Bu Xiao, Lin Jing-Yuan, Cheng Jing, Yang Dong, Duan Chang-Qing, Koffas Mattheos, Yan Guo-Liang
Centre for Viticulture and Enology, College of Food Science and Nutritional Engineering, China Agricultural University, 17 East Tsinghua Rd, Beijing, 100083 China.
Key Laboratory of Viticulture and Enology, Ministry of Agriculture and Rural Affairs, Beijing, 100083 China.
Biotechnol Biofuels. 2020 Oct 10;13:168. doi: 10.1186/s13068-020-01809-6. eCollection 2020.
Product toxicity is one of the bottlenecks for microbial production of biofuels, and transporter-mediated biofuel secretion offers a promising strategy to solve this problem. As a robust microbial host for industrial-scale production of biofuels, contains a powerful transport system to export a wide range of toxic compounds to sustain survival. The aim of this study is to improve the secretion and production of the hydrophobic product (β-carotene) by harnessing endogenous ABC transporters combined with physiological engineering in .
Substrate inducibility is a prominent characteristic of most endogenous transporters. Through comparative proteomic analysis and transcriptional confirmation, we identified five potential ABC transporters (Pdr5p, Pdr10p, Snq2p, Yor1p, and Yol075cp) for β-carotene efflux. The accumulation of β-carotene also affects cell physiology in various aspects, including energy metabolism, mitochondrial translation, lipid metabolism, ergosterol biosynthetic process, and cell wall synthesis. Here, we adopted an inducible GAL promoter to overexpress candidate transporters and enhanced the secretion and intracellular production of β-carotene, in which Snq2p showed the best performance (a 4.04-fold and a 1.33-fold increase compared with its parental strain YBX-01, respectively). To further promote efflux capacity, two strategies of increasing ATP supply and improving membrane fluidity were following adopted. A 5.80-fold increase of β-carotene secretion and a 1.71-fold increase of the intracellular β-carotene production were consequently achieved in the engineered strain YBX-20 compared with the parental strain YBX-01.
Overall, our results showcase that engineering endogenous plasma membrane ABC transporters is a promising approach for hydrophobic product efflux in . We also highlight the importance of improving cell physiology to enhance the efficiency of ABC transporters, especially energy status and cell membrane properties.
产物毒性是微生物生产生物燃料的瓶颈之一，而转运蛋白介导的生物燃料分泌为解决这一问题提供了一种有前景的策略。作为工业规模生产生物燃料的强大微生物宿主，含有一个强大的转运系统来输出多种有毒化合物以维持生存。本研究的目的是通过利用内源性ABC转运蛋白并结合生理工程来提高疏水性产物（β-胡萝卜素）的分泌和产量。
底物诱导性是大多数内源性转运蛋白的一个突出特征。通过比较蛋白质组学分析和转录确认，我们鉴定出了五个潜在的用于β-胡萝卜素外排的ABC转运蛋白（Pdr5p、Pdr10p、Snq2p、Yor1p和Yol075cp）。β-胡萝卜素的积累也在各个方面影响细胞生理，包括能量代谢、线粒体翻译、脂质代谢、麦角固醇生物合成过程和细胞壁合成。在此，我们采用可诱导的GAL启动子来过表达候选转运蛋白，并增强了β-胡萝卜素的分泌和细胞内产量，其中Snq2p表现出最佳性能（与亲本菌株YBX-01相比，分别增加了4.04倍和1.33倍）。为了进一步提高外排能力，随后采用了增加ATP供应和改善膜流动性的两种策略。与亲本菌株YBX-01相比，工程菌株YBX-20中β-胡萝卜素的分泌增加了5.80倍，细胞内β-胡萝卜素产量增加了1.71倍。
总体而言，我们的结果表明，工程化内源性质膜ABC转运蛋白是在中实现疏水性产物外排的一种有前景的方法。我们还强调了改善细胞生理以提高ABC转运蛋白效率的重要性，特别是能量状态和细胞膜特性。
