The gram-positive bacterium Bacillus licheniformis exhibits obvious selective utilization on carbon sources.This process is mainly governed by the global regulator catabolite control protein A(CcpA),which can recogniz...The gram-positive bacterium Bacillus licheniformis exhibits obvious selective utilization on carbon sources.This process is mainly governed by the global regulator catabolite control protein A(CcpA),which can recognize and bind to multiple target genes that are widely distributed in metabolic pathways.Although the DNA-binding domain of CcpA has been predicted,the infuence of key amino acids on target gene recognition and binding has yet to be uncovered.In this study,the impact of Lys31,Ile42 and Leu56 on in vitro protein–DNA interactions and in vivo carbon source selective utilization was investigated.The results showed that alanine substitution of Lys31 and Ile42,located within the 3rd helices of the DNAbinding domain,signifcantly weakened the binding strength between CcpA and target genes.These mutations also lead to alleviated repression of xylose utilization in the presence of glucose.On the other hand,the Leu56Arg mutant in the 4th helices exhibited enhanced binding afnity compared with that of the wild-type one.When this mutant was used to replace the native one in B.licheniformis cells,the selective utilization of glucose over xylose increased.This study provides a new strategy for understanding the relationship between the function and structure of regulatory proteins.This study also used a new strategy was used to regulate carbon source utilization beyond CCR engineering.展开更多
Presently,Saccharomyces cerevisiae demonstrates proficient co-fermentation of glucose and xylose,marking a significant advancement in second-generation fuel ethanol production.However,the presence of high concen-trati...Presently,Saccharomyces cerevisiae demonstrates proficient co-fermentation of glucose and xylose,marking a significant advancement in second-generation fuel ethanol production.However,the presence of high concen-trations of inhibitors in industrial lignocellulose hydrolysates and post-glucose effect caused by glucose con-sumption hinders severely impedes yeast robustness and xylose utilization for ethanol fermentation.Even worse,the antagonism between xylose utilization ability and strain robustness was observed,which proposes a difficult challenge in the production of second-generation fuel ethanol by S.cerevisiae.This review introduces the effect of engineering transcriptional regulatory networks on enhancing xylose utilization,improving strain robustness,alleviating antagonism between xylose utilization and strain robustness,and reducing post-glucose effect.Additionally,we provide an outlook on the developmental trends in this field,offering insights into future di-rections for increasing the production of second-generation fuel ethanol in S.cerevisiae.展开更多
Promoters are critical elements to control gene expression but could behave differently under various growth conditions. Here we report the construction of a genome-wide promoter library, in which each native promoter...Promoters are critical elements to control gene expression but could behave differently under various growth conditions. Here we report the construction of a genome-wide promoter library, in which each native promoter in Saccharomyces cerevisiae was cloned upstream of a yellow fluorescent protein （YFP） reporter gene. Nine libraries were arbitrarily defined and assembled in bacteria. The resulting pools of promoters could be prepared and transformed into a yeast strain either as centromeric plasmids or integrated into a genomic locus upon enzymatic treatment. Using fluorescence activated cell sorting, we classified the yeast strains based on YFP fluorescence intensity and arbitrarily divided the entire library into 12 bins, representing weak to strong promoters. Several strong promoters were identified from the most active bins and their activities were assayed under different growth conditions. Finally, these promoters were applied to drive the expression of genes in xylose utilization to improve fermentation efficiency. Together, this library could provide a quick solution to identify and utilize desired promoters under user-defined growth conditions.展开更多
基金This work was supported by National Key Research&Development Program of China(2018YFA0900504,2020YFA0907700 and 2018YFA0900300)the National Natural Foundation of China(31401674)+1 种基金the National First-Class Discipline Program of Light Industry Technology and Engineering(LITE2018-22)the Topnotch Academic Programs Project of Jiangsu Higher Education Institutions.
文摘The gram-positive bacterium Bacillus licheniformis exhibits obvious selective utilization on carbon sources.This process is mainly governed by the global regulator catabolite control protein A(CcpA),which can recognize and bind to multiple target genes that are widely distributed in metabolic pathways.Although the DNA-binding domain of CcpA has been predicted,the infuence of key amino acids on target gene recognition and binding has yet to be uncovered.In this study,the impact of Lys31,Ile42 and Leu56 on in vitro protein–DNA interactions and in vivo carbon source selective utilization was investigated.The results showed that alanine substitution of Lys31 and Ile42,located within the 3rd helices of the DNAbinding domain,signifcantly weakened the binding strength between CcpA and target genes.These mutations also lead to alleviated repression of xylose utilization in the presence of glucose.On the other hand,the Leu56Arg mutant in the 4th helices exhibited enhanced binding afnity compared with that of the wild-type one.When this mutant was used to replace the native one in B.licheniformis cells,the selective utilization of glucose over xylose increased.This study provides a new strategy for understanding the relationship between the function and structure of regulatory proteins.This study also used a new strategy was used to regulate carbon source utilization beyond CCR engineering.
基金supported by the National Key Research and Development Project of China(2018YFB1501401)the Key innovation Project of Qilu University of Technology(Shandong Academy of Sciences)(2024ZDZX03)+1 种基金the Shandong Provincial Technical Innovation Boot Program(02055183)the National Natural Science Foundation of China(31870063).
文摘Presently,Saccharomyces cerevisiae demonstrates proficient co-fermentation of glucose and xylose,marking a significant advancement in second-generation fuel ethanol production.However,the presence of high concen-trations of inhibitors in industrial lignocellulose hydrolysates and post-glucose effect caused by glucose con-sumption hinders severely impedes yeast robustness and xylose utilization for ethanol fermentation.Even worse,the antagonism between xylose utilization ability and strain robustness was observed,which proposes a difficult challenge in the production of second-generation fuel ethanol by S.cerevisiae.This review introduces the effect of engineering transcriptional regulatory networks on enhancing xylose utilization,improving strain robustness,alleviating antagonism between xylose utilization and strain robustness,and reducing post-glucose effect.Additionally,we provide an outlook on the developmental trends in this field,offering insights into future di-rections for increasing the production of second-generation fuel ethanol in S.cerevisiae.
基金We thank Jianhuo Fang at DNA sequencing facility in Tsinghua University for providing the sequencing service. This work was supported by the National Natural Science Foundation of China （Grant No. 31471254）, Chinese Ministry of Science and Technology grant 2012CB725201 and Tsinghua University Initiative grant 20161080088.
文摘Promoters are critical elements to control gene expression but could behave differently under various growth conditions. Here we report the construction of a genome-wide promoter library, in which each native promoter in Saccharomyces cerevisiae was cloned upstream of a yellow fluorescent protein （YFP） reporter gene. Nine libraries were arbitrarily defined and assembled in bacteria. The resulting pools of promoters could be prepared and transformed into a yeast strain either as centromeric plasmids or integrated into a genomic locus upon enzymatic treatment. Using fluorescence activated cell sorting, we classified the yeast strains based on YFP fluorescence intensity and arbitrarily divided the entire library into 12 bins, representing weak to strong promoters. Several strong promoters were identified from the most active bins and their activities were assayed under different growth conditions. Finally, these promoters were applied to drive the expression of genes in xylose utilization to improve fermentation efficiency. Together, this library could provide a quick solution to identify and utilize desired promoters under user-defined growth conditions.
