Heparin,a glycosaminoglycan,is a stable source of carbon that supports the growth of microorganisms in the human intestine.It is also a commonly used anticoagulant drug in clinical practice,with significant therapeuti...Heparin,a glycosaminoglycan,is a stable source of carbon that supports the growth of microorganisms in the human intestine.It is also a commonly used anticoagulant drug in clinical practice,with significant therapeutic effects.Low molecular weight heparin(LMWH)is a highly active low molecular weight fragment obtained via enzymatic reaction or the chemical degradation of heparin.LMWH has been applied globally in the prevention and treatment of venous thromboembolism in thrombosis patients.Simultaneously,as a potential prebiotic,because of its low molecular weight,LMWH can be well degraded by the gut microbiota to maintain intestinal balance.Enzymatic heparin degradation has recently emerged as a viable disposal method for LMWH preparation;however,only very few benchmark enzymes have been thoroughly described and subjected to protein engineering to improve their properties over the past few years.The commercialization of enzymes will require the development of robustly engineered enzymes that meet the demands of industrial processes.Herein,we report a rational protein engineering strategy that includes molecular dynamic simulations of flexible amino acid mutations and disulfide bond screening.Several Bacteroides thetaiotaomicron heparanase I(Bt-HepI)mutants were obtained and screened for high thermal stability.We obtained the Bt-HepI^(D204C/K208C/H189W/Q198R)variant,which features a stabilized protein surface structure,with a 1.3-fold increase in catalytic constant/michaelis-menten constant(k_(cat)/K_(m)),a 2.44-fold increase in thermal stability at 50℃,and a 1.8-fold decrease in the average molecular weight of LMWH produced at 40℃compared with that seen with Bt-HepI^(WT).Our study establishes a strategy to engineer thermostable HepI to underpin its industrial applications.展开更多
A 61-kb biosynthetic gene cluster(BGC),which is accountable for the biosynthesis of hibarimicin(HBM)B from Microbispora rosea subsp.hibaria TP-A0121,was heterologously expressed in Streptomyces coelicolor M1154,which ...A 61-kb biosynthetic gene cluster(BGC),which is accountable for the biosynthesis of hibarimicin(HBM)B from Microbispora rosea subsp.hibaria TP-A0121,was heterologously expressed in Streptomyces coelicolor M1154,which generated a trace of the target products but accumulated a large amount of shunt products.Based on rational analysis of the relevant secondary metabolism,directed engineering of the biosynthetic pathways resulted in the high production of HBM B,as well as new HBM derivates with improved antitumor activity.These results not only establish a biosynthetic system to effectively synthesize HBMs-a class of the largest and most complex Type-Ⅱpolyketides,with a unique pseudo-dimeric structure-but also set the stage for further engineering and deep investigation of this complex biosynthetic pathway toward potent anticancer drugs.展开更多
Cas12h1 is a compact CRISPR-associated nuclease from functionally diverse type V CRISPR-Cas effectors and recognizes a purine-rich protospacer adjacent motif(PAM)distinct from that of other type V Cas effectors.Here,w...Cas12h1 is a compact CRISPR-associated nuclease from functionally diverse type V CRISPR-Cas effectors and recognizes a purine-rich protospacer adjacent motif(PAM)distinct from that of other type V Cas effectors.Here,we report the nickase preference of Cas12h1,which predominantly cleaves the nontarget strand(NTS)of a double-stranded DNA(dsDNA)substrate.In addition,Cas12h1 acts as a nickase in human cells.We further determined the cryo-EM structures of Cas12h1 in the surveillance,R-loop formation,and interference states,revealing the molecular mechanisms involved in the crRNA maturation,target recognition,R-loop formation,nuclease activation and target degradation.Cas12h1 notably recognizes a broad 5’-DHR-3’PAM(D is A,G,or T;H is A,C,or T;R is A or G)both in vitro and in human cells.In addition,Cas12h1 utilizes a distinct activation mechanism that the lid motif undergoes a“flexible to stable”transition to expose the catalytic site to the substrate.A high-fidelity nucleic acid detector,Cas12h1hf,was developed through rational engineering,which distinguishes single-base mismatches and retains comparable on-target activities.Our results shed light on the molecular mechanisms underlying Cas12h1 nickase,improve the understanding of type V Cas effectors,and expand the CRISPR toolbox for genome editing and molecular diagnosis.展开更多
