Chung Daehwan, Verbeke Tobin J, Cross Karissa L, Westpheling Janet, Elkins James G
BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6341 USA.
Department of Genetics, University of Georgia, Athens, GA 30602-7223 USA.
Biotechnol Biofuels. 2015 Jul 22;8:102. doi: 10.1186/s13068-015-0287-y. eCollection 2015.
Compounds such as furfural and 5-hydroxymethylfurfural (5-HMF) are generated through the dehydration of xylose and glucose, respectively, during dilute-acid pretreatment of lignocellulosic biomass and are also potent microbial growth and fermentation inhibitors. The enzymatic reduction of these furan aldehydes to their corresponding, and less toxic, alcohols is an engineering approach that has been successfully implemented in both Saccharomyces cerevisiae and ethanologenic Escherichia coli, but has not yet been investigated in thermophiles relevant to biofuel production through consolidated bioprocessing (CBP). Developing CBP-relevant biocatalysts that are either naturally resistant to such inhibitors, or are amenable to engineered resistance, is therefore, an important component in making biofuels production from lignocellulosic biomass feasible.
A butanol dehydrogenase encoding gene from Thermoanaerobacter pseudethanolicus 39E (Teth39_1597), previously shown to have furfural and 5-HMF reducing capabilities, was cloned into a suicide plasmid, pDCW171 and transformed into a lactate dehydrogenase mutant of Caldicellulosiruptor bescii. Integration of the gene into the C. bescii chromosome was verified via PCR amplification and stable expression was observed up to 75°C. Heterologous expression of the NADPH-dependent BdhA enzyme conferred increased resistance of the engineered strain to both furfural and 5-HMF relative to the wild-type and parental strains. Further, when challenged with 15 mM concentrations of either furan aldehyde, the ability to eliminate furfural or 5-HMF from the culture medium was significantly improved in the engineered strain.
A genetically engineered strain of C. bescii (JWCB044) has been constructed that shows both an improved tolerance to furan aldehydes and an improved ability to eliminate furfural and 5-HMF from the culture medium. The work presented here represents the first example of engineering furan aldehyde resistance into a CBP-relevant thermophile and further validates C. bescii as being a genetically tractable microbe of importance for lignocellulosic biofuel production.
在木质纤维素生物质的稀酸预处理过程中，分别通过木糖和葡萄糖的脱水生成糠醛和5-羟甲基糠醛（5-HMF）等化合物，它们也是强效的微生物生长和发酵抑制剂。将这些呋喃醛酶促还原为相应的、毒性较小的醇是一种工程方法，已在酿酒酵母和产乙醇的大肠杆菌中成功实施，但尚未在与通过联合生物加工（CBP）生产生物燃料相关的嗜热菌中进行研究。因此，开发对这类抑制剂具有天然抗性或易于进行工程抗性改造的与CBP相关的生物催化剂，是使从木质纤维素生物质生产生物燃料变得可行的重要组成部分。
来自嗜热栖热放线菌39E（Teth39_1597）的一个编码丁醇脱氢酶的基因，先前已显示具有糠醛和5-HMF还原能力，被克隆到自杀质粒pDCW171中，并转化到嗜热栖热放线菌的乳酸脱氢酶突变体中。通过PCR扩增验证该基因已整合到嗜热栖热放线菌染色体中，并观察到在高达75°C时能稳定表达。与野生型和亲本菌株相比，依赖NADPH的BdhA酶的异源表达赋予了工程菌株对糠醛和5-HMF更高的抗性。此外，当受到15 mM浓度的任何一种呋喃醛挑战时，工程菌株从培养基中消除糠醛或5-HMF的能力显著提高。
构建了一种嗜热栖热放线菌的基因工程菌株（JWCB044），它对呋喃醛的耐受性提高，并且从培养基中消除糠醛和5-HMF的能力也得到改善。此处展示的工作代表了将呋喃醛抗性工程化引入与CBP相关的嗜热菌的首个实例，并进一步验证了嗜热栖热放线菌是对木质纤维素生物燃料生产具有重要意义的可遗传操作的微生物。
