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Identification of neutral genome integration sites with high expression and high integration efficiency in Fusarium venenatum TB01

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Synthetic and Systems biotechnology 2023年第1期8卷 141-147页

作者： Sheng Tong Kexin An Wuxi Chen Mengdan Chai Yuanxia Sun Qinhong Wang Demao Li tianjin Key Laboratory for industrial Biological Systems and Bioprocessing Engineering Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjin300308China National Innovation Centre for Synthetic Biology Tianjin300308China

CRISPR/Cas9-mediated homology-directed recombination is an efficient method to express target genes.Based on the above method,providing ideal neutral integration sites can ensure the reliable,stable,and high expression of target genes.In this study,we obtained a fluorescent transformant with neutral integration and high expression of the GFP expression cassette from the constructed GFP expression library and named strain FS.The integration site mapped at 4886 bp upstream of the gene FVRRES_00686 was identified in strain FS based on a Y-shaped adaptor-dependent extension,and the sequence containing 600 bp upstream and downstream of this site was selected as the candidate region for designing sgRNAs(Sites)for CRISPR/Cas9-mediated homology-directed recombination.PCR analysis showed that the integration efficiency of CRISPR/Cas9-mediated integration of target genes in designed sites reached 100%.Further expression stability and applicability analysis revealed that the integration of the target gene into the above designed sites can be stably inherited and expressed and has no negative effect on the growth of F.venenatum TB01.These results indicate the above designed neutral sites have the potential to accelerate the development of F.venenatum TB01 through overexpression of target genes in metabolic engineering.

关键词： GFP expression library Neutral integration site Y-shaped adaptor-dependent extension CRISPR/Cas9 Fusarium venenatum

Melatonin biosynthesis pathways in nature and its production in engineered microorganisms

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Synthetic and Systems biotechnology 2022年第1期7卷 544-553页

作者： Xiaotong Xie Dongqin Ding Danyang Bai Yaru Zhu Wei Sun Yumei Sun Dawei Zhang Dalian Polytechnic University Dalian116000PR China tianjin institutes of industrial biotechnology Chinese Academy of SciencesTianjin300308PR China Biodesign Center Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjin300308PR China tianjin University of science and technology Tianjin300308PR China

Melatonin is a biogenic amine that can be found in plants,animals and microorganism.The metabolic pathway of melatonin is different in various organisms,and biosynthetic endogenous melatonin acts as a molecular signal and antioxidant protection against external stress.Microbial synthesis pathways of melatonin are similar to those of animals but different from those of plants.At present,the method of using microorganism fermentation to produce melatonin is gradually prevailing,and exploring the biosynthetic pathway of melatonin to modify microorganism is becoming the mainstream,which has more advantages than traditional chemical synthesis.Here,we review recent advances in the synthesis,optimization of melatonin pathway.L-tryptophan is one of the two crucial precursors for the synthesis of melatonin,which can be produced through a four-step reaction.Enzymes involved in melatonin synthesis have low specificity and catalytic efficiency.Site-directed mutation,directed evolution or promotion of cofactor synthesis can enhance enzyme activity and increase the metabolic flow to promote microbial melatonin production.On the whole,the status and bottleneck of melatonin biosynthesis can be improved to a higher level,providing an effective reference for future microbial modification.

关键词： Melatonin Biosynthesis Plants Animals Microorganisms Metabolic modification Enzyme catalysis mechanism

Origin and evolution of the main starch biosynthetic enzymes

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Synthetic and Systems biotechnology 2023年第3期8卷 462-468页

作者： Hong Chang Jie Bai Hejian Zhang Rong Huang Huanyu Chu Qian Wang Hao Liu Jian Cheng Huifeng Jiang College of biotechnology Tianjin University of Science and TechnologyTianjin300457China Key Laboratory of Engineering Biology for Low-carbon Manufacturing Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjin300308China National Center of Technology Innovation for Synthetic Biology Tianjin300308China

