Gambacorta Francesca V, Dietrich Joshua J, Baerwald Justin J, Brown Stephanie J, Su Yun, Pfleger Brian F
DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, United States.
Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI, United States.
Front Bioeng Biotechnol. 2022 Dec 2;10:1080024. doi: 10.3389/fbioe.2022.1080024. eCollection 2022.
is the dominant fermentative producer of ethanol in industry and a preferred host for production of other biofuels. That said, rewiring the metabolism of to produce other fermentation products, such as isobutanol, remains an academic challenge. Many studies report aerobic production of isobutanol, but ethanol remains a substantial by-product under these conditions due to the Crabtree effect. These studies indicate that the native isobutanol pathway is incapable of carrying sufficient flux to displace ethanol. In this report, we screened a combinatorial library of pathway enzymes to identify an isobutanol pathway cassette capable of supporting the growth of a non-ethanol producing . We began by identifying a diverse set of isobutanol pathway enzyme homologs and combined each open reading frame with varied-strength promoters in a combinatorial, pooled fashion. We applied a growth-coupled screen where a functional isobutanol pathway restored NAD regeneration during glucose catabolism that is otherwise repressed the Crabtree effect. Using this screen, we isolated a cassette consisting of a mosaic of bacterial and cytosol-localized fungal enzymes that conferred under aerobic conditions the ability to produce 364 mg/L isobutanol (8.8% of the theoretical maximum yield). We next shifted the cofactor usage of the isolated ketol-acid reductoisomerase enzyme in the cassette from NADPH to NADH-preferring to improve redox balance. The approach used herein isolated isobutanol producing strains that approach the best in the literature without producing substantial ethanol titers. Still, the best isolated cassette was insufficient to support anaerobic growth in the absence of ethanol fermentation - indicating the presence of further fundamental gaps in our understanding of yeast fermentation.
是工业中乙醇的主要发酵生产菌，也是生产其他生物燃料的理想宿主。话虽如此，改变其代谢以生产其他发酵产物，如异丁醇，仍然是一个学术挑战。许多研究报道了异丁醇的需氧生产，但由于克勒勃屈利效应，在这些条件下乙醇仍然是大量的副产物。这些研究表明，天然的异丁醇途径无法携带足够的通量来取代乙醇。在本报告中，我们筛选了途径酶的组合文库，以鉴定能够支持非乙醇生产菌生长的异丁醇途径盒。我们首先鉴定了一组多样的异丁醇途径酶同源物，并以组合、汇集的方式将每个开放阅读框与不同强度的启动子相结合。我们应用了一种生长偶联筛选方法，其中功能性异丁醇途径在葡萄糖分解代谢过程中恢复了NAD再生，否则该过程会受到克勒勃屈利效应的抑制。通过这种筛选，我们分离出了一个由细菌和胞质溶胶定位的真菌酶组成的嵌合体盒，该盒在有氧条件下赋予了产生364mg/L异丁醇的能力（理论最大产量的8.8%）。接下来，我们将盒中分离出的酮醇酸还原异构酶的辅因子使用从NADPH转变为更倾向于NADH，以改善氧化还原平衡。本文使用的方法分离出了异丁醇生产菌株，这些菌株在文献中接近最佳水平，且不产生大量乙醇滴度。然而，最好的分离盒不足以支持在没有乙醇发酵的情况下进行厌氧生长——这表明我们对酵母发酵的理解仍存在进一步的基本差距。
