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metabolic engineering of Bacillus amyloliquefaciens for efficient production ofα-glucosidase inhibitor1-deoxynojirimycin

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Synthetic and Systems Biotechnology 2023年第3期8卷 378-385页

作者： Xujie Li Meng Zhang Yu Lu Ningyang Wu Jian’gang Chen Zhixia Ji Yangyang Zhan Xin Ma Junyong Chen Dongbo Cai Shouwen Chen State Key Laboratory of Biocatalysis and Enzyme engineering Environmental Microbial Technology Center of Hubei ProvinceCollege of Life SciencesHubei UniversityWuhan430062China State Key Laboratory of Agricultural Microbiology Huazhong Agricultural UniversityWuhan430070China Department of Urology Zhuhai People’s Hospital(Zhuhai Hospital Affiliated to Jinan University)Zhuhai519000China Wuhan Jun’an Biotechnology Co. Ltd.Wuhan430070China

Owing to the feature of strongα-glucosidase inhibitory activity,1-deoxynojirimycin(1-DNJ)has broad application prospects in areas of functional food,biomedicine,etc.,and this research wants to construct an efficient strain for 1-DNJ production,basing on Bacillus amyloliquefaciens ***,using the temperature-sensitive shuttle plasmid T2(2)-Ori,gene ptsG in phosphotransferase system(PTS)was weakened by homologous recombination,and non-PTS pathway was strengthened by deleting its repressor gene iolR,and 1-DNJ yield of resultant strain HZ-S2 was increased by 4.27-fold,reached 110.72 mg/***,to increase precursor fructose-6-phosphate(F-6-P)supply,phosphofructokinase was weaken,fructose phosphatase GlpX and 6-phosphate glucose isomerase Pgi were strengthened by promoter replacement,moreover,regulator gene nanR was deleted,1-DNJ yield was further increased to 267.37 mg/L by ***,promoter of 1-DNJ synthetase cluster was optimized,as well as 5′-UTRs of downstream genes in synthetase cluster,and 1-DNJ produced by the final strain reached 478.62 mg/*** but not the least,1-DNJ yield of 1632.50 mg/L was attained in 3 L fermenter,which was the highest yield of 1-DNJ reported to *** together,our results demonstrated that metabolic engineering was an effective strategy for 1-DNJ synthesis,this research laid a foundation for industrialization of functional food and drugs based on 1-DNJ.

关键词： 1-Deoxynojirimycin Bacillus amyloliquefaciens metabolic engineering Glucose transport pathway Fructose 6-phosphate 1-DNJ synthetase Gene cluster

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metabolic engineering of an industrial bacterium Zymomonas mobilis for anaerobic l-serine production

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Synthetic and Systems Biotechnology 2024年第2期9卷 349-358页

作者： Zhen Wang Xia Wang Xiongying Yan Haixia Yi Shuche He Haoyu Zhang Xinli Zhou Qiaoning He Shihui Yang State Key Laboratory of Biocatalysis and Enzyme engineering and School of Life Sciences Hubei UniversityWuhan430062China

Due to the complicated metabolic and regulatory networks of l-serine biosynthesis and degradation,microbial cell factories for l-serine production using non-model microorganisms have not been *** this study,a combination of synthetic biology and process optimization were applied in an ethanologenic bacterium Zymomonas mobilis for l-serine *** blocking the degradation pathway while introducing an exporter EceamA from ***,l-serine titer in recombinant *** was increased from 15.30 mg/L to 62.67 mg/*** was further increased to 260.33 mg/L after enhancing the l-serine biosynthesis ***,536.70 mg/L l-serine was achieved by removing feedback inhibition with a SerA mutant,and an elevated titer of 687.67 mg/L was further obtained through increasing serB copies while enhancing the ***,855.66 mg/L l-serine can be accumulated with the supplementation of the glutamate *** work thus not only constructed an l-serine producer to help understand the bottlenecks limiting l-serine production in *** for further improvement,but also provides guidance on engineering non-model microbes to produce biochemicals with complicated pathways such as amino acids or terpenoids.

