Bursac Thea, Gralnick Jeffrey A, Gescher Johannes
Department of Applied Biology, Institute for Applied Biosciences, Karlsruhe Institute of Technology, Karlsruhe, Germany.
BioTechnology Institute and Department of Microbiology, University of Minnesota, Twin Cities, St. Paul, Minnesota.
Biotechnol Bioeng. 2017 Jun;114(6):1283-1289. doi: 10.1002/bit.26243. Epub 2017 Feb 15.
This study describes the realization of an anoxic acetoin production process using the proteobacterium Shewanella oneidensis. Fermentative processes are of high biotechnