碳分解代谢物阻遏(carbon catabolite repression,CCR)是指微生物在混合碳源发酵时优先利用速效碳源(通常为葡萄糖),且该碳源的代谢产物会抑制其他非速效碳源代谢相关的基因表达和蛋白活性,从而影响非速效碳源利用的现象。在低GC含量革...碳分解代谢物阻遏(carbon catabolite repression,CCR)是指微生物在混合碳源发酵时优先利用速效碳源(通常为葡萄糖),且该碳源的代谢产物会抑制其他非速效碳源代谢相关的基因表达和蛋白活性,从而影响非速效碳源利用的现象。在低GC含量革兰氏阳性菌中,CCR效应的关键调控因子为分解代谢物控制蛋白CcpA(catabolite control protein A)。该调控蛋白具有多效性功能,除参与CCR外,还与中心碳、氮代谢的调控、生物被膜的形成和毒性基因的表达等多种生理过程相关。综述了近年来有关CcpA蛋白的功能、作用机制及分子结构的研究进展。展开更多
The nitrogen source requirements for riboflavin production by ccpA mutant Bacillus subtilis 24A1/pMX45 were optimized using linear regression. The optimal medium components considered included 8% glucose as carbon sou...The nitrogen source requirements for riboflavin production by ccpA mutant Bacillus subtilis 24A1/pMX45 were optimized using linear regression. The optimal medium components considered included 8% glucose as carbon source, 2% yeast powder, 0.05% MgSO4 ·7H2O, and four types of nitrogen sources ： 0.1% yeast extract, 2% soybean powder, 1% corn plasm, and 0.2% （ NH4 ） 2 HPO4 in shake flask tests. Predictive ellipsoid was applied to determining the response values under the optimal levels for riboflavin production and glucose consumption. The optimal concentrations of the four types of nitrogen sources can remedy ammonium assimilative defection of ccpA mutant. Under the optimal conditions, the riboflavin yield increases to more than 5.0 g/L and 8%, glucose can be consumed completely after 60 h.展开更多
For increased and various communication requirements of modem applications on embedded systems, general purpose protocol stacks and protocol models are not efficient because they are fixed to execute in the static mod...For increased and various communication requirements of modem applications on embedded systems, general purpose protocol stacks and protocol models are not efficient because they are fixed to execute in the static mode. We present the Component-Based Communication Protocol Architecture （CCPA） to make communication dynamic and configurable. It can develop, test and store the customized components for flexible reuse. The protocols are implemented by component assembly and support by configurable environments. This leads to smaller memory, more flexibility, more reconfiguration ability, better concurrency, and multiple data channel support.展开更多
Previous research has confirmed the involvement of the lac1 gene cluster in lactose metabolism in Bacillus licheniformis,which is able to co-utilize lactose and low concentrations of glucose or certain carbon sources....Previous research has confirmed the involvement of the lac1 gene cluster in lactose metabolism in Bacillus licheniformis,which is able to co-utilize lactose and low concentrations of glucose or certain carbon sources.However,the internal regulatory mechanisms underlying specific lactose utilization remain elusive.In this study,we conducted a comprehensive investigation to elucidate the modulation mechanism of the lac1 gene cluster.We discovered a 162 bp bidirectional promoter located between the lacDCAB and lacR genes,which encodes a GntR family transcriptional regulatory factor.A 38 bp perfectly palindromic sequence(BOX1)within this promoter is critical for inducing lac1 expression.LacR and CcpA were shown to bind specifically to BOX1,and their novel binding motifs were identified.In BL2ΔlacR,BL2ΔccpA,and BL2ΔlacRΔccpA mutant strains,we confirmed the differential inhibitory levels of LacR and CcpA on bidirectional promoters.Furthermore,it was demonstrated that TnrA also binds BOX1 and exerts a positive regulatory effect on the lac1 cluster.Finally,we proposed a novel model to unravel the complex molecular interactions controlling the expression of the lac1 gene.These insights provide a foundation for exploring rational design strategies to optimize lactose fermentation processes and improve the efficiency of industrial lactose utilization.展开更多
