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Engineering of cofactor preference and catalytic activity of methanol dehydrogenase by growth-coupled directed evolution

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Green Carbon 2024年第2期2卷 242-251页

作者： Jinxing Yang Liwen Fan Guimin Cheng Tao Cai Jibin Sun Ping Zheng Shuang Li Yu Wang School of Biology and Biological Engineering South China University of TechnologyGuangzhou 510006China Haihe Laboratory of Synthetic Biology Tianjin 300308China Key Laboratory of Engineering Biology for Low-Carbon Manufacturing Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjin 300308China National Technology Innovation Center of Synthetic Biology Tianjin 300308China College of Biotechnology Tianjin University of Science and TechnologyTianjin 300222China

Methanol,produced from carbon dioxide,natural gas,and biomass,has drawn increasing attention as a promising green carbon feedstock for biomanufacturing due to its sustainable and energy-rich *** adenine dinucleotide(NAD^(+))-dependent methanol dehydrogenase(MDH)catalyzes the oxidation of methanol to formaldehyde via NADH generation,providing a highly active C1 intermediate and reducing power for subsequent ***,the unsatisfactory catalytic efficiency and cofactor bias of MDH significantly impede methanol valorization,especially in nicotinamide adenine dinucleotide phosphate(NADP^(+))-dependent ***,we employed synthetic NADH and NADPH auxotrophic Escherichia coli strains as growth-coupled selection platforms for the directed evolution of MDH from Bacillus stearothermophilus DSM *** or NADPH generated by MDH-catalyzed methanol oxidation enabled the growth of synthetic cofactor auxotrophs,establishing a positive correlation between the cell growth rate and MDH *** this principle,MDH mutants exhibiting a 20-fold improvement in catalytic efficiency(k_(cat)/K_(m))and a 90-fold cofactor specificity switch from NAD^(+)to NADP+without a decrease in specific enzyme activity,were efficiently screened from random and semi-rationally designed *** envision that these mutants will advance methanol valorization and that the synthetic cofactor auxotrophs will serve as versatile selection platforms for the evolution of NAD(P)^(+)-dependent enzymes.

关键词： Growth-coupled screening Methanol dehydrogenase Cofactor engineering directed evolution C1 bioconversion

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directed evolution of L-Aspartase by Mobility of Domains

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Chemical Research in Chinese Universities 2004年第1期20卷 50-54页

作者： GOUXiao-jun LIShuang KONGXiang-duo LIUWei SUNYan-hong ZHANGJin TheJointLaboratoryofMolecularevolutionandEngineering DepartmentofBioengineeringChengduUniversityChengdu610106P.R.China KeyLaboratoryforMolecularEnzymologyandEngineeringofMinistryofEducation JilinUniversityChangchun130023P.R.China

To enhance the relative movement of domains, we inserted a random sequence of fifteen-peptide into the three domains of L-aspartase. By means of directed screening, the three isoforms of monomeric, dimmeric and tetrameric enzymes were obtained. Compared to the wild-type tetrameric L-asparease, these mutants remained 19.7%, 42.3%, and 92% of the enzyme activity, respectively. Moreover, the examination of enzyme properties revealed that their k_ cat and K_M changed in varying degrees, and the optimum pH shifted towards acidic pH, while the dependence of the activity of enzyme on Mg 2+ concentration and thermostability increased. Therefore this strategy provides a novel approach to directed evolution of enzymes.

关键词： directed evolution Domain L-aspartase Mobility

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directed evolution of Human Cu,Zn-SOD Under Artificial Oxidant Pressures

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Chemical Research in Chinese Universities 2002年第3期18卷 294-298页

作者： GOU Xiao jun LI Chun lei KONG Xiang duo ZHU Shi zhen ZHANG Hong ying WANG Xiao ping ZHANG Jin LIU Tian yun Key Lab for Molecular Enzymology and Engineering of Ministry of Education Jilin UniversityChangchun 130023P.R.China The People's Hospital of Tongjiang Country Tongjiang 635700P.R.China

