Improved n-Butanol Production from Clostridium cellulovorans by Integrated Metabolic and Evolutionary Engineering
Improved n-Butanol Production from Clostridium cellulovorans by Integrated Metabolic and Evolutionary Engineering
复制标题
通过综合代谢和进化工程改进纤维素梭菌的正丁醇生产
DOI：
10.1128/aem.02560-18
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发表时间：
2019-01
影响因子：
4.4
通讯作者：
Yang Sheng
中科院分区：
文献类型：
--
作者：
Wen Zhigiang;Ledesma Amaro Rodrigo;Lin Jianping;Jiang Yu;Yang Sheng
由于缺乏遗传工具，Clostridium cellulovorans DSM 743B 尚未被全面探索作为通过综合生物加工 (CBP) 生产正丁醇的推定菌株平台。基于之前对遗传工具的研究，本研究展示了用于开发 CBP 的微生物底盘的 C. cellulovorans 菌株工程。代谢工程和进化工程相结合，提高了利用低成本可再生农业废料碱提取去壳玉米芯（AECC）生产纤维素纤维的正丁醇。 AECC 的正丁醇产量增加了 138 倍，从不足 0.025 克/升增加到 3.47 克/升，这是迄今为止 CBP 报道的使用单一重组梭菌菌株生产正丁醇的最高滴度。该工程菌株可作为 CBP 从木质纤维素生产正丁醇的有前途的基础。摘要 纤维素梭菌 DSM 743B 具有作为通过综合生物加工 (CBP) 进行生物质精炼的基础菌株的潜力。然而，其从木质纤维素生物质生产正丁醇的方法尚未得到证实。本研究通过引入来自丙酮丁醇梭菌 ATCC 824 的 adhE1 和 ctfA-ctfB-adc 基因，证明了在纤维素纤维梭菌中构建了辅酶 A (CoA) 依赖性丙酮-丁醇-乙醇 (ABE) 途径，使其能够利用丰富且低成本的农业废料碱提去壳玉米棒子 (AECC) 作为唯一碳源来生产正丁醇。然后，采用一种新的适应性实验室进化（ALE）方法来增强C. cellulovorans的正丁醇耐受性，以充分利用其正丁醇输出潜力。为了进一步提高正丁醇的产量，将代谢工程和进化工程结合起来，利用进化的菌株作为代谢工程的宿主。工程改造的纤维素纤维AECC的正丁醇产量增加了138倍，从低于0.025克/升增加到3.47克/升。该方法代表了 CBP 使用单一重组梭菌菌株生产正丁醇的里程碑。该工程菌株为木质纤维素发酵正丁醇提供了一种有前景的 CBP 微生物底盘。重要性 由于缺乏遗传工具，Clostridium cellulovorans DSM 743B 尚未被全面探索作为通过综合生物加工 (CBP) 生产正丁醇的推定菌株平台。基于之前对遗传工具的研究，本研究展示了用于开发 CBP 的微生物底盘的 C. cellulovorans 菌株工程。代谢工程和进化工程相结合，提高了利用低成本可再生农业废料碱提取去壳玉米芯（AECC）生产纤维素纤维的正丁醇。 AECC 的正丁醇产量增加了 138 倍，从不足 0.025 克/升增加到 3.47 克/升，这是迄今为止 CBP 报道的使用单一重组梭菌菌株生产正丁醇的最高滴度。该工程菌株可作为 CBP 从木质纤维素生产正丁醇的有前途的基础。
Due to a lack of genetic tools, Clostridium cellulovorans DSM 743B has not been comprehensively explored as a putative strain platform for n-butanol production by consolidated bioprocessing (CBP). Based on the previous study of genetic tools, strain engineering of C. cellulovorans for the development of a CBP-enabling microbial chassis was demonstrated in this study. Metabolic engineering and evolutionary engineering were integrated to improve the n-butanol production of C. cellulovorans from the low-cost renewable agricultural waste of alkali-extracted, deshelled corn cobs (AECC). The n-butanol production from AECC was increased 138-fold, from less than 0.025 g/liter to 3.47 g/liter, which represents the highest titer of n-butanol produced using a single recombinant clostridium strain by CBP reported to date. This engineered strain serves as a promising chassis for n-butanol production from lignocellulose by CBP. ABSTRACT Clostridium cellulovorans DSM 743B offers potential as a chassis strain for biomass refining by consolidated bioprocessing (CBP). However, its n-butanol production from lignocellulosic biomass has yet to be demonstrated. This study demonstrates the construction of a coenzyme A (CoA)-dependent acetone-butanol-ethanol (ABE) pathway in C. cellulovorans by introducing adhE1 and ctfA-ctfB-adc genes from Clostridium acetobutylicum ATCC 824, which enabled it to produce n-butanol using the abundant and low-cost agricultural waste of alkali-extracted, deshelled corn cobs (AECC) as the sole carbon source. Then, a novel adaptive laboratory evolution (ALE) approach was adapted to strengthen the n-butanol tolerance of C. cellulovorans to fully utilize its n-butanol output potential. To further improve n-butanol production, both metabolic engineering and evolutionary engineering were combined, using the evolved strain as a host for metabolic engineering. The n-butanol production from AECC of the engineered C. cellulovorans was increased 138-fold, from less than 0.025 g/liter to 3.47 g/liter. This method represents a milestone toward n-butanol production by CBP, using a single recombinant clostridium strain. The engineered strain offers a promising CBP-enabling microbial chassis for n-butanol fermentation from lignocellulose. IMPORTANCE Due to a lack of genetic tools, Clostridium cellulovorans DSM 743B has not been comprehensively explored as a putative strain platform for n-butanol production by consolidated bioprocessing (CBP). Based on the previous study of genetic tools, strain engineering of C. cellulovorans for the development of a CBP-enabling microbial chassis was demonstrated in this study. Metabolic engineering and evolutionary engineering were integrated to improve the n-butanol production of C. cellulovorans from the low-cost renewable agricultural waste of alkali-extracted, deshelled corn cobs (AECC). The n-butanol production from AECC was increased 138-fold, from less than 0.025 g/liter to 3.47 g/liter, which represents the highest titer of n-butanol produced using a single recombinant clostridium strain by CBP reported to date. This engineered strain serves as a promising chassis for n-butanol production from lignocellulose by CBP.
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影响因子：
6.3
作者：
Willson BJ;Kovács K;Wilding-Steele T;Markus R;Winzer K;Minton NP
通讯作者：
Minton NP
影响因子：
8.4
作者：
Sillers, Ryan;Chow, Alison;Papoutsakis, Eleftherios T.
通讯作者：
Papoutsakis, Eleftherios T.
影响因子：
32.5
作者：
Brethauer, Simone;Studer, Michael Hanspeter
通讯作者：
Studer, Michael Hanspeter
影响因子：
8.4
作者：
Lin, Paul P.;Mi, Luo;Liao, James C.
通讯作者：
Liao, James C.
DOI：
10.1073/pnas.89.8.3483
发表时间：
1992-04-15
影响因子：
11.1
作者：
SHOSEYOV, O;TAKAGI, M;DOI, RH
通讯作者：
DOI, RH