摘要
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
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)
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).
