Functional Evaluation And Molecular Design Of C1 Domain,A Key Enzyme For Acyl Modification In Lipopeptide Antibiotics Biosynthesis

Lipopeptide antibiotics are active substances synthesized by nonribosomal peptide synthetase(NRPS)with broad-spectrum antibacterial,antiviral,and antitumor functions.They are composed of peptide skeleton and fatty acid tails,having broad application prospects in biotechnology and pharmaceutical fields.The activity and toxicity of lipopeptide antibiotics are closely related to the nature of the N-terminal fatty acid chain.Therefore,it is of great significance to achieve specific fatty acid chain modification through rational design.For example,the important antibacterial drug daptomycin is a trace component of the multi-component mixture A21978 C.Its N-terminal is connected to n-decanoyl group and it has optimal activity.Breaking through the "bottleneck" of n-decanoyl loading through enzyme modification is expected to achieve high production and excellent yield of daptomycin.The acyl modification of lipopeptide antibiotics includes fatty acid activation and loading,in which the first condensation domain(C1 domain)of NRPS is responsible for fatty acyl loading.Therefore,the substrate selectivity of C1 determines the loading efficiency of different fatty acyl groups.Our work takes the C1 domain of lipopeptide antibiotics as the research object,and comprehensively applies a variety of techniques such as enzymology,protein engineering and computer simulation.We aim to explore the important area of the C1 domain,including substrate-binding pocket and the protein-protein interface.The important residues related to enzyme activity and substrate selectivity are determined,aiming to reveal the molecular mechanism of C1’s substrate selectivity at the molecular level and guide the rational design and modification of the C1 domain.First,we established multi-enzyme mixed reaction system of C1 in antibacterial lipopeptide A54145(lptC1)in vitro.Furthermore,we determined four key residues in the lptC1 substrate-binding pocket through molecular docking.A369 D and L397 Y lost their condensation activity,and the selectivity of A152 G and A386 S for fatty acid substrates was changed.Also,through protein-protein docking,we located five key residues at the protein-protein interface between lptC1 and its partners,acyl carrier protein and aminoacyl carrier protein.Single point mutations realized the change of substrate preference or catalytic activity.Based on the research of lptC1,we further rationally designed the C1 domain of daptomycin(dptC1)with a view to achieving an improvement in the catalytic efficiency of target n-decanoyl substrate.Our work provides a new perspective for the optimization of lipopeptide natural products biosynthesis,and provides new resources to enrich its diversity and increase the output of important components.It has important theoretical and practical significance.At the same time,it can provide theoretical guidance and technical support for the complex and efficient biosynthesis of other NRPS.And it can obtain more effective or new antibiotic drugs through the optimization of biosynthetic pathways.