Bacillus licheniformis is an industrially significant microorganism known for its broad carbon source utilization compared to other bacteria.However,the mechanisms underlying this utilization are tightly controlled by...Bacillus licheniformis is an industrially significant microorganism known for its broad carbon source utilization compared to other bacteria.However,the mechanisms underlying this utilization are tightly controlled by global regulatory proteins such as CodY,and the details of these mechanisms remain elusive.This poses challenges for metabolic engineering efforts.In this study,we used the urease encoding gene as a reporter to establish a CRISPRi system based on conditional dCas9 expression in B.licheniformis.The induction with mannose resulted in an 84%transcriptional inhibition of ureA,and a 57%reduction in urease activity,confirming the system's successful construction.We designed three different sgRNA sites within the 5'-end coding region of the codY gene to achieve varying degrees of protein expression knockdown.The results showed that a 10-75%knockdown of codY led to a 23-87%decrease in the maximum specific uptake rates of glucose and maltose.Concurrently,the accumulation of carbon overflow metabolites such as 2,3-butanediol(2,3-BDO)and acetate decreased by 38%and 26%,respectively.These findings enhance our understanding of CodY’s regulatory role in catabolism and metabo-lism.The CRISPRi system with conditional dCas9 expression developed here serves as an effective synthetic biology tool for metabolic pathway engineering.展开更多
In Lactococcus lactis, the global transcriptional regulatory factor CodY can interact with the promoter DNA to regulate the growth, metabolism, environmental adaptation and other biological activities of the strains. ...In Lactococcus lactis, the global transcriptional regulatory factor CodY can interact with the promoter DNA to regulate the growth, metabolism, environmental adaptation and other biological activities of the strains. In order to study the mechanism of interaction between CodY and its target DNA, molecular docking and molecular dynamics simulations were used to explore the binding process at molecular level. Through the calculations of the free energy of binding, hydrogen bonding and energy decomposition, nine key residues of CodY were identified, corresponding to SERI84, SERI 86, SER20& THR217, ARG21 & SER219, ASN223, LYS242 and GLY243, among which SERI86, ARG218 and LYS242 play a vital role in DNA binding. Our research results provide important theoretical guidance for using wet-lab methods to study and optimize the metabolic network regulated by CodY.展开更多
To thrive in nature,bacteria have to rapidly proliferate in favorable conditions while constantly adapt to the fluctuating nutrient environments.However,the molecular players that ensure rapid growth of bacteria in fa...To thrive in nature,bacteria have to rapidly proliferate in favorable conditions while constantly adapt to the fluctuating nutrient environments.However,the molecular players that ensure rapid growth of bacteria in favorable conditions remain poorly understood.Here,we focus on the growth physiology of Bacillus subtilis and find that codY knockout strongly compromises cell growth in rich medium.Global proteome allocation analysis has shown that codY knockout causes a"waste"of cellular resources by stimulating unnecessary expression of many proteins,further reducing the cellular investment on translation machinery.Therefore,CodY-dependent repression is crucial in ensuring rapid growth of B.subtilis in rich medium.On the other hand,the relief of CodY-dependent repression could promote the bacterial adaption during transition from rich medium to minimal medium by shifting resource allocation from ribosome synthesis to amino acid biosynthesis.In addition,the relief of CodY-dependent repression in minimal medium also stimulates pathways of alternative functions such as motility and biosynthesis of secondary metabolites.Our study has thus revealed the pivotal role of CodY in bacterial growth control via governing the condition-dependent resource allocation of B.subtilis,further shedding light on the fundamental molecular strategy of bacteria to achieve fitness maximization.展开更多
基金funded by the National Key Research&Development Program of China(2020YFA0907700,2018YFA0900504 and 2018YFA0900300)the National Natural Foundation of China(32172174,31401674)+1 种基金the National First-Class Discipline Program of Light Industry Technology and Engineering(LITE2018-22)the Top-notch Academic Programs Project of Jiangsu Higher Education Institutions.
文摘Bacillus licheniformis is an industrially significant microorganism known for its broad carbon source utilization compared to other bacteria.However,the mechanisms underlying this utilization are tightly controlled by global regulatory proteins such as CodY,and the details of these mechanisms remain elusive.This poses challenges for metabolic engineering efforts.In this study,we used the urease encoding gene as a reporter to establish a CRISPRi system based on conditional dCas9 expression in B.licheniformis.The induction with mannose resulted in an 84%transcriptional inhibition of ureA,and a 57%reduction in urease activity,confirming the system's successful construction.We designed three different sgRNA sites within the 5'-end coding region of the codY gene to achieve varying degrees of protein expression knockdown.The results showed that a 10-75%knockdown of codY led to a 23-87%decrease in the maximum specific uptake rates of glucose and maltose.Concurrently,the accumulation of carbon overflow metabolites such as 2,3-butanediol(2,3-BDO)and acetate decreased by 38%and 26%,respectively.These findings enhance our understanding of CodY’s regulatory role in catabolism and metabo-lism.The CRISPRi system with conditional dCas9 expression developed here serves as an effective synthetic biology tool for metabolic pathway engineering.
基金the National Natural Science Foundation of China (Grant No. 31570049).
文摘In Lactococcus lactis, the global transcriptional regulatory factor CodY can interact with the promoter DNA to regulate the growth, metabolism, environmental adaptation and other biological activities of the strains. In order to study the mechanism of interaction between CodY and its target DNA, molecular docking and molecular dynamics simulations were used to explore the binding process at molecular level. Through the calculations of the free energy of binding, hydrogen bonding and energy decomposition, nine key residues of CodY were identified, corresponding to SERI84, SERI 86, SER20& THR217, ARG21 & SER219, ASN223, LYS242 and GLY243, among which SERI86, ARG218 and LYS242 play a vital role in DNA binding. Our research results provide important theoretical guidance for using wet-lab methods to study and optimize the metabolic network regulated by CodY.
基金supported by the National Natural Science Foundation of China(32270034,32370044,and 32470038)the National Key Research and Development Program of China(2022YFF1000400)+3 种基金Changjiang Young Scholar Program of Chinese Ministry of Education,Natural Science Funds for Distinguished Young Scholar of Hubei Province(2022CFA044)Hubei Major Special Project for Agricultural Microbiology Industry(NYWSWZX2025-2027-01)Funding of Innovation Joint Laboratory of Bio-Health Industry in Jiangxia District,Wuhan Science and Technology Major Project(2023020302020708)the Fundamental Research Funds for the Central Universities.
文摘To thrive in nature,bacteria have to rapidly proliferate in favorable conditions while constantly adapt to the fluctuating nutrient environments.However,the molecular players that ensure rapid growth of bacteria in favorable conditions remain poorly understood.Here,we focus on the growth physiology of Bacillus subtilis and find that codY knockout strongly compromises cell growth in rich medium.Global proteome allocation analysis has shown that codY knockout causes a"waste"of cellular resources by stimulating unnecessary expression of many proteins,further reducing the cellular investment on translation machinery.Therefore,CodY-dependent repression is crucial in ensuring rapid growth of B.subtilis in rich medium.On the other hand,the relief of CodY-dependent repression could promote the bacterial adaption during transition from rich medium to minimal medium by shifting resource allocation from ribosome synthesis to amino acid biosynthesis.In addition,the relief of CodY-dependent repression in minimal medium also stimulates pathways of alternative functions such as motility and biosynthesis of secondary metabolites.Our study has thus revealed the pivotal role of CodY in bacterial growth control via governing the condition-dependent resource allocation of B.subtilis,further shedding light on the fundamental molecular strategy of bacteria to achieve fitness maximization.