Cu,Zn SOD is a highly conserved enzyme and the controversy about its evolutionary possibility in the near future has been lively. In order to further our understanding of the future fate of human Cu,Zn SOD, we adopted a strategy relating to the directed evolution to study how the mutants of human Cu,Zn SOD respond to different oxidative stress. After five rounds of screening, we found a mutant that can survive under harsh pressures and DNA sequencing proves that it shows a mutation responsible for the phenomenon. However, under natural pressure, our screening comes to nothing. Then we may draw the following conclusions: the evolution of biological macromolecules in some respect depends on their surroundings and if they are too familiar with a certain environment, they may embody evolutionary inertia.

关键词： Human Cu Zn SOD directed evolution Screening

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directed evolution of the fusion enzyme for improving astaxanthin biosynthesis in Saccharomyces cerevisiae

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Synthetic and Systems Biotechnology 2023年第1期8卷 46-53页

作者： Yong-Wen Ding Chuan-Zhen Lu Yan Zheng Han-Zhang Ma Jin Jin Bin Jia Ying-Jin Yuan Frontier Science Center for Synthetic Biology(Ministry of Education) School of Chemical Engineering and TechnologyTianjin UniversityTianjin300072China

catalyzed byβ-carotene hydroxylase(crtZ)andβ-carotene ketolase(crtW)decreases the content of the ***,we exploited directed evolution of the fusion of crtZ and crtW for improving astaxanthin biosynthesis in Saccharomyces *** results demonstrated that the fusion enzyme of crtZ-crtW with 2 X GGGGS peptides linker can effectively reduce the accumulation of intermediates and improves the content of *** with the control strain,the fusion enzyme of ketase and hydroxylase reduced zeaxanthin and canthaxanthin by 7 and 14 times and increased astaxanthin by 1.6 times,***,9 variant fusion mutants with improved astaxanthin production were generated through directed *** these dominant mutants generated a variant,L95S+I206L,which increased the astaxanthin content of 3.8 times than the control *** AlphaFold2 assisted structural analysis indicated that these two mutations alter the interaction between the substrate and the enzymes *** research provided an efficient idea to reduce the accumulation of the intermediate products in complex biosynthesis pathway.

关键词： Astaxanthin directed evolution Fusion enzyme Saccharomyces cerevisiae Synthetic biology

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directed evolution of a neutrophilic and mesophilic methanol dehydrogenase based on high-throughput and accurate measurement of formaldehyde

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Synthetic and Systems Biotechnology 2023年第3期8卷 386-395页

作者： Jin Qian Liwen Fan Jinxing Yang Jinhui Feng Ning Gao Guimin Cheng Wei Pu Wenjuan Zhou Tao Cai Shuang Li Ping Zheng Jibin Sun Depei Wang Yu Wang College of Biotechnology Tianjin University of Science and TechnologyTianjin300222China Haihe Laboratory of Synthetic Biology Tianjin300308China Key Laboratory of Engineering Biology for Low-carbon Manufacturing Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjin300308China National Technology Innovation Center of Synthetic Biology Tianjin300308China University of Chinese Academy of Sciences Beijing100049China School of Biology and Biological Engineering South China University of TechnologyGuangzhou510006China

Methanol is a promising one-carbon feedstock for biomanufacturing,which can be sustainably produced from carbon dioxide and natural ***,the efficiency of methanol bioconversion is limited by the poor catalytic properties of nicotinamide adenine dinucleotide(NAD^(+))-dependent methanol dehydrogenase(Mdh)that oxidizes methanol to ***,the neutrophilic and mesophilic NAD^(+)-dependent Mdh from Bacillus stearothermophilus DSM 2334(Mdh_(Bs))was subjected to directed evolution for enhancing the catalytic *** combination of formaldehyde biosensor and Nash assay allowed high-throughput and accurate measurement of formaldehyde and facilitated efficient selection of desired ***_(Bs)variants with up to 6.5-fold higher K_(cat)/K_(M)value for methanol were screened from random mutation *** T153 residue that is spatially proximal to the substrate binding pocket has significant influence on enzyme *** beneficial T153P mutation changes the interaction network of this residue and breaks theα-helix important for substrate binding into two shortα-*** the interaction network of T153 with surrounding residues may represent a promising strategy to further improve Mdh_(Bs),and this study provides an efficient strategy for directed evolution of Mdh.

