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Engineering CrtW and CrtZ for improving biosynthesis of astaxanthin in Escherichia coli

Chinese Journal of Natural Medicines 2020年第9期18卷 666-676页

作者： LI Di LI Yang XU Jiao-Yang LI Qing-Yan TANG Jin-Lei JIA Shi-Ru BI Chang-Hao DAI Zhu-Bo ZHU Xin-Na ZHANG Xue-Li College of biotechnology Tianjin University of Science and TechnologyTianjin 310018China Tianjin Institute of Industrial biotechnology Chinese of Academy of SciencesTianjin 300308China Key Laboratory of Systems microbial biotechnology.Chinese Academy of Sciences Tianjin 300308China University of Chinese Academy of Sciences.Beijing 100071 China National Technology Innovation Center of Synthetic Biology Tianjin 300308China

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 A6 T, T105 A and L239 M 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^(-1) 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.

关键词： Astaxanthin RBS library Metabolic engineering β-Carotene ketolase Cre-loxP Escherichia coli

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Characterization of the positive SARP family regulator PieR for improving piericidin A1 production in Streptomyces piomogeues ***

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Synthetic and Systems biotechnology 2019年第1期4卷 16-24页

作者： Yan Li Lingxin Kong Jufang Shen Qing Wang Qian Liu Weinan Yang Zixin Deng Delin You State Key Laboratory of microbial Metabolism Joint International Research Laboratory of Metabolic and Developmental SciencesSchool of Life Sciences&BiotechnologyShanghai Jiao Tong UniversityShanghaiChina

Piericidin A1,a member ofɑ-pyridone antibiotic,exhibits various biological activities such as antimicrobial,antifungal,and antitumor properties and possesses potent respiration-inhibitory activity against insects due to its competitive binding capacity to mitochondrial complex *** biosynthetic pathway of piericidin A1 has been reported in Streptomyces piomogeues ***,while the regulatory mechanism remains poorly *** this study,a Streptomyces antibiotic regulatory protein(SARP)family transcriptional regulator PieR was *** disruption and complementation manipulations revealed that PieR positively regulated the production of piericidin ***,the overexpression of pieR contributed to the improvement of piericidin A1 *** real-time quantitative PCR(RT-qPCR)was carried out and the data showed that pieR stimulated the transcription of all the biosynthesis-related genes for piericidin *** order to explore the regulatory mechanism,electrophoresis mobility shift assays(EMSA)and DNase I footprinting experiments have been conducted.A protected region covering 50 nucleotides within the upstream region of pieR was identified and two 5-nt direct repeat sequences(5′-CCGGA-3′)in the protected region were *** findings,taken together,set stage for transcriptional control engineering in the view of optimizing piericidin A1 production and thus provide a viable potent route for the construction of strains with high productivity.

关键词： ɑ-pyridone antibiotic Transcriptional regulation SARP family regulator Secondary metabolic Streptomyces

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m4C DNA methylation regulates biosynthesis of daptomycin in Streptomyces roseosporus L30

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Synthetic and Systems biotechnology 2022年第4期7卷 1013-1023页

作者： Jiao-Le Fang Wen-Li Gao Wei-Feng Xu Zhong-Yuan Lyu Lie Ma Shuai Luo Xin-Ai Chen Xu-Ming Mao Yong-Quan Li Institute of Pharmaceutical biotechnology Zhejiang University School of MedicineHangzhou310058PR China Zhejiang Provincial Key Laboratory for microbial Biochemistry and Metabolic Engineering 310058HangzhouPR China

Despite numerous studies on transcriptional level regulation by single genes in drug producing Actinomyces,the global regulation based on epigenetic modification is not well explored.N4-methylcytosine(m4C),an abundant epigenetic marker in Actinomycetes’genome,but its regulatory mechanism remains *** this study,we identify a m4C methyltransferase(SroLm3)in Streptomyces roseosporus L30 and multi-omics studies were performed and revealed SroLm3 as a global regulator of secondary ***,three BGCs inΔsroLm3 strain exhibited decreased expression compared to wild ***-frame deletion of sroLm3 in *** L30 further revealed its role in enhancing daptomycin *** summary,we characterized a m4C methyltransferase,revealed the function of m4C in secondary metabolism regulation and biosynthesis of red pigment,and mapped a series of novel regulators for daptomycin biosynthesis dominated by m4C *** research further indicated that m4C DNA methylation may contribute to a metabolic switch from primary to secondary metabolism in Actinomyces.

