Latimer Luke N, Lee Michael E, Medina-Cleghorn Daniel, Kohnz Rebecca A, Nomura Daniel K, Dueber John E
Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Energy Biosciences Institute, Berkeley, CA 94720, USA.
Energy Biosciences Institute, Berkeley, CA 94720, USA; The UC Berkeley & UCSF Graduate Program in Bioengineering, Berkeley, CA 94720, USA; Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA.
Metab Eng. 2014 Sep;25:20-9. doi: 10.1016/j.ymben.2014.06.002. Epub 2014 Jun 13.
Fermentation of xylose, a major constituent of lignocellulose, will be important for expanding sustainable biofuel production. We sought to better understand the effects of intrinsic (genotypic) and extrinsic (growth conditions) variables on optimal gene expression of the Scheffersomyces stipitis xylose utilization pathway in Saccharomyces cerevisiae by using a set of five promoters to simultaneously regulate each gene. Three-gene (xylose reductase, xylitol dehydrogenase (XDH), and xylulokinase) and eight-gene (expanded with non-oxidative pentose phosphate pathway enzymes and pyruvate kinase) promoter libraries were enriched under aerobic and anaerobic conditions or with a mutant XDH with altered cofactor usage. Through characterization of enriched strains, we observed (1) differences in promoter enrichment for the three-gene library depending on whether the pentose phosphate pathway genes were included during the aerobic enrichment; (2) the importance of selection conditions, where some aerobically-enriched strains underperform in anaerobic conditions compared to anaerobically-enriched strains; (3) improved growth rather than improved fermentation product yields for optimized strains carrying the mutant XDH compared to the wild-type XDH.
木糖是木质纤维素的主要成分，其发酵对于扩大可持续生物燃料生产具有重要意义。我们试图通过使用一组五个启动子同时调控每个基因，来更好地理解内在（基因型）和外在（生长条件）变量对酿酒酵母中树干毕赤酵母木糖利用途径最佳基因表达的影响。在需氧和厌氧条件下，或使用辅因子使用发生改变的突变木糖醇脱氢酶（XDH），对三基因（木糖还原酶、木糖醇脱氢酶（XDH）和木酮糖激酶）和八基因（扩展了非氧化戊糖磷酸途径酶和丙酮酸激酶）启动子文库进行富集。通过对富集菌株的表征，我们观察到：（1）三基因文库启动子富集存在差异，这取决于需氧富集过程中是否包含戊糖磷酸途径基因；（2）选择条件的重要性，一些需氧富集菌株在厌氧条件下的表现不如厌氧富集菌株；（3）与野生型XDH相比，携带突变XDH的优化菌株生长得到改善，但发酵产物产量并未提高。