The eps gene cluster(20 genes,LC2W_2170-LC2W_2189)is responsible for exopolysaccharides(EPS)biosynthesis in Lacticaseibacillus casei LC2W,but its transcriptional regulatory mechanism remains unclear.Catabolite control...The eps gene cluster(20 genes,LC2W_2170-LC2W_2189)is responsible for exopolysaccharides(EPS)biosynthesis in Lacticaseibacillus casei LC2W,but its transcriptional regulatory mechanism remains unclear.Catabolite control protein(CcpA)and pur operon repressor(PurR)were identified as pivotal regulators to modulate EPS biosynthesis through DNA affinity pull-down assay.Electrophoretic mobility shift assay(EMSA)revealed that CcpA and PurR both bound two eps promoters P2169-2170 and P2189-2190.Overexpression of ccpA significantly downregulated the expression of LC2W_2170,LC2W_2171,and LC2W_2172,resulting in an 18%decrease in the EPS titer to 122.27 mg/L.Conversely,purR knockout led to an obvious reduction in the transcription levels of LC2W_2187,LC2W_2188,and LC2W_2189,resulting in a decreased EPS titer to 123.77 mg/L.Combining EMSA and Regprecise prediction suggested two putative CcpA-binding sites,motif-C1(GTCAAATCGTTTTTTG)and motif-C2(TTGTTAACGATTTGCA),within the promoter P2169-2170,and the sole putative PurR-binding site,motif-P1(GCAATGCAACTTT),on promoter P2189-2190.To our knowledge,it is the first time to uncover the regulatory role of PurR in EPS biosynthesis.In summary,CcpA and PurR respectively function as transcriptional repressor and activator to regulate EPS biosynthesis,which expand the understanding of EPS regulatory mechanism in L.casei.展开更多
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
原核表达猪链球菌2型分解代谢控制蛋白A(catabolite control protein A,ccpA)并进行纯化,通过免疫印迹反应初步鉴定重组蛋白的抗原性和特异性。采用PCR方法,扩增ccpA基因序列,克隆至原核表达载体pET-28a中,转入大肠杆菌BL21中进行诱导...原核表达猪链球菌2型分解代谢控制蛋白A(catabolite control protein A,ccpA)并进行纯化,通过免疫印迹反应初步鉴定重组蛋白的抗原性和特异性。采用PCR方法,扩增ccpA基因序列,克隆至原核表达载体pET-28a中,转入大肠杆菌BL21中进行诱导、表达和亲和层析纯化;经Western blotting初步评价ccpA的抗原性。重组原核表达质粒pET-28a-ccpA经双酶切及测序证明构建正确;表达的重组蛋白相对分子质量约38000,能被相关抗体所识别。因此,能成功在大肠杆菌中表达猪链球菌2型ccpA,为猪链球菌2型相关调节蛋白的免疫学研究奠定基础。展开更多
代谢调控蛋白A(catabolite control protein A,CcpA)是革兰阳性菌重要的多效调节因子,在磷酸烯醇丙酮酸依赖的磷酸转移酶系统辅助下,能与靶基因上的特异性DNA序列——代谢反应元件结合,促进或阻遏靶基因表达,从而调控细菌的生理过程和...代谢调控蛋白A(catabolite control protein A,CcpA)是革兰阳性菌重要的多效调节因子,在磷酸烯醇丙酮酸依赖的磷酸转移酶系统辅助下,能与靶基因上的特异性DNA序列——代谢反应元件结合,促进或阻遏靶基因表达,从而调控细菌的生理过程和毒力。CcpA通过介导微生物碳分解代谢物阻遏效应,控制细菌摄取与利用外界环境中营养物质,保障其高效利用能源。CcpA通过直接或间接调控细菌的代谢过程和毒力基因表达,将细菌的代谢与毒力偶联。该文介绍了CcpA的作用机制及其对细菌多种代谢途径和致病性调控的研究进展。展开更多
文摘碳分解代谢物阻遏(carbon catabolite repression,CCR)是指微生物在混合碳源发酵时优先利用速效碳源(通常为葡萄糖),且该碳源的代谢产物会抑制其他非速效碳源代谢相关的基因表达和蛋白活性,从而影响非速效碳源利用的现象。在低GC含量革兰氏阳性菌中,CCR效应的关键调控因子为分解代谢物控制蛋白CcpA(catabolite control protein A)。该调控蛋白具有多效性功能,除参与CCR外,还与中心碳、氮代谢的调控、生物被膜的形成和毒性基因的表达等多种生理过程相关。综述了近年来有关CcpA蛋白的功能、作用机制及分子结构的研究进展。
文摘The nitrogen source requirements for riboflavin production by ccpA mutant Bacillus subtilis 24A1/pMX45 were optimized using linear regression. The optimal medium components considered included 8% glucose as carbon source, 2% yeast powder, 0.05% MgSO4 ·7H2O, and four types of nitrogen sources ： 0.1% yeast extract, 2% soybean powder, 1% corn plasm, and 0.2% （ NH4 ） 2 HPO4 in shake flask tests. Predictive ellipsoid was applied to determining the response values under the optimal levels for riboflavin production and glucose consumption. The optimal concentrations of the four types of nitrogen sources can remedy ammonium assimilative defection of ccpA mutant. Under the optimal conditions, the riboflavin yield increases to more than 5.0 g/L and 8%, glucose can be consumed completely after 60 h.
基金Project (No. 2002AA1Z2306) supported by the Hi-Tech Researchand Development Program (863) of China
文摘For increased and various communication requirements of modem applications on embedded systems, general purpose protocol stacks and protocol models are not efficient because they are fixed to execute in the static mode. We present the Component-Based Communication Protocol Architecture （CCPA） to make communication dynamic and configurable. It can develop, test and store the customized components for flexible reuse. The protocols are implemented by component assembly and support by configurable environments. This leads to smaller memory, more flexibility, more reconfiguration ability, better concurrency, and multiple data channel support.