关键词： N4-methylcytosine DNA methyltransferase Daptomycin Transcriptional regulator Secondary metabolism

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New xylose transporters support the simultaneous consumption of glucose and xylose in Escherichia coli

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mLife 2022年第2期1卷 156-170页

作者： Xinna Zhu Feiyu Fan Huanna Qiu Mengyao Shao Di Li Yong Yu Changhao Bi Xueli Zhang Tianjin Institute of Industrial biotechnology Chinese Academy of SciencesTianjinChina Key Laboratory of Systems microbial biotechnology Chinese Academy of SciencesTianjinChina College of biotechnology Tianjin University of Sciences and TechnologyTianjinChina University of Chinese Academy of Sciences BeijingChina

Glucose and xylose are two major components of *** consumption of glucose and xylose is critical for engineered microorganisms to produce fuels and chemicals from lignocellulosic *** many production limitations have been resolved,glucose‐induced inhibition of xylose transport remains a *** this study,a cell growthbased screening strategy was designed to identify xylose transporters uninhibited by *** glucose pathway was genetically blocked in Escherichia coli so that glucose functions only as an inhibitor and cells need xylose as the carbon source for *** adaptive evolution,omics analysis and reverse metabolic engineering,a new phosphoenolpyruvate:carbohydrate phosphotransferase system(PTS)galactitol transporter(GalABC,encoded by EcolC_1640,EcolC_1641,and EcolC_1642 genes)that is not inhibited by glucose was *** of adenylate cyclase led to increased expression of the EcolC_1642 gene,and a point mutation in gene EcolC_1642(N13S)further enhanced xylose *** the second round of gene mining,AraE and a new ABC transporter(AraFGH)of xylose were identified.A point mutation in the transcription regulator araC(L156I)caused increased expression of araE and araFGH genes without arabinose induction,and a point mutation in araE(D223Y)further enhanced xylose *** newly identified xylose transporters can support the simultaneous consumption of glucose and xylose and have potential use in producing chemicals from lignocellulose.

关键词： ABC transporter glucose PTS xylose xylose transporter

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Probiotics and Their Metabolites Ameliorate Inflammatory Bowel Disease:A Critical Review

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Infectious Microbes & Diseases 2021年第1期3卷 4-13页

作者： Ying Liang Manman Liu Jiaqian Pu Zichun Zhu Zining Gao Qingqing Zhou Qing Gu Ping Li Key Laboratory for Food microbial Technology of Zhejiang Province College of Food Science and BiotechnologyZhejiang Gongshang UniversityHangzhouZhejiangChina.

Crohn disease and ulcerative colitis are the two main manifestations of inflammatory bowel disease(IBD),and both are highly ***,the precise etiology of IBD is still unknown and effective therapeutics are yet to be *** is becoming increasingly clear that a combination of factors,including genetic background,host immune response,and microbial reduced diversity status are related to *** cardinal symptom of IBD patients is the imbalance of the intestinal *** to previous studies,both probiotics and symbiotic microbiota can play a protective role through intestinal micro-ecosystem regulation,epithelial barrier integrity enhancement,and inflammation ***,probiotics can provide an alternative or auxiliary method to traditional IBD ***,we reviewed the possible pathogenesis of IBD,summarized the possible mechanisms of probiotics modulation of IBD,and emphasized the prevention and treatment targets of probiotics-mediated IBD,with the aim to provide theoretical support for the treatment of IBD patients by probiotics in clinical trials.