关键词： Zymomonas mobilis metabolic engineering l-serine Feedback inhibition Anaerobic fermentation

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metabolic engineering of yeasts for green and sustainable production of bioactive ginsenosides F2 and 3β,20S-Di-O-Glc-DM

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Acta Pharmaceutica Sinica B 2022年第7期12卷 3167-3176页

作者： Fenglin Jiang Chen Zhou Yan Li Haidong Deng Ting Gong Jingjing Chen Tianjiao Chen Jinling Yang Ping Zhu State Key Laboratory of Bioactive Substance and Function of Natural Medicines NHC Key Laboratory of Biosynthesis of Natural ProductsCAMS Key Laboratory of Enzyme and Biocatalysis of Natural DrugsInstitute of Materia MedicaChinese Academy of Medical Sciences&Peking Union Medical CollegeBeijing 100050China

Both natural ginsenoside F2 and unnatural ginsenoside 3β,20S-Di-O-Glc-DM were reported to exhibit anti-tumor *** approaches for producing them rely on direct extraction from Panax ginseng,enzymatic catalysis or chemical synthesis,all of which result in low yield and high *** engineering of microbes has been recognized as a green and sustainable biotechnology to produce natural and unnatural *** we engineered the complete biosynthetic pathways of F2 and 3β,20S-Di-OGlc-DM in Saccharomyces cerevisiae via the CRISPR/Cas9 *** titers of F2 and 3β,20S-Di-O-GlcDM were increased from 1.2 to 21.0 mg/L and from 82.0 to 346.1 mg/L at shake flask level,respectively,by multistep metabolic engineering ***,pharmacological evaluation showed that both F2and 3β,20S-Di-O-Glc-DM exhibited anti-pancreatic cancer activity and the activity of 3β,20S-Di-O-GlcDM was even ***,the titer of 3β,20S-Di-O-Glc-DM reached 2.6 g/L by fed-batch fermentation in a 3 L *** our knowledge,this is the first report on demonstrating the anti-pancreatic cancer activity of F2 and 3β,20S-Di-O-Glc-DM,and achieving their de novo biosynthesis by the engineered *** work presents an alternative approach to produce F2 and 3β,20S-Di-O-Glc-DM from renewable biomass,which lays a foundation for drug research and development.

关键词： Ginsenoside UDP-glycosyltransferase metabolic engineering Synthetic biology Saccharomyces cerevisiae CRISPR/Cas9 system Microbial cell factory Anti-pancreatic cancer activity

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metabolic engineering in woody plants: challenges, advances, and opportunities

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aBIOTECH 2021年第3期2卷 299-313页

作者： Shu Yu Cody S.Bekkering Li Tian Department of Plant Sciences Mail Stop 3University of CaliforniaDavisCA 95616USA

Woody plant species represent an invaluable reserve of biochemical diversity to which metabolic engineering can be applied to satisfy the need for commodity and specialty chemicals,pharmaceuticals,and renewable *** plants are particularly promising for this application due to their low input needs,high biomass,and immeasurable ecosystem ***,existing challenges have hindered their widespread adoption in metabolic engi neering efforts,such as long generation times,large and highly heterozygous genomes,and difficulties in transfor mation and *** advances in omics approaches,systems biology modeling and plant transformation and regeneration methods provide effective approaches in overcoming these outstanding *** brought by developments in this space are steadily opening the door to widespread metabolic engineering of woody pl ants to meet the global need for a wide range of sustainably sourced chemicals and materials.

关键词： Woody plant metabolic engineering Bioeconomy Sustainability

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metabolic engineering of Streptomyces coelicolor for enhanced prodigiosins (RED) production

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Science China(Life Sciences) 2017年第9期60卷 948-957页

作者： Panpan Liu Hong Zhu Guosong Zheng Weihong Jiang Yinhua Lu Key Laboratory of Synthetic Biology Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200032 China University of Chinese Academy of Sciences Beijing 100039 China jiangsu National Synergetic Innovation Center for Advanced Materials SICAM Nanjing 210009 China College of Life and Environment Sciences Shanghai Normal University Shanghai 200234 China