关键词： Methanol dehydrogenase Formaldehyde biosensor directed evolution C1 bioconversion Methanol oxidation Methylotrophy

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Advances in the study of directed evolution for cellulases

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Frontiers of Environmental Science & Engineering 2011年第4期5卷 519-525页

作者： Hailong LIN Weiguang LI Changhong GUO Sihang QU Nanqi REN State Key Laboratory of Urban Water Resource and Environment Harbin Institute of TechnologyHarbin 150090China College of Life Science and Technology Harbin Normal UniversityHarbin 150080China

If cellulose can be effectively hydrolyzed intoglucose by cellulase,the production costs of hydrogen,ethanol or other chemicals from cellulosic materials will begreatly decreased,and economically viable production ofbiohydrogen and bioethanol will become *** degraded into glucoses by multi-component *** cellulases are widely used in brewing,food,bioenergy,fodder,textiles,paper,pharmaceuticals,environmental protection and other ***,existing cellulases have several problems that limit theirwider applications,including the low turnover number forsolid cellulosic materials,and low stability in adapting tovarious application *** example,high temperature,low pH,and so *** of directedevolution technology may be one of the most effectiveways for improving the characteristics of *** presents a brief review of the cellulases hydrolysismechanism by cellulase,advances in cellulases(endoglucanaseandβ-glucosidase)improvement by directedevolution for several characteristics(for instance,thermalstability,pH adaptability and enzyme activity),limitationsof directed evolution for cellulases,and the outlook fordirected evolution for cellulase.

关键词： biohydrogen bioethanol cellulase cellulose directed evolution

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Enhancement of the thermostability of β-1,3-1,4-glucanase by directed evolution

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Journal of Zhejiang University-Science A(Applied Physics & Engineering) 2006年第11期7卷 1948-1955页

作者： ZHANG Xiu-yan RUAN Hui MU Lin HE Guo-qing TANG Xing-jun CHEN Qi-he Department of Food Science and Nutrition Zhejiang University Hangzhou 310029 China

In order to improve the thermostability of β- 1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the β-1,3-1,4-glucanase from Bacillus subtilis ZJF-1A5. The process involves random mutation by error-prone PCR and DNA shuffling followed by screening on the filter-based assay. Two mutants, EGsl and EGs2, were found to have four and five amino acid substitutions, respectively. These substitutions resulted in an increase in melting temperature from Tm=62.5℃ for the wild-type enzyme to Tm=65.5℃ for the mutant EGsl and 67.5℃ for the mutant EGs2. However, the two mutated enzymes had opposite approaches to produce reducing sugar from lichenin with either much higher （28%） for the former or much lower （21.6%） for the latter in comparison with their parental enzymes. The results demonstrate that directed evolution is an effective approach to improve the thermostability of a mesophilic enzyme.

关键词： directed evolution Error-prone PCR DNA shuffling β- 1 3-1 4-glucanase Thermostability

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Enhancing the thermostability of D-allulose 3-epimerase from Clostridium cellulolyticum H10 via directed evolution

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Systems Microbiology and Biomanufacturing 2022年第4期2卷 685-694页