Starch,a semi-crystalline energy storage form primarily found in plant plastids plays a crucial role in various food or no-food applications.Despite the starch biosynthetic pathway’s main enzymes have been characterized,their origin and evolution remained a subject of debate.In this study,we conducted the comprehensive phylogenetic and structural analysis of three types of starch biosynthetic enzymes:starch synthase(SS),starch branching enzyme(SBE)and isoamylase-type debranching enzyme(ISA)from 51,151 annotated genomes.Our findings provide valuable insights into the possible scenario for the origin and evolution of the starch biosynthetic pathway.Initially,the ancestor of SBE can be traced back to an unidentified bacterium that existed before the formation of the last eukaryotic common ancestor(LECA)via horizontal gene transfer(HGT).This transfer event likely provided the eukaryote ancestor with the ability to synthesize glycogen.Furthermore,during the emergence of Archaeplastida,one clade of SS was transferred from Deltaproteobacteria by HGT,while ISA and the other clade of SS originated from Chlamydiae through endosymbiosis gene transfer(EGT).Both these transfer events collectively contributed to the establishment of the original starch biosynthetic pathway.Subsequently,after the divergence of Viridiplantae from Rhodophyta,all three enzymes underwent multiple duplications and N-terminus extension domain modifications,resulting in the formation of functionally specialized isoforms and ultimately leading to the complete starch biosynthetic pathway.By shedding light on the evolutionary origins of key enzymes involved in the starch biosynthetic pathway,this study provides important insights into the evolutionary events of plants.

关键词： Origin Evolution Starch biosynthesis Starch synthase Starch branching enzyme Isoamylase-type debranching enzyme

Modification of the substrate specificity of leucine dehydrogenase by site‑directed mutagenesis based on biocomputing strategies

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Systems Microbiology and Biomanufacturing 2023年第2期3卷 384-392页

作者： Jiapeng Lu Zengyu Wang Yingying Jiang Zhoutong Sun Wei Luo The Key Laboratory of Carbohydrate Chemistry and biotechnology Ministry of Education School of BiotechnologyJiangnan UniversityWuxi 214000Jiangsu ProvinceChina Tianjin Institute of Industrial Biotechnology Chinese Academy of SciencesTianjin 300308China

Synthesis of chiral amines by amine dehydrogenase has the advantages of environmental friendliness,high stereoselectivity,and mild reaction conditions.However,amine dehydrogenase has low catalytic activity,which greatly limits its application in the large-scale synthesis of chiral amines.In this study,a novel amine dehydrogenase was obtained by modifying the substrate specificity of leucine dehydrogenase via computer-aided protein engineering strategy.Furthermore,conservation analysis,homology modeling and molecular docking analysis were carried out via biocomputing strategy to select the mutation sites,and the mutants L52S and T143C were obtained.The enzyme activities of the two mutants to 2-pentanone were 1.55 U/mg and 2.06 U/mg,respectively.The enzyme activity of the latter was 188%and the Tm value was 2.55°Chigher than those of the original mutant,which laid a foundation for the efficient preparation of chiral amines by using this novel enzyme.

关键词： Amine dehydrogenase L-leucine dehydrogenase Semi-rational design Protein engineering Substrate binding pocket

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 lignocellulose.Simultaneous consumption of glucose and xylose is critical for engineered microorganisms to produce fuels and chemicals from lignocellulosic biomass.Although many production limitations have been resolved,glucose‐induced inhibition of xylose transport remains a challenge.In this study,a cell growthbased screening strategy was designed to identify xylose transporters uninhibited by glucose.The 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 survival.Through 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 identified.Inactivation of adenylate cyclase led to increased expression of the EcolC_1642 gene,and a point mutation in gene EcolC_1642(N13S)further enhanced xylose transport.During 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 transport.These 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

Development of a 16S r RNA gene-based microarray for the detection of marine bacterioplankton community

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Acta Oceanologica Sinica 2017年第10期36卷 106-114页

作者： ZHAO Wei WANG Jingjing LIANG Yajie HUANG Zhiyong tianjin Key Laboratory of industrial Biological Systems and Process Engineering Tianjin Institute of Industrial Biotechnology Chinese Academy of Sciences

A better understanding of bacterioplankton community shifts following change in marine environments is critical to predict the marine ecosystem function. In order to get a snapshot of the microbial taxonomy profiling of a wide range marine area, a quick, convenient and low cost method would be favorable. In this study, we developed a 16S rRNA gene-based microarray using ARB software, which contained 447 probes targeting 160 families of marine bacteria. The specificity, sensitivity and quantitative capability of this microarray were assessed by single cloned16S rRNA genes. The reliability of this microarray was tested by eight environmental samples. The results showed that the microarray was specific, only 1.16% false results were detected in five single-clone hybridization tests. The microarray could detect DNA samples as few as 1 ng/μL and the signal intensity could reflect the relative abundance of the bacteria in the range of 1 ng/μL to 100 ng/μL of DNA concentration. Hybridization with environmental samples showed that it can discriminate bacterioplankton communities by sites and time. High throughput sequencing results from the eight samples confirmed the hybridization results. It indicated that this developed microarray could be used as a convenient tool to monitor the bacterioplankton community in marine environment.

