Marine College, Shandong University, Weihai, 264209, Shandong, China.
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, China.
J Ind Microbiol Biotechnol. 2020 Dec;47(12):1141-1154. doi: 10.1007/s10295-020-02315-2. Epub 2020 Sep 29.
A breeding approach combining genome shuffling with multiple antibiotic-resistance including gentamicin, rifampin and lincomycin, was developed in this research to improve the poly-γ-L-diaminobutanoic acid (γ-PAB) production in Bacillus pumilus LS-1. By this unique strategy, recombinants from the third round of genome shuffling could tolerate higher concentration of compound antibiotics and exhibited higher γ-PAB production as 392.4 mg/L in shake-flask fermentation, tenfold over the parent. In batch fermentation, B. pumilus GS3-M7 could produce γ-PAB as high as 2316.4 mg/L in two days, 5.4-fold higher than the control, which was the highest productivity ever reported. In addition, the optimal pH in B. pumilus for γ-PAB synthesis was decreased after ARTP mutagenesis and protoplast fusion, because the lower pH environment is favorable for accumulation of intracellular ATP. Some key enzymes in GS3-M7 showed higher activities than those in the parent, suggesting a greater flux to TCA circle and DAP pathway, which was a reason for enhanced γ-PAB production.
本研究采用基因组改组与多种抗生素抗性（包括庆大霉素、利福平、林可霉素）相结合的育种方法，提高了地衣芽孢杆菌 LS-1 中聚-γ-L-二氨基丁酸（γ-PAB）的产量。通过这种独特的策略，第三轮基因组改组的重组体能耐受更高浓度的复合抗生素，并在摇瓶发酵中表现出更高的γ-PAB 产量，达到 392.4mg/L，是亲本的 10 倍。在分批发酵中，B. pumilus GS3-M7 可以在两天内产生高达 2316.4mg/L 的γ-PAB，比对照提高了 5.4 倍，这是迄今为止报道的最高生产力。此外，经过 ARTP 诱变和原生质体融合，B. pumilus 中合成γ-PAB 的最佳 pH 值降低，因为较低的 pH 值环境有利于细胞内 ATP 的积累。GS3-M7 中的一些关键酶的活性高于亲本，表明 TCA 循环和 DAP 途径的通量增加，这是增强γ-PAB 产量的一个原因。