Ectoine is a high-value stabilizer and protective agent with various applications in enzyme industry,cosmetics,and biomedi-cine.In this study,rational engineering strategies have been implemented in Escherichia coli t...Ectoine is a high-value stabilizer and protective agent with various applications in enzyme industry,cosmetics,and biomedi-cine.In this study,rational engineering strategies have been implemented in Escherichia coli to efficiently produce ectoine.First,the synthetic pathway of ectoine was constructed in E.coli MG1655 by introducing an artificial thermal switch system harboring the ectABC cluster from Halomonas elongate,and the resulting strain produced 1.95 g/L ectoine.Second,crr encoding the glucose-specific enzyme II domain A of phosphotransferase system and iclR encoding the glyoxylate shunt transcriptional repressor were deleted in E.coli for enhancing the oxaloacetate supply,leading to the increasement of the ectoine titer to 9.09 g/L.Third,thrA encoding the bifunctional aspartokinase/homoserine dehydrogenase was removed from the genome to weaken the competitive pathway;simultaneously,an endogenous feedback-resistant lysC was overexpressed to complement the enzymatic activity deficiency of the aspartate kinase,leading to 30.36%increase of ectoine titer.Next,the expression of phosphoenolpyruvate carboxylase was modulated with varying gradient strength promoters to accelerate the biosynthesis efficiency of ectoine.Finally,aspDH encoding aspartate dehydrogenase from Pseudomonas aeruginosa PAO1 was overexpressed to further improve the biosynthesis of ectoine.The final strain MWZ003/pFT28-ectABC-EclysC^(*)-aspDH-ppc3 produced 30.37 g/L ectoine after 36-h fed-batch fermentation with a yield of 0.132 g/g glucose and a productivity of 0.844 g/(L h).展开更多
High fructose corn syrup has been industrially produced by converting glucose to fructose by glucose isomerases,tetrameric metalloenzymes widely used in industrial biocatalysis.Advances in enzyme engineering and comme...High fructose corn syrup has been industrially produced by converting glucose to fructose by glucose isomerases,tetrameric metalloenzymes widely used in industrial biocatalysis.Advances in enzyme engineering and commercial production of glucose isomerase have paved the way to explore more efficient variants of these enzymes.The 5-hydroxymethylfurfural can be produced from high fructose corn syrup catalytic dehydration,and it can be further converted into various furanic compounds chemically or biologically for various industrial applications as a promising platform chemical.Although the chemical conversion of 5-hydroxymethylfurfural into furanic compounds has been extensively investigated in recent years,bioconversion has shown promise for its mild conditions due to the harsh chemical reaction conditions.This review discusses pro-tein engineering potential for improving glucose isomerase production and recent advancements in bioconversion of 5-hydroxymethylfurfural into value-added furanic derivatives.It suggests bi-ological strategies for the industrial transformation of 5-hydroxymethylfurfural.展开更多
基金supported by the Natural Science Foundation of Jiangsu Province(BE2021623,BK20220155)Natural Science Foundation of Jiangsu Province(BE2021623)+4 种基金National Natural Science Foundation of China(32001665,U1903205,32021005)the National Key Research and Development Program of China(2017YF0400303)the Key Scientific and Technological Research Projects in the Key Areas of the Xinjiang Production and Construction Corps(2018AB010)the Key Research and Development 303 Program of Ningxia(2020BFG02012)Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province。
文摘Heparin,a glycosaminoglycan,is a stable source of carbon that supports the growth of microorganisms in the human intestine.It is also a commonly used anticoagulant drug in clinical practice,with significant therapeutic effects.Low molecular weight heparin(LMWH)is a highly active low molecular weight fragment obtained via enzymatic reaction or the chemical degradation of heparin.LMWH has been applied globally in the prevention and treatment of venous thromboembolism in thrombosis patients.Simultaneously,as a potential prebiotic,because of its low molecular weight,LMWH can be well degraded by the gut microbiota to maintain intestinal balance.Enzymatic heparin degradation has recently emerged as a viable disposal method for LMWH preparation;however,only very few benchmark enzymes have been thoroughly described and subjected to protein engineering to improve their properties over the past few years.The commercialization of enzymes will require the development of robustly engineered enzymes that meet the demands of industrial processes.Herein,we report a rational protein engineering strategy that includes molecular dynamic simulations of flexible amino acid mutations and disulfide bond screening.Several Bacteroides thetaiotaomicron heparanase I(Bt-HepI)mutants were obtained and screened for high thermal stability.We obtained the Bt-HepI^(D204C/K208C/H189W/Q198R)variant,which features a stabilized protein surface structure,with a 1.3-fold increase in catalytic constant/michaelis-menten constant(k_(cat)/K_(m)),a 2.44-fold increase in thermal stability at 50℃,and a 1.8-fold decrease in the average molecular weight of LMWH produced at 40℃compared with that seen with Bt-HepI^(WT).Our study establishes a strategy to engineer thermostable HepI to underpin its industrial applications.
基金supported in part by grants from the National Key Research and Development Program of China(2018YFA0901900)the National Natural Science Foundation of China(22137009)the China Postdoctoral Science Foundation(2020M671271).