基金the National Key Research&Development Program of China(2020YFA0907700,2018YFA0900504,and 2018YFA0900300)the National Natural Science Foundation of China(32172174,31401674)the National First-Class Discipline Program of Light Industry Technology and Engineering(LITE2018-22).
文摘Previous research has confirmed the involvement of the lac1 gene cluster in lactose metabolism in Bacillus licheniformis,which is able to co-utilize lactose and low concentrations of glucose or certain carbon sources.However,the internal regulatory mechanisms underlying specific lactose utilization remain elusive.In this study,we conducted a comprehensive investigation to elucidate the modulation mechanism of the lac1 gene cluster.We discovered a 162 bp bidirectional promoter located between the lacDCAB and lacR genes,which encodes a GntR family transcriptional regulatory factor.A 38 bp perfectly palindromic sequence(BOX1)within this promoter is critical for inducing lac1 expression.LacR and CcpA were shown to bind specifically to BOX1,and their novel binding motifs were identified.In BL2ΔlacR,BL2ΔccpA,and BL2ΔlacRΔccpA mutant strains,we confirmed the differential inhibitory levels of LacR and CcpA on bidirectional promoters.Furthermore,it was demonstrated that TnrA also binds BOX1 and exerts a positive regulatory effect on the lac1 cluster.Finally,we proposed a novel model to unravel the complex molecular interactions controlling the expression of the lac1 gene.These insights provide a foundation for exploring rational design strategies to optimize lactose fermentation processes and improve the efficiency of industrial lactose utilization.
基金supported by National Science Fund for Distinguished Young Scholars(grant No.32025029)Key Special Project of Synthetic Biology in Shanghai“Science and Technology Innovation Action Plan”(grant No.23HC1400900)+3 种基金Shanghai Education Committee Scientific Research Innovation Project(grant no.2101070007800120)Academician/Expert Workstation in Yunnan,China(No.202205AF150065)Yunnan Provincial Key Laboratory of Applied Technology for Special Forest Fruits(202305AG070001)Shanghai Engineering Research Center of Food Microbiology(grant No.19DZ2281100).
文摘The eps gene cluster(20 genes,LC2W_2170-LC2W_2189)is responsible for exopolysaccharides(EPS)biosynthesis in Lacticaseibacillus casei LC2W,but its transcriptional regulatory mechanism remains unclear.Catabolite control protein(CcpA)and pur operon repressor(PurR)were identified as pivotal regulators to modulate EPS biosynthesis through DNA affinity pull-down assay.Electrophoretic mobility shift assay(EMSA)revealed that CcpA and PurR both bound two eps promoters P2169-2170 and P2189-2190.Overexpression of ccpA significantly downregulated the expression of LC2W_2170,LC2W_2171,and LC2W_2172,resulting in an 18%decrease in the EPS titer to 122.27 mg/L.Conversely,purR knockout led to an obvious reduction in the transcription levels of LC2W_2187,LC2W_2188,and LC2W_2189,resulting in a decreased EPS titer to 123.77 mg/L.Combining EMSA and Regprecise prediction suggested two putative CcpA-binding sites,motif-C1(GTCAAATCGTTTTTTG)and motif-C2(TTGTTAACGATTTGCA),within the promoter P2169-2170,and the sole putative PurR-binding site,motif-P1(GCAATGCAACTTT),on promoter P2189-2190.To our knowledge,it is the first time to uncover the regulatory role of PurR in EPS biosynthesis.In summary,CcpA and PurR respectively function as transcriptional repressor and activator to regulate EPS biosynthesis,which expand the understanding of EPS regulatory mechanism in L.casei.
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.
文摘原核表达猪链球菌2型分解代谢控制蛋白A(catabolite control protein A,ccpA)并进行纯化,通过免疫印迹反应初步鉴定重组蛋白的抗原性和特异性。采用PCR方法,扩增ccpA基因序列,克隆至原核表达载体pET-28a中,转入大肠杆菌BL21中进行诱导、表达和亲和层析纯化;经Western blotting初步评价ccpA的抗原性。重组原核表达质粒pET-28a-ccpA经双酶切及测序证明构建正确;表达的重组蛋白相对分子质量约38000,能被相关抗体所识别。因此,能成功在大肠杆菌中表达猪链球菌2型ccpA,为猪链球菌2型相关调节蛋白的免疫学研究奠定基础。
文摘代谢调控蛋白A(catabolite control protein A,CcpA)是革兰阳性菌重要的多效调节因子,在磷酸烯醇丙酮酸依赖的磷酸转移酶系统辅助下,能与靶基因上的特异性DNA序列——代谢反应元件结合,促进或阻遏靶基因表达,从而调控细菌的生理过程和毒力。CcpA通过介导微生物碳分解代谢物阻遏效应,控制细菌摄取与利用外界环境中营养物质,保障其高效利用能源。CcpA通过直接或间接调控细菌的代谢过程和毒力基因表达,将细菌的代谢与毒力偶联。该文介绍了CcpA的作用机制及其对细菌多种代谢途径和致病性调控的研究进展。
