum and muscle TG.FFA welc deceased(all P<0.01).insulin sensitivity was increased(P<0.05)and the flux of HBP[GFAT mRNA 0.27±0.03,UDP-GIcNAc(2.62±0.32)nmol/g]was reduced(all P<0.05)as compared with high fat group. Conclusions High fat diet-induced insulin resistance in rats is correlated with the increased flux of HBP in skeletaI muscle.which is decreased by rosiglitazone.

The Study on Gene Engineering Strains Producing Lycopene / 中国生物工程杂志

Lycopene—a kind of important active compound of caroteinoids, is greatly beneficial to human health with its diverse biological functions. With the elucidation of lycopene biosynthetic pathway and cloning genes of relative enzymes from microorganisms, it is possible to regulate lycopene biosynthesis via genetic engineering. The biosynthesis pathways of lycopene and gene cloning of lycopene biosynthetic enzymes in microorganisms were reviewed, and gene engineering strains documented in previous works including: E.coli and yeast constructed by genetic recombination, mold strains enhanced the ability of producing lycopene by gene manipulation were summarized. At last, compared with the present methods, the problems existed in the process of construction were pointed out.

The effect of glucose transporter 1 on hexosamine biosynthesis pathway in rat glomerular mesangial cells / 中华内分泌代谢杂志

Objective To investigate the role of hexosamine biosynthesis pathway (HBP) in the functionalalterationofmesangialcellstransinfectedwith glucose transporter 1 (GLUT1) gene. Methods Rat mesangial cells were transinfected with the human GLUT1 gene (MCGT1) by retrovirus vector. Mesangial cells transinfected with bacterial ?-galactosidase (MCLacZ) were used as control. Glucose uptake was detected with 2-deoxy-〔 3H〕-D-glucose (2-DG). Cell size, RNA/DNA ratio, protein/DNA ratio and the synthesis of fibronectin were evaluated by flow cytometry. The activity of glutamine: fructose-6-P aminotransferase (GFAT), which is the key enzyme of HBP, was assayed by spectrophotometry. The expression of GFAT gene was analyzed by RT-PCR. Results MCGT1 demonstrated higher 2-DG uptake than MCLacZ 〔(741.0?60.5)dpm/?g protein vs (92.2?9.0)dpm/?g protein,P