关键词： microarray bacterioplankton community 16S rRNA gene marine environment

A new era for paclitaxel biosynthesis is coming

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Molecular Plant 2024年第3期17卷 370-371页

作者： Xiaonan Liu Xiaoxi Zhu Jian Cheng Huifeng Jiang Key Laboratory of Engineering Biology for Low-carbon Manufacturing Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjin 300308China National Center of Technology Innovation for Synthetic Biology Tianjin 300308China

Paclitaxel(Taxol)stands out as a tetracyclic diterpenoid natural product derived from the endangered plant Taxus.Recognized as a pivotal broad-spectrum anticancer drug,it has garnered widespread attention due to its low yield,intricate structure,unique anticancer mechanism,and remarkable efficacy(Tong et al.,2021).Although chemists achieved the total synthesis of paclitaxel 30 years ago,after decades of research,the natural biosynthetic pathway for its production remains an enigma(Ajikumar et al.,2010;Malci et al.,2023).A recent breakthrough,published in Molecular Plant by Alisdair R.Fernie's team,unveils a minimal gene set of 18 genes required for paclitaxel biosynthesis(Zhang et al.,2023),representing a significant mark toward unraveling the entire biosynthetic pathway and enhancing paclitaxel production efficiency.

关键词： breakthrough representing synthesis

An in vitro synthetic biology platform for emerging industrial biomanufacturing:Bottom-up pathway design

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Synthetic and Systems biotechnology 2018年第3期3卷 186-195页

作者： Ting Shi Pingping Han Chun You Yi-Heng P.Job Zhang Tianjin Institute of Industrial Biotechnology Chinese Academy of Sciences32 West 7th AvenueTianjin Airport Economic AreaTianjin300308China

Although most in vitro(cell-free)synthetic biology projects are usually used for the purposes of fundamental research or the formation of high-value products,in vitro synthetic biology platform,which can implement complicated biochemical reactions by the in vitro assembly of numerous enzymes and coenzymes,has been proposed for low-cost biomanufacturing of bioenergy,food,biochemicals,and nutraceuticals.In addition to the most important advantage-high product yield,in vitro synthetic biology platform features several other biomanufacturing advantages,such as fast reaction rate,easy product separation,open process control,broad reaction condition,tolerance to toxic substrates or products,and so on.In this article,we present the basic bottom-up design principles of in vitro synthetic pathway from basic building blocks-BioBricks(thermoenzymes and/or immobilized enzymes)to building modules(e.g.,enzyme complexes or multiple enzymes as a module)with specific functions.With development in thermostable building blocks-BioBricks and modules,the in vitro synthetic biology platform would open a new biomanufacturing age for the cost-competitive production of biocommodities.

关键词： Biomanufacturing Coenzyme regeneration Pathway design In vitro synthetic biology

Sequence motifs and prediction model of GBE editing outcomes based on target library analysis and machine learning

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Journal of Genetics and Genomics 2022年第3期49卷 254-257页

作者： Bo Li Ya-Qiu Li Dongdong Zhao Jie Yang Yan-He Ma Chang-Hao Bi Xue-Li Zhang Tianjin Institute of Industrial Biotechnology Chinese Academy of SciencesTianjin 300308China University of Chinese Academy of Sciences Beijing 100049China Key Laboratory of Systems Microbial biotechnology Chinese Academy of SciencesTianjin 300308China National Technology Innovation Center of Synthetic Biology Tianjin 300308China

Base editor techniques were developed for precise base conversion without requiring double-stranded DNA breaks(DSBs) or an editing template(Komor et al., 2016;Nishida et al., 2016).

关键词： prediction editor