Zhang Yanfei, Lane Stephan, Chen Jhong-Min, Hammer Sarah K, Luttinger Jake, Yang Lifeng, Jin Yong-Su, Avalos José L
1Department of Chemical and Biological Engineering, Princeton University, 101 Hoyt Laboratory, William Street, Princeton, NJ 08544 USA.
4Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL USA.
Biotechnol Biofuels. 2019 Sep 20;12:223. doi: 10.1186/s13068-019-1560-2. eCollection 2019.
Branched-chain higher alcohols (BCHAs), including isobutanol and 2-methyl-1-butanol, are promising advanced biofuels, superior to ethanol due to their higher energy density and better compatibility with existing gasoline infrastructure. Compartmentalizing the isobutanol biosynthetic pathway in yeast mitochondria is an effective way to produce BCHAs from glucose. However, to improve the sustainability of biofuel production, there is great interest in developing strains and processes to utilize lignocellulosic biomass, including its hemicellulose component, which is mostly composed of the pentose xylose.
In this work, we rewired the xylose isomerase assimilation and mitochondrial isobutanol production pathways in the budding yeast . We then increased the flux through these pathways by making gene deletions of , , and , to develop a strain (YZy197) that produces as much as 4 g/L of BCHAs (3.10 ± 0.18 g isobutanol/L and 0.91 ± 0.02 g 2-methyl-1-butanol/L) from xylose. This represents approximately a 28-fold improvement on the highest isobutanol titers obtained from xylose previously reported in yeast and the first report of 2-methyl-1-butanol produced from xylose. The yield of total BCHAs is 57.2 ± 5.2 mg/g xylose, corresponding to ~ 14% of the maximum theoretical yield. Respirometry experiments show that xylose increases mitochondrial activity by as much as 7.3-fold compared to glucose.
The enhanced levels of mitochondrial BCHA production achieved, even without disrupting ethanol byproduct formation, arise mostly from xylose activation of mitochondrial activity and are correlated with slow rates of sugar consumption.
支链高级醇（BCHA），包括异丁醇和2-甲基-1-丁醇，是很有前景的先进生物燃料，由于其较高的能量密度以及与现有汽油基础设施更好的兼容性，优于乙醇。将异丁醇生物合成途径分隔在酵母线粒体中是从葡萄糖生产BCHA的有效方法。然而，为了提高生物燃料生产的可持续性，人们对开发利用木质纤维素生物质（包括其半纤维素成分，主要由戊糖木糖组成）的菌株和工艺非常感兴趣。
在这项工作中，我们在出芽酵母中重新构建了木糖异构酶同化和线粒体异丁醇生产途径。然后，我们通过缺失、和基因来增加这些途径的通量，以开发出一种菌株（YZy197），该菌株能从木糖中生产高达4 g/L的BCHA（3.10±0.18 g异丁醇/L和0.91±0.02 g 2-甲基-1-丁醇/L）。这比之前报道的酵母中从木糖获得的最高异丁醇滴度提高了约28倍，也是首次报道从木糖生产2-甲基-1-丁醇。总BCHA的产量为57.2±5.2 mg/g木糖，约为最大理论产量的14%。呼吸测定实验表明，与葡萄糖相比，木糖可使线粒体活性提高多达7.3倍。
即使不破坏乙醇副产物的形成，线粒体BCHA产量的提高主要源于木糖对线粒体活性的激活，并且与缓慢的糖消耗速率相关。