作者： Shuhan Liu Yifan Wang Demin Kong Jing Wu Zhanzhi Liu State Key Laboratory of Food Science and Technology Jiangnan University1800 Lihu AvenueWuxi 214122Jiangsu ProvinceChina School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education Jiangnan University1800 Lihu AvenueWuxi 214122Jiangsu ProvinceChina International Joint Laboratory On Food Safety Jiangnan University1800 Lihu AvenueWuxi 214122Jiangsu ProvinceChina

d-allulose,the epimer at C-3 position of d-fructose,is a low-calorie functional rare sugar,which is regarded as one of the most potential *** present,the main production method of d-allulose is epimerization of d-fructose by d-allulose 3-epimerase(DAE).However,industrial applications of DAE are still limited by its poor ***,directed evolution was applied to improve the thermostability of dAE from Clostridium cellulolyticum H10(CcDAE).Two optimal mutants D281G and C289R,exhibiting 13.80-fold and 13.88-fold t_(1/2 )values as that of wild type at 65℃,respectively,were *** further enhance the thermostability,the triple mutant A107P/D281G/C289R was constructed after combina-tion of mutants D281G,C289R,and previously identified thermostability-enhanced mutant *** T_(m) and optimal temperature of triple mutant were increased by 14.39℃and 5℃,respectively,compared to the wild type,meanwhile,the half-life of triple mutant was 58.85-fold as that of wild type at 65℃.Furthermore,the conversion rate of triple mutant was increased from 24.76%of wild type to 27.53%using 300 g/L d-fructose as substrate at 70℃.The effectiveness of directed evolution was verified and the triple mutant with enhanced thermostability had great application value in the large-scale production of d-allulose.

关键词： D-allulose 3-epimerase directed evolution Ultra-high throughput screening Thermostability

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Enhancement of the thermostability of β-1,3-1,4-glucanase by directed evolution

Enhancement of the thermostability of β-1,3-1,4-glucanase b...

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中国食品科学技术学会第五届年会暨第四届东西方食品业高层论坛

作者： ZHANG Xiu-yan RUAN Hui MU Lin HE Guo-qing TANG Xing-jun CHEN Qi-he (Department of Food Science and Nutrition,Zhejiang University,Hangzhou 310029,China)

In order to improve the thermostability of β-1,3-1,4-glucanase,evolutionary molecular engineering was used to evolve the β-1,3-1,4-glucanase from Bacillus subtilis *** process involves random mutation by error-prone PCR and DNA shuffling followed by screening on the filter-based *** mutants,EGsl and EGs2,were found to have four and five amino acid substitutions,*** substitutions resulted in an increase in melting temperature from Tm=62.5℃ for the wild-type enzyme to Tm=65.5℃ for the mutant EGsl and 67.5℃ for the mutant ***,the two mutated enzymes had opposite approaches to produce reducing sugar from lichenin with either much higher(28%)for the former or much lower (21.6%)for the latter in comparison with their parental *** results demonstrate that directed evolution is an effective approach to improve the thermostability of a mesophilic enzyme.

关键词： directed evolution Error-prone PCR DNA shuffling β-1,3-1,4-glucanase Thermostability

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Biochemical characterization of a polyethylene terephthalate hydrolase and design of high-throughput screening for its directed evolution

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Engineering Microbiology 2022年第2期2卷 1-9页

作者： Xiaotong Wang Chaoyi Song Qingsheng Qi Youming Zhang Ruijuan Li Liujie Huo State Key Laboratory of Microbial Technology Shandong UniversityQingdao 266237China Helmholtz International Laboratory Shandong University-Helmholtz Institute of BiotechnologyQingdao 266237China

Polyethylene terephthalate(PET),one of the most widely used plastics in the world,causes serious environmental ***,researchers have focused their efforts on enzymatic degradation of PET,which is an attractive way of degrading and recycling *** this work,PET hydrolase Sb PETase from Schlegelella brevitalea *** biochemically characterized,and rational design was performed based on its sequence similarity with the previ-ously reported Is PETase from Ideonella sakaiensis,resulting in a triple mutant with increased ***,using a sec-dependent signal peptide PeIB and colicin release protein Kil,we set up a high-efficiency secretion system of PETase in Escherichia coli BL21(DE3),enabling higher PETase *** this secretion system,we established a high-throughput screening method named SecHTS(sec retion-based h igh-throughput s creening)and performed directed evolution of Is PETase and Sb PETase through DNA ***,we generated a mutant Is PETase S139T with increased activity from the mutant library.

关键词： PETase Protein secretion directed evolution DNA shuffling High-throughput screening

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