Promdonkoy Peerada, Siripong Wiparat, Downes Joe James, Tanapongpipat Sutipa, Runguphan Weerawat
National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Paholyothin Road, Klong 1, Klong Luang, Pathumthani, 12120, Thailand.
University of Kent, Canterbury, Kent, UK.
AMB Express. 2019 Oct 10;9(1):160. doi: 10.1186/s13568-019-0885-3.
As the importance of reducing carbon emissions as a means to limit the serious effects of global climate change becomes apparent, synthetic biologists and metabolic engineers are looking to develop renewable sources for transportation fuels and petroleum-derived chemicals. In recent years, microbial production of high-energy fuels has emerged as an attractive alternative to the traditional production of transportation fuels. In particular, the Baker's yeast Saccharomyces cerevisiae, a highly versatile microbial chassis, has been engineered to produce a wide array of biofuels. Nevertheless, a key limitation of S. cerevisiae is its inability to utilize xylose, the second most abundant sugar in lignocellulosic biomass, for both growth and chemical production. Therefore, the development of a robust S. cerevisiae strain that is able to use xylose is of great importance. Here, we engineered S. cerevisiae to efficiently utilize xylose as a carbon source and produce the advanced biofuel isobutanol. Specifically, we screened xylose reductase (XR) and xylose dehydrogenase (XDH) variants from different xylose-metabolizing yeast strains to identify the XR-XDH combination with the highest activity. Overexpression of the selected XR-XDH variants, a xylose-specific sugar transporter, xylulokinase, and isobutanol pathway enzymes in conjunction with the deletions of PHO13 and GRE3 resulted in an engineered strain that is capable of producing isobutanol at a titer of 48.4 ± 2.0 mg/L (yield of 7.0 mg/g D-xylose). This is a 36-fold increase from the previous report by Brat and Boles and, to our knowledge, is the highest isobutanol yield from D-xylose in a microbial system. We hope that our work will set the stage for an economic route for the production of advanced biofuel isobutanol and enable efficient utilization of lignocellulosic biomass.
随着将减少碳排放作为限制全球气候变化严重影响的一种手段的重要性日益凸显，合成生物学家和代谢工程师正在寻求开发用于运输燃料和石油衍生化学品的可再生资源。近年来，微生物生产高能燃料已成为传统运输燃料生产的一种有吸引力的替代方案。特别是，面包酵母酿酒酵母作为一种用途广泛的微生物底盘，已被改造用于生产多种生物燃料。然而，酿酒酵母的一个关键限制是它无法利用木糖（木质纤维素生物质中第二丰富的糖类）进行生长和化学品生产。因此，开发一种能够利用木糖的强大酿酒酵母菌株具有重要意义。在此，我们对酿酒酵母进行工程改造，使其能够高效利用木糖作为碳源并生产先进生物燃料异丁醇。具体而言，我们从不同的木糖代谢酵母菌株中筛选木糖还原酶（XR）和木糖脱氢酶（XDH）变体，以鉴定具有最高活性的XR - XDH组合。所选XR - XDH变体、木糖特异性糖转运蛋白、木酮糖激酶和异丁醇途径酶的过表达，同时缺失PHO13和GRE3，产生了一种工程菌株，该菌株能够以48.4±2.0 mg/L的滴度生产异丁醇（产量为7.0 mg/g D - 木糖）。这比Brat和Boles之前的报告提高了36倍，据我们所知，这是微生物系统中从D - 木糖获得的最高异丁醇产量。我们希望我们的工作将为先进生物燃料异丁醇的经济生产路线奠定基础，并实现木质纤维素生物质的高效利用。