关键词： inflammatory bowel disease metabolites probiotics the regulatory mechanism

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Nucleotide Recognition by a Guanidinocalixarene Receptor in Aqueous Solution

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Chemical Research in Chinese Universities 2023年第1期39卷 144-150页

作者： GENG Wenchao ZHENG Zhe JIANG Huifeng GUO Dongsheng Key Laboratory of Systems microbial biotechnology Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjin300308P.R.China College of Chemistry Key Laboratory of Functional Polymer Materials of Ministry of EducationState Key Laboratory of Elemento-Organic ChemistryNankai UniversityTianjin300071P.R.China

Nucleotides participate in various physiological processes through their supramolecular interactions with ***,the molecular recognition of nucleotides became an important topic in supramolecular chemistry and exhibited many biomedical *** showed very strong binding affinities towards nucleotides,even reaching the nanomolar *** this work,we systematically determined the binding constants between a typical guanidinocalixarene(guanidinium-modified calix[5]arene,GC5A)and various nucleotides and revealed the driving forces behind the molecular recognition using theoretical *** electrostatic interactions and hydrogen bonding provided by the phosphate groups of the nucleotides dominated the binding between the nucleotides and *** lower rim alkyl chains and the skeleton of GC5A provide preorganized cavity and upper guanidinium *** difference in the type of nucleobase is also attributed to the different binding *** work provides insight into the molecular recognition of nucleotides and facilitates the development of new supramolecular hosts for nucleotides and related biological applications.

关键词： Calixarene Nucleotide Molecular recognition Indicator displacement assay Theoretical calculation

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A New Indicator Cell Line Established to Monitor Bovine Foamy Virus Infection

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Virologica Sinica 2011年第5期26卷 315-323页

作者： Hong-yan Guo Zhi-bin Liang Yue Li Juan Tan Qi-min Chen Wen-tao Qiao Key Laboratory of Molecular Microbiology and biotechnology Ministry of Education and Key Laboratory of Microbial Functional Genomics of Tianjin College of Life Sciences Nankai University Tianjin 300071 China

In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro,we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter (LTR,from-7 to 1012).The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the *** clone,designated BFVL,was selected from ten neomycin-resistant *** showed a specific and inducible dose-and time-dependent luciferase activity in response to BFV *** the changes in luciferase activity of BFVL peaked at 84 h post infection,it was possible to differentiate infected and uninfected cells at 48 h post infection.A linear relationship was established between the multiplicity of infection (MOI) of BFV and the activated ratio of luciferase expression in ***,the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post *** findings suggest that the BFVL-based assay is rapid,easy,sensitive,quantitative and specific for detection of BFV infection.

关键词： Bovine foamy virus Firefly luciferase Indicator cell line

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Metabolic engineering of a methyltransferase for production of drug precursors demecycline and demeclocycline in Streptomyces aureofaciens

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Synthetic and Systems biotechnology 2020年第3期5卷 121-130页

作者： Weinan Yang Lingxin Kong Qing Wang Zixin Deng Delin You State Key Laboratory of microbial Metabolism Joint International Research Laboratory of Metabolic and Developmental SciencesSchool of Life Sciences&BiotechnologyShanghai Jiao Tong UniversityShanghai200030China

Demecycline(DMTC)and demeclocycline(DMCTC)are C6-demethylated derivatives of tetracycline(TC)and chlortetracycline(CTC),*** are precursors of minocycline and tigecycline,which showed remarkable bioactivity against TC-resistant bacteria and have been used clinically for *** order to biosynthesize drug precursors DMTC and DMCTC,the function of a possible C-methyltransferase encoding gene ctcK was studied systematically in the CTC high-yielding industrial strain Streptomyces aureofaciens ***ΔctcK mutant accumulated two new products,which were turned out to be DMTC and ***,timecourse analysis of the fermentation products detected the epimers of DMTC and DMCTC transformed ***,an engineering strain with higher productivity of DMCTC was constructed by deleting ctcK and overexpressing ctcP of three extra copies *** of these two engineering strains not only served as a successful example of synthesizing required products through metabolic engineering,but also provided original strains for following elaborate engineering to synthesize more effective tetracycline derivatives.

