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, Saccharomyces cerevisiae 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 S. cerevisiae. 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 S. cerevisiae. We also highlight the importance of improving cell physiology to enhance the efficiency of ABC transporters, especially energy status and cell membrane properties.

中文翻译：

---

改善 ATP 供应和膜柔韧性的工程内源性 ABC 转运蛋白增强酿酒酵母中 β-胡萝卜素的分泌

产品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途的策略。作为工业规模生产生物燃料的强大微生物宿主，酿酒酵母包含强大的运输系统，可以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 转运蛋白结合酿酒酵母的生理工程来改善疏水产物（β-胡萝卜素）的分泌和生产。底物诱导性是大多数内源性转运蛋白的显着特征。通过比较蛋白质组学分析和转录确认，我们确定了五种潜在的 ABC 转运蛋白（Pdr5p、Pdr10p、Snq2p、Yor1p 和 Yol075cp）用于 β-胡萝卜素外流。β-胡萝卜素的积累也会影响细胞生理的各个方面，包括能量代谢、线粒体翻译、脂质代谢、麦角甾醇生物合成过程和细胞壁合成。在这里，我们采用诱导型 GAL 启动子过表达候选转运蛋白并增强 β-胡萝卜素的分泌和细胞内产生，其中 Snq2p 表现出最佳性能（与其亲本菌株 YBX-01 相比分别增加 4.04 倍和 1.33 倍） ， 分别）。为了进一步提高外排能力，采用了增加ATP供应和提高膜流动性两种策略。与亲本菌株 YBX-01 相比，工程菌株 YBX-20 的 β-胡萝卜素分泌增加 5.80 倍，细胞内 β-胡萝卜素产量增加 1.71 倍。总体，我们的结果表明，工程内源性质膜 ABC 转运蛋白是酿酒酵母中疏水性产物流出的一种有前途的方法。我们还强调了改善细胞生理学以提高 ABC 转运蛋白效率的重要性，尤其是能量状态和细胞膜特性。