Study On Production Of A82846B By Strain Breeding And Process Optimization

Oritavancin is a new type of semi-synthetic glycopeptide antibiotic developed in recent year,after the first generation of glycopeptide antibiotics such as vancomycin and teicolanin,which is used for the treatment of acute bacterial skin and skin structural infections（ABSSSIs）caused by gram-positive bacteria（including methicillin-resistant staphylococcus aureus,MRSA）.It has the advantages of being effective against drug-resistant bacteria,long half-life,convenient administration and so on.The synthesis of oritavancin need the A82846B which is mainly synthesized by microbial fermentation,the producing bacteria including Amycolatopsis orientalis and Kibdelosporangium aridum,however,the yield of the two strians both is low so far,in addition,two structure similar by-product:A82846A and A82846C would synthesis in fermentation process,the only difference on structure between them is the chlorine atoms number,which would cause the difficulty of separation in next step,increase the cost of separation and purification.In this paper,a strain of Kibdelosporangium aridum ZH04 in the laboratory was taken as the research object,and the methods of strain screnning and process optimization were used to further improve the fermentation efficiency and the yield of target products.The main contents of this paper are as follows:1.Using ZH04 as the parent strain,two high-yield strains ZH04-PC-21 and ZH04-PC-13 were selected through UV mutagenesis,atmospheric pressure plasma mutagenesis（ARTP）,natural screening,resistance screening and ribosome engineering.After shake flask fermentation and HPLC testing,it was found that strain ZH04-PC-21had a 110%increase in the yield of the target product A82846B compared to the parent strain.Among the two structurally similar by-products,A82846A and A82846C increased by 160%and 52%respectively compared to the parent strain,ZH04-PC-13increased A82846B by 61%,A82846C and A82846A increased by 36%and 130%respectively compared to the parent strain.Compared with the target product,the yield of the monochlorinated by-product A82846A increased the most,which can establish the strain foundation for the subsequent molecular transformation of chlorination pathway in laboratory.2.Through the optimization of the fermentation process,compared with the shaking flask fermentation unit of the strain before optimization,the yield of A82846B components increased by 128%reached to 373.20 mg/L,;the A82846A component increased by 176%reached to 968.72 mg/L,and the A82846C component increased by163%reached to 159.07 mg/L.The effect of carbon source,nitrogen source,trace elements,inoculation time,initial p H,filling volume,inoculation volume,shearing force were studied in shake flaske.The optimal component of the culture medium were:glucose,corn starch,soybean meal,yeast extract,molasses,calcium carbonate,Cu SO₄·7H₂O,and the optimal shake flask fermentation culture conditions were:initial p H 7.5,inoculation time 48 h,inoculation amount 10%,filling volume 25 m L,culture temperature 28°C,shaker speed 220 rpm.and the strain ZH04 was found very sensitive to shear force,Excessive shear will affect the synthesis of secondary metabolites,which can be used to guide the scale-up fermentation.In addition,preliminary studies of 5-L scale-up fermentation also demonstrated that the strain ZH04 is sensitive to dissolved oxygen and stirring shear.3.Ribosomal mutation site study of high-yielding strains was adopted to study the mutation point of genes such as rps L and rsm G of mutant strains.After gene sequencing,a mutation occurred in the rsp L gene of a mutant strain ZH04-PC-21,the base of position 184 was mutated from G to A,and the encoded amino acid was mutated from glutamic acid to lysine,it is speculated that this mutation may affect the related regulatory proteins of secondary metabolite synthesis,resulting in the increase of product yield.No mutations were detected in the remaining mutant strains,it need further in-depth analysis.