Consolidated bioprocessing of cellulose to isobutanol using Clostridium thermocellum
Consolidated bioprocessing of cellulose to isobutanol using Clostridium thermocellum
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DOI：
10.1016/j.ymben.2015.07.001
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发表时间：
2015-09-01
影响因子：
8.4
通讯作者：
Liao, James C.
中科院分区：
文献类型：
--
作者：
Lin, Paul P.;Mi, Luo;Liao, James C.
固化生物处理（CBP）有可能通过同时处理纤维素水解和发酵而不添加预先制造的纤维素酶来降低生物燃料或生化生产成本。特别是热胞梭菌，由于其高纤维素分解率，是一种很有前途的嗜热CBP宿主。本文报道了C. thermocellum生产异丁醇的工程技术。C型热细胞生产异丁醇的代谢工程受到克隆过程中的酶毒性、耗时的途径工程程序和生产试验的缓慢周转的阻碍。在这项工作中，我们首先克隆了不同启动子下的异丁醇途径必需基因，在大肠杆菌中构建了不同的质粒结构。然后，将这些构建体转化到C. thermocellurn中进行测试。在这些工程菌株中，筛选出最佳异丁醇产菌，并对其生产条件进行了优化。我们通过mRNA的表达量证实了过表达基因的表达。我们还确定了天然的酮异戊酸氧化还原酶（KOR）和异种表达的酮异戊酸脱羧酶（MVO）都负责异丁醇的产生。我们进一步发现质粒通过单次交叉整合到染色体中。所得菌株稳定，无抗生素选择压力。该菌株在50℃的最小培养基中，在75 h内产生5.4 g/L的异丁醇角纤维素，达到理论产量的41%。(C) 2015年国际代谢工程学会。Elsevier Inc.出版。版权所有。
Consolidated bioprocessing (CBP) has the potential to reduce bioluel or biochemical production costs by processing cellulose hydrolysis and fermentation simultaneously without the addition of pre manufactured cellulases. In particular, Clostridium thertnocellum is a promising thermophilic CBP host because of its high cellulose decomposition rate. Here we report the engineering of C. thermocellum to produce isobutanol. Metabolic engineering for isobutanol production in C thermocellurn is hampered by enzyme toxicity during cloning, time consuming pathway engineering procedures, and slow turnaround in production tests. In this work, we first cloned essential isobutanol pathway genes under different promoters to create various plasmid constructs in Escherichiu coli. Then, these constructs were transformed and tested in C. thermocellurn. Among these engineered strains, the best isobutanol producer was selected and the production conditions were optimized. We confirmed the expression of the overexpressed genes by their mRNA quantities. We also determined that both the native ketoisovalerate oxidoreductase (KOR) and the heterologous ketoisovalerate decarboxylase (MVO) expressed were responsible for isobutanol production. We further found that the plasmid was integrated into the chromosome by single crossover. The resulting strain was stable without antibiotic selection pressure. This strain produced 5.4 g/L of isobutanol horn cellulose in minimal medium at 50 C within 75 h, Coffesponding to 41% of theoretical yield. (C) 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.