Research And Engineering Of Post-modification Enzymes For Milbemycins Overproduction In Streptomyces Bingchenggensis

Milbemycins and their semisynthetic derivatives are eco-friendly acaricides and pesticides,which are produced by Streptomyces bingchenggensis.Among the tens of secondary metabolites produced by Streptomyces bingchenggensis,several by-products such as nanchangmycin and bingchamides are produced simultaneously with the milbemycin A3/A4.These compounds as well as other analogues of milbemycin A3/A4,including milbemycin B1,B2,?9,?10,?1,?2 and other ?-family milbemycins bring difficulty to the isolation and purification of milbemycin A3/A4.Notably,it is hard to abolish these analogues by physicochemical separation approaches.Since the by-products compete with milbemycin A3/A4 for precursors,the present of these analogues inhibit further titer and yield improvement of milbemycin A3/A4.Consequently,it is difficult to reduce the production costs of milbemycins,which limits the broad applications in the agricultural fied.Our group has been dedicating to strain improvement of S.bingchenggensis,we have obtained the mutant strain incapable of producing milbemycin B1,B2,?1,?2.Despite of these achievements,the by-products milbemycin ?9/?10 and ?-family milbemycins are still existed in fermentation broth.To block the formation of milbemycin ?9/?10 and ?-family milbemycins and further optimize the milbemycin A3/A4-producing strain,this study explored post-modification enzymes that participate in post-PKS steps of milbemycin ?9/?10 and ?-family milbemycins biosynthesis.cyp41?sbi₀7711?encode a cytochrome P450 enzyme Cyp41.Cyp41,a CYP268 A subfamily cytochrome P450 enzyme,is the only one found in Streptomyces sp.strain in the CYP268 family of Nelson bacteria P450 database.It was identified to catalyze the oxidization at C26 of milbemycin A3/A4 to generate milbemycin ?26/?27,the precursors of milbemycin ?9/?10,by disruption and complementation of cyp41 and feeding experiments.In-frame deletion of cyp41 was conducted in Streptomyces bingchenggensis BC04,yielding the mutant strain B4 DP.The production of milbemycin ?9/?10 is eliminated and the titer of milbemycin A3/A4 is increased from 2382.5 mg/L to 2625.6 mg/L in the Streptomyces bingchenggensis B4 DP.In addition,we have researched a CYP171 family cytochrome P450 enzyme MilE by disruption and complementation of milE and feeding experiments.MilE was identified to be responsible for the generation of the furan ring between C6 and C8 a of milbemycins converting ?-family milbemycins to ?-family milbemycins.Overexpression of milE in Streptomyces bingchenggensis B4 DP,the titer of ?-family milbemycins was reduced by 77.2% and the production of milbemycin A3/A4 was increased by 39.0%.Meanwhile,overexpression of milE increased the transcriptional levels of milbemycin biosynthetic genes in varying degrees by qRT-PCR.Furthermore,overexpression of milC responsible for the spiroketal formation of milbemycins increased milbemycin A3/A4 titer by 13.5%.Based on the above results,the high-yielding strain BPSEC was obtained by rational genetic engineering,in which milbemycin A3/A4 titer was increased by 57.69% to 3727.21 mg/L compared with the strain BC04 with reduced production of milbemycin-like by-products.In all,the following conclusions have been obtained in this study:?1?We found and identified a previously unreported CYP450 that is responsible for the oxidization at C26 of milbemycin A3/A4.?2?Overexpression of milE can significantly increase the transcriptional levels of milbemycin biosynthetic genes.?3?We constructed a high-production strain BPSEC producing milbemycin A3/A4 without milbemycin ?9/?10 production and reduced ?-family milbemycins production.?4?We provided an efficient strategy of engineering post-modification steps to reduce analogous by-products and improve the production of milbemycin A3/A4.