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 amber mutation sites of 6 purR(am) mutants were determined by cloning and DMA sequencing. The results showed that the mutations were distributed at three different sites in PurR coding region, G721(→A), C933(→T)...The amber mutation sites of 6 purR(am) mutants were determined by cloning and DMA sequencing. The results showed that the mutations were distributed at three different sites in PurR coding region, G721(→A), C933(→T) and C1155(→T), which respectively turn Trp-147,Gln-218 and Gln-292 of PurR into TAG terminal codon. To determine the effect of the three amino acid residues on regulatory function of PurR protein 5 different kinds of tRNA suppressor genes, Su3, Su4, Su6, Su7 and Su9 were used for creating the PurR protein variants with single amino acid substitution. The results indicated that Cys, Glu, Gly, His and Arg which substituted Trp-147 respectively all could not recover the regulation function of PurR. It confirmed that Trp-147 is a critical amino acid for the PurR function. Gln-292 substituted respectively by the same amino acids also could not recover the PurR function, demonstrating that Gln-292 is also an important amino acid residue in PurR.展开更多
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the National Natural Science Foundation of China (Grant No. 39770011).
文摘The amber mutation sites of 6 purR(am) mutants were determined by cloning and DMA sequencing. The results showed that the mutations were distributed at three different sites in PurR coding region, G721(→A), C933(→T) and C1155(→T), which respectively turn Trp-147,Gln-218 and Gln-292 of PurR into TAG terminal codon. To determine the effect of the three amino acid residues on regulatory function of PurR protein 5 different kinds of tRNA suppressor genes, Su3, Su4, Su6, Su7 and Su9 were used for creating the PurR protein variants with single amino acid substitution. The results indicated that Cys, Glu, Gly, His and Arg which substituted Trp-147 respectively all could not recover the regulation function of PurR. It confirmed that Trp-147 is a critical amino acid for the PurR function. Gln-292 substituted respectively by the same amino acids also could not recover the PurR function, demonstrating that Gln-292 is also an important amino acid residue in PurR.