Bacterial prodigiosins are red-colored secondary metabolites with multiple activities,such as anticancer,antimalarial and immunosuppressive,which hold great potential for medical *** this study,dramatically enhanced prodigiosins(RED) production in Streptomyces coelicolor was achieved by combinatorial metabolic engineering,including inactivation of the repressor gene ohkA,deletion of the actinorhodin(ACT) and calcium-dependent antibiotic(CDA) biosynthetic gene clusters(BGCs) and multi-copy chromosomal integration of the RED *** results showed that ohkA deletion led to a 1-fold increase of RED production over the wild-type strain ***,the ACT and CDA BGCs were deleted successively based on the AohkA mutant(SBJ101).To achieve multi-copy RED BGC integration,artificial ΦC31 attB site(s) were inserted simultaneously at the position where the ACT and CDA BGCs were *** resulting strains SBJ102(with a single deletion of the ACT BGC and insertion of one artificial attB site) and SBJ103(with the deletion of both BGCs and insertion of two artificial attB sites) produced 1.9-and 6-fold higher RED titers than M145,***,the entire RED BGC was introduced into mutants from SBJ101 to SBJ103,generating three mutants(from SBJ104 to SBJ106) with chromosomal integration of one to three copies of the RED *** highest RED yield was from SBJ106,which produced a maximum level of 96.8 mg g^(-1) cell dry weight,showing a 12-fold increase relative to ***,the metabolic engineering strategies employed in this study are very efficient for the construction of high prodigiosin-producing strains.

关键词： Streptomyces coelicolor prodigiosins metabolic engineering multi-copy integration

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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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metabolic engineering and genome editing strategies for enhanced lipid production in microalgae

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BIOCELL 2024年第8期48卷 1181-1195页

作者： ANJANI DEVI CHINTAGUNTA SAMUDRALA PRASHANT JEEVAN KUMAR NUNE SATYA SAMPATH KUMAR Department of Biotechnology Vignan’s Foundation ScienceTechnology and ResearchVadlamudiAndhra Pradesh522213India ICAR-Directorate of Floricultural Research Pune412307India

Depleting global petroleum reserves and skyrocketing prices coupled with succinct supply have been a grave concern,which needs alternative sources to conventional *** microalgae have been explored for enhanced lipid production,leading towards biodiesel *** microalgae have short life cycles,require less labor,and space,and are easy to scale ***,the primary source of lipids needed to produce biodiesel,is accumulated by most *** article focuses on different types of oleaginous microalgae,which can be used as a feedstock to produce *** biosynthesis in microalgae occurs through fatty acid synthesis and TAG synthesis ***-depth discussions are held regarding other efficient methods for enhancing fatty acid and TAG synthesis,regulating TAG biosynthesis bypass methods,blocking competing pathways,multigene approach,and genome *** most potential targets for gene transformation are hypothesized to be a malic enzyme and diacylglycerol acyltransferase while lowering phosphoenolpyruvate carboxylase activity is reported to be advantageous for lipid synthesis.

关键词： Oleaginous microalgae Biodiesel TAG synthesis metabolic engineering Genome editing

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metabolic engineering for the microbial production of isoprenoids:Carotenoids and isoprenoid-based biofuels

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Synthetic and Systems Biotechnology 2017年第3期2卷 167-175页

作者： Fu-Xing Niu Qian Lu Yi-Fan Bu Jian-Zhong Liu Biotechnology Research Center and Biomedical Center School of Life SciencesSun Yat-sen UniversityGuangzhou 510275China South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center School of Life SciencesSun Yat-sen UniversityGuangzhou 510275China

Isoprenoids are the most abundant and highly diverse group of natural *** isoprenoids have been used for pharmaceuticals,nutraceuticals,flavors,cosmetics,food additives and *** and isoprenoid-based biofuels are two classes of important *** isoprenoids have been produced microbially through metabolic engineering and synthetic biology ***,we briefly review the engineered biosynthetic pathways in well-characterized microbial systems for the production of carotenoids and several isoprenoid-based biofuels.