文摘A 61-kb biosynthetic gene cluster(BGC),which is accountable for the biosynthesis of hibarimicin(HBM)B from Microbispora rosea subsp.hibaria TP-A0121,was heterologously expressed in Streptomyces coelicolor M1154,which generated a trace of the target products but accumulated a large amount of shunt products.Based on rational analysis of the relevant secondary metabolism,directed engineering of the biosynthetic pathways resulted in the high production of HBM B,as well as new HBM derivates with improved antitumor activity.These results not only establish a biosynthetic system to effectively synthesize HBMs-a class of the largest and most complex Type-Ⅱpolyketides,with a unique pseudo-dimeric structure-but also set the stage for further engineering and deep investigation of this complex biosynthetic pathway toward potent anticancer drugs.
基金supported by the National Key Research and Development Program of China(2021YFC2301403)National Natural Science Foundation of China grants(82225028 and 82172287)Natural Science Foundation of Fujian Province of China(2022J01638).
文摘Cas12h1 is a compact CRISPR-associated nuclease from functionally diverse type V CRISPR-Cas effectors and recognizes a purine-rich protospacer adjacent motif(PAM)distinct from that of other type V Cas effectors.Here,we report the nickase preference of Cas12h1,which predominantly cleaves the nontarget strand(NTS)of a double-stranded DNA(dsDNA)substrate.In addition,Cas12h1 acts as a nickase in human cells.We further determined the cryo-EM structures of Cas12h1 in the surveillance,R-loop formation,and interference states,revealing the molecular mechanisms involved in the crRNA maturation,target recognition,R-loop formation,nuclease activation and target degradation.Cas12h1 notably recognizes a broad 5’-DHR-3’PAM(D is A,G,or T;H is A,C,or T;R is A or G)both in vitro and in human cells.In addition,Cas12h1 utilizes a distinct activation mechanism that the lid motif undergoes a“flexible to stable”transition to expose the catalytic site to the substrate.A high-fidelity nucleic acid detector,Cas12h1hf,was developed through rational engineering,which distinguishes single-base mismatches and retains comparable on-target activities.Our results shed light on the molecular mechanisms underlying Cas12h1 nickase,improve the understanding of type V Cas effectors,and expand the CRISPR toolbox for genome editing and molecular diagnosis.
基金This study was supported by the National Key R&D Program of China(2018YFA0900300)the National First-class Discipline Program of Light Industry Technology and Engineering(LITE2018-10)+1 种基金the Key Technology Project of Inner Mongolia Autonomous Region in China(2019GG302)the Collaborative Innovation Center of Jiangsu Modern Industrial Fermentation.
文摘Ectoine is a high-value stabilizer and protective agent with various applications in enzyme industry,cosmetics,and biomedi-cine.In this study,rational engineering strategies have been implemented in Escherichia coli to efficiently produce ectoine.First,the synthetic pathway of ectoine was constructed in E.coli MG1655 by introducing an artificial thermal switch system harboring the ectABC cluster from Halomonas elongate,and the resulting strain produced 1.95 g/L ectoine.Second,crr encoding the glucose-specific enzyme II domain A of phosphotransferase system and iclR encoding the glyoxylate shunt transcriptional repressor were deleted in E.coli for enhancing the oxaloacetate supply,leading to the increasement of the ectoine titer to 9.09 g/L.Third,thrA encoding the bifunctional aspartokinase/homoserine dehydrogenase was removed from the genome to weaken the competitive pathway;simultaneously,an endogenous feedback-resistant lysC was overexpressed to complement the enzymatic activity deficiency of the aspartate kinase,leading to 30.36%increase of ectoine titer.Next,the expression of phosphoenolpyruvate carboxylase was modulated with varying gradient strength promoters to accelerate the biosynthesis efficiency of ectoine.Finally,aspDH encoding aspartate dehydrogenase from Pseudomonas aeruginosa PAO1 was overexpressed to further improve the biosynthesis of ectoine.The final strain MWZ003/pFT28-ectABC-EclysC^(*)-aspDH-ppc3 produced 30.37 g/L ectoine after 36-h fed-batch fermentation with a yield of 0.132 g/g glucose and a productivity of 0.844 g/(L h).
基金supported by the Natural Sciences and Engineering Research Council of Canada(Grant number RGPIN-2017-05366)to WQ.
文摘High fructose corn syrup has been industrially produced by converting glucose to fructose by glucose isomerases,tetrameric metalloenzymes widely used in industrial biocatalysis.Advances in enzyme engineering and commercial production of glucose isomerase have paved the way to explore more efficient variants of these enzymes.The 5-hydroxymethylfurfural can be produced from high fructose corn syrup catalytic dehydration,and it can be further converted into various furanic compounds chemically or biologically for various industrial applications as a promising platform chemical.Although the chemical conversion of 5-hydroxymethylfurfural into furanic compounds has been extensively investigated in recent years,bioconversion has shown promise for its mild conditions due to the harsh chemical reaction conditions.This review discusses pro-tein engineering potential for improving glucose isomerase production and recent advancements in bioconversion of 5-hydroxymethylfurfural into value-added furanic derivatives.It suggests bi-ological strategies for the industrial transformation of 5-hydroxymethylfurfural.
