Genomics Analysis And Fermentation Process Research Of A82846B-Producing Strain Kibdelosporangium Aridum ZH04

As a naturally-derived glycopeptide antibiotic,A82846B has a structure similar to vancomycin,from which the second-generation glycopeptide antibiotic oritavancin can be produced by one-step chemical reaction.Oritavancin has been approved by the US Food and Drug Administration in 2014 for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive bacteria.With multiple antibiotic action mechanisms and better antibacterial activity against troublesome drug-resistant pathogens（such as vancomycin-resistant Enterococcus and vancomycin-resistant Staphylococcus aureus）,Oritavancin only needs to be administered in a single dose.As a key intermediate in the synthesis of oritavancin,A82846B is rather crucial.Two similar impurity components,A82846A and A82846C,will appear in the biosynthesis process,with a difference of only 0-2chlorine atoms in structure.The fermentation level of A82846B in the existing producing bacteria is low,and the content of impurity components is higher than the target component,which is not conducive to the industrial production of A82846B,and the extraction and purification cost is relatively high.Kibdelosporangium aridum contains abundant secondary metabolite synthesis gene clusters,and has the potential to synthesize glycopeptide antibiotics and other multiple active metabolites.At present,there are few researches on this strain,especially lack of related bioinformatics analysis.Based on the current status of A82846B’s fermentation,research on its producing strain,K.aridum ZH04,is carried out to understand the synthesis of the product A82846B from the genetic level.In this paper,we performed whole genome sequencing of the strain of K.aridum ZH04,conducted gene prediction,annotated functions of encoding genes and predicted secondary metabolite synthesis gene clusters etc.to understand the genetic background of the producing bacterium.The genetic manipulation system of this strain was established,and the conjugative transfer method suitable for K.aridum ZH04 was also explored.This method can be used to genetically engineer the original strain.The synthesis of A82846B was also optimized from mutation breeding and fermentation conditions.Details are as described below:1.PacBio RS Ⅱ single-molecule real-time sequencing and Illumina sequencing were performed on the A82846B-producing strain K.aridum ZH04.The total length of this strain’s genome is 12475688 bp,the average GC content is 66.27%,and 11,900 coding genes are predicted.Using antiSMASH 5.0 software,it is predicted that there are 47secondary metabolite synthesis gene clusters in the genome,which has rich potential for the synthesis of secondary metabolites,and it is possible to synthesize compounds such as melanin,ectoine and geosmin at the genome level.There are 33 synthesis-related genes of A82846B in cluster32,which are consecutive genes（gene07839-7871）.The protein sequence of gene07859 has high homology with the halogenase protein sequences of vancomycin and balimycin,and is predicted to be responsible for the halogenase gene in the synthesis of A82846B.2.This paper set up a simple and feasible genetic manipulation system for K.aridum ZH04,and explored the method of conjugative transfer.Using spores as receptors for conjugal transfer,MS medium was filtrated to be suitable for spore production of K.aridum ZH04,and mature spores could be obtained on the 7th day of culture.Screening antibiotics,coverage concentration and time,the results indicated that the strain was very sensitive to apramycin,the coverage concentration of antibiotics is identified as 50μg/m L,as well as the optimal result of covering antibiotics achieved with spread plate to breed it for 18 hours.Using the optimized conjugal transfer method,the plasmid containing the A82846B synthesis-related mbt H-like gene gene07864 was transferred into K.aridum ZH04.The fermentation results showed that overexpression of the gene could promote the synthesis of A82846,and the titer of A82846increased by 26.42%.3.The synthesis of A82846B was optimized from the perspective of mutation breeding and fermentation process optimization.Using the ARTP mutagenesis method and high-concentration Na Cl medium to breed and evolve the high-titer strain PC36,a mutagenic strain PC3636 strain was selected,and the A82846B titer of PC3636 increased by 15.97%.Adding different compounds to the fermentation medium,screening the precursor substances L-tyrosine,XCl₂ and NH₄Cl can promote the synthesis of A82846.Optimize the added concentration of L-tyrosine,and the results show that 4 g/L L-tyrosine has the best effect,and the titer of A82846 increased by 32.28%.Among the two chlorides of XCl₂ and NH₄Cl,the promotion effect of XCl₂ is better than that of NH₄Cl.The effect of adding XCl₂ with a chlorine concentration of 5 mg/L during the fermentation for 24 h is the best,the output of A82846 increased by 22.76%and A828246B 19.72%compared with control group.The utilization of carbon sources in the fermentation process was tracked,and the utilization speed and utilization rate of the strains for glucose,maltodextrin,and corn starch gradually decreased.In the fermentation medium formula,glucose and maltodextrin were used as mixed carbon sources.In this research project,the whole genome sequence and analysis of K.aridum ZH04,which produces the intermediate A82846B of oritavancin,was carried out to further understand the genetic information of this strain.The formation of a genetic manipulation system suitable for this strain is of great importance for ensuing genetic engineering.Additionally,approaches like genetic engineering,mutation breeding,and fermentation optimization have been applied to multiply the output of A82846B in various ways,which is conducive to the industrial production of oritavancin.