关键词： Carotenoids Isoprenoid-based biofuel metabolic engineering Synthetic biology

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metabolic engineering and flux analysis of Corynebacterium glutamicum for L-serine production

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Science China(Life Sciences) 2012年第4期55卷 283-290页

作者： LAI ShuJuan ZHANG Yun LIU ShuWen LIANG Yong SHANG XiuLing CHAI Xin WEN TingYi Department of lndustrial Microbiology and Biotechnology Institute of Microbiology Chinese Academy of Sciences Beijing 100101 China Graduate University of Chinese Academy of Sciences Beijing 100049 China

L-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of L-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engineered de novo by blocking and at- tenuating the conversion of L-serine to pyruvate and glycine, releasing the feedback inhibition by L-serine to 3-phosphoglycerate dehydrogenase （PGDH）, in combination with the co-expression of 3-phosphoglycerate kinase （PGK） and feedback-resistant PGDH （PGDHr）. The resulting strain, SER-8, exhibited a lower specific growth rate and significant differ- ences in L-serine levels from Phase I to Phase V as determined for fed-batch fermentation. The intracellular L-serine pool reached （14.22_＋1.41） ~trnol gcoM-1, which was higher than glycine pool, contrary to fermentation with the wild-type strain. Furthermore, metabolic flux analysis demonstrated that the over-expression of PGK directed the flux of the pentose phosphate pathway （PPP） towards the glycolysis pathway （EMP）, and the expression of PGDHr improved the L-serine biosynthesis pathway. In addition, the flux from L-serine to glycine dropped by 24%, indicating that the deletion of the activator GlyR re- sulted in down-regulation of serine hydroxymethyltransferase （SHMT） expression. Taken together, our findings imply that L-serine pool management is fundamental for sustaining the viability of C. glutamicum, and improvement of C1 units genera- tion by introducing the glycine cleavage system （GCV） to degrade the excessive glycine is a promising target for L-serine pro- duction in C. glutamicum.

关键词： Corynebacterium glutamicum L-serine intracellular metabolites metabolic engineering elementary mode analysis

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metabolic engineering of Corynebacterium glutamicum S9114 based on whole-genome sequencing for efficient N-acetylglucosamine synthesis

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Synthetic and Systems Biotechnology 2019年第3期4卷 120-129页

作者： Chen Deng Xueqin Lv Yanfeng Liu Jianghua Li Wei Lu Guocheng Du Long Liu Key Laboratory of Carbohydrate Chemistry and Biotechnology Ministry of EducationJiangnan UniversityWuxi214122China Key Laboratory of Industrial Biotechnology Ministry of EducationJiangnan UniversityWuxi214122China Shandong Runde Biotechnology CO. LTDTaian271200China

Glucosamine(GlcN)and its acetylated derivative N-acetylglucosamine(GlcNAc)are widely used in the pharmaceutical ***,we attempted to achieve efficient production of GlcNAc via genomic engineering of Corynebacterium ***,we ligated the GNA1 gene,which converts GlcN-6-phosphate to GlcNAc-6-phosphate by transferring the acetyl group in Acetyl-CoA to the amino group of GlcN-6-phosphate,into the plasmid pJYW4 and then transformed this recombinant vector into the *** ATCC 13032,ATCC 13869,ATCC 14067,and S9114 strains,and we assessed the GlcNAc titers at 0.5 g/L,1.2 g/L,0.8 g/L,and 3.1 g/L from each strain,*** suggested that there were likely to be significant differences among the key genes in the glutamate and GlcNAc synthesis pathways of these *** ***,we performed whole genome sequencing of the S9114 strain,which has not been previously published,and found that there are many differences among the genes in the glutamate and GlcNAc synthesis pathways among the four strains ***,nagA(encoding GlcNAc-6-phosphate deacetylase)and gamA(encoding GlcN-6-phosphate deaminase)were deleted in *** S9114 to block the catabolism of intracellular GlcNAc,leading to a 54.8%increase in GlcNAc production(from 3.1 to 4.8 g/L)when grown in a shaker *** addition,lactate synthesis was blocked by knockout of ldh(encoding lactate dehydrogenase);thus,further increasing the GlcNAc titer to 5.4 g/***,we added a key gene of the GlcN synthetic pathway,glmS,from different sources into the expression vector pJYW-4-ceN,and the resulting recombinant strain CGGN2-GNA1-CgglmS produced the GlcNAc titer of 6.9 g/*** is the first report concerning the metabolic engineering of ***,and the results of this study provide a good starting point for further metabolic engineering to achieve industrial-scale production of GlcNAc.

关键词： C.glutamicum Complete genome N-acetylglucosamine metabolic engineering

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