关键词： Demecycline Demeclocycline Streptomyces aureofaciens F3 Metabolic engineering

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Glycosyltransferases:Mining,engineering and applications in biosynthesis of glycosylated plant natural products

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Synthetic and Systems biotechnology 2022年第1期7卷 602-620页

作者： Bo He Xue Bai Yumeng Tan Wentao Xie Yan Feng Guang-Yu Yang State Key Laboratory of microbial Metabolism Joint International Research Laboratory of Metabolic&Developmental SciencesSchool of Life Sciences and BiotechnologyShanghai Jiao Tong UniversityShanghai200240China

UDP-Glycosyltransferases(UGTs)catalyze the transfer of nucleotide-activated sugars to specific acceptors,among which the GT1 family enzymes are well-known for their function in biosynthesis of natural product *** GT function represents necessary step in metabolic engineering of aglycone glycosylation to produce drug leads,cosmetics,nutrients and *** this review,we systematically summarize the phylogenetic distribution and catalytic diversity of plant *** also discuss recent progress in the identifi-cation of novel GT candidates for synthesis of plant natural products(PNPs)using multi-omics technology and deep learning predicted *** also highlight recent advances in rational design and directed evolution engineering strategies for new or improved GT ***,we cover recent breakthroughs in the appli-cation of GTs for microbial biosynthesis of some representative glycosylated PNPs,including flavonoid glycosides(fisetin 3-O-glycosides,astragalin,scutellarein 7-O-glucoside),terpenoid glycosides(rebaudioside A,ginseno-sides)and polyketide glycosides(salidroside,polydatin).

关键词： Glycosyltransferases Glycosylated plant natural products Enzyme mining Protein engineering Biosynthesis

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Hepatitis C Virus RNA Strands Detection in Peripheral Blood Mononuclear Cells Legitimizes Virus Eradication in Negative Serum PCR Naive and Post-treatment Patients

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Journal of Clinical and Translational Hepatology 2017年第1期5卷 1-8页

作者： Mohamed Darwish Ahmed Abd Alla Mostafa Kamel El Awady Tropical Medicine Department Faculty of MedicineAl-Azhar UniversityCairoEgypt Department of microbial biotechnology National Research CenterCairoEgypt

Background and Aims:Hepatitis C virus(HCV)hepatotropism is associated with intra-peripheral blood mononuclear cell(PBMC)infection that causes post-treatment relapse in RNA seronegative *** understanding of the association of non-viremic hepatic fibrosis with positive anti-HCV IgG antibodies and active hepatocellular damage might be increased by PBMCs screening for intracellular ***,the goals of this study included evaluation of PBMCs PCR for diagnosing HCV infection,addressing PBMCs plus serum real-time(SRT)PCR benefits over SRT-PCR alone,studying intraPBMCs distribution of RNA sense and antisense strands,and identifying treatment feasibility in solitary intracellular ***:Enzyme-linked immunosorbent assay,SRT-PCR and PBMCs PCR were used to evaluate HCV infection in 401 *** patients were classified into groups of negative controls(n=30),positive controls(n=63),non-viremia post-treatment(experienced;n=166)and na(i)ve(n=49)cases,and non-viremia positive PBMCs PCR na(i)ve(n=35)and experienced(n=58)***:The diagnosis of true positive and negative by PBMCs PCR and SRT-PCR had 100%and 96.7%compatibility *** PCR detected intracellular HCV infection in 49 out of 215 non-viremia patients;among them,na(i)ve cirrhotics had significantly higher number of intracellular infection than the na(i)ve non-cirrhotic(P<0.001)and experienced patients(P<0.0001).Antisense and sense strands were respectively recognized in na(i)ve and experienced cases(P=0.01218).Intracellular HCV strands were detected in 18.02%of experienced *** of intracellular RNA strands showed significant decline in experienced compared to na(i)ve patients(P<0.05).Conclusion:PBMCs PCR is a valid diagnostic test that can diagnose intracellular HCV when SRT-PCR is *** and sense strands are respectively recognized more often in na(i)ve and experienced *** expected overall relapsing rate in our cohort was 18.02%.Int

关键词： HCV PBMCs RNA Strands

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