Jian Pei  ,  Yaohui Hou  ,  Yanwei Li ,  Kai Liu ,  Li Dai  ,  Zongchao Huo  ,  Chenyang Tong  ,  Chengyan Gao  ,  Hailong Wang ,  Bingyin Peng  ,  Dongying Zhao  ,  Jiafeng Yu ,  Binghai Du ,  Chengqiang Wang

Abstract 

Paenibacillus polymyxa is a plant growth-promoting rhizobacterium widely employed as a chassis organism in biomanufacturing. In this study, inducible regulatory systems and polymyxin efflux mechanisms were investigated in P. polymyxa to enhance polymyxin production. By employing a heterologous xylose-inducible promoter, PxylA, to drive the expression of the pmxABCDE operon, xylose-dependent polymyxin synthesis was achieved, with delayed induction resulting in improved production. Deletion of transporter genes pmxCD reduced polymyxin P production by approximately 50%, and complementation with pmxD partially restored productivity. A pathogen-responsive endogenous promoter PlipA1 was further identified to express pmxD, and a polymyxin secretion research model was also constructed. Alanine-scanning mutagenesis identified T38 as a critical residue for PmxD-mediated polymyxin efflux activity. Subsequent saturation mutagenesis at position 38 showed that the T38W and T38Y variants enhanced polymyxin P production by nearly fourfold compared to the wild-type. To reduce industrial production costs, a non-detoxified composite medium composed of cellulose hydrolysate and sugarcane molasses was firstly developed for polymyxin production. Under these low-cost conditions, the strain expressing the T38W mutant exhibited a statistically significant increase in polymyxin P production-from 70.31 mg/L in the native PmxD-expressing strain to 87.66 mg/L-corresponding to a 24.68% improvement in titer. Collectively, our findings demonstrate that inducible promoter systems and rational engineering of efflux transporters are effective strategies for optimizing polymyxin biosynthesis and secretion in P. polymyxa, even when utilizing cost-effective substrates.

Paper Linkage:https://www.sciencedirect.com/science/article/pii/S0960852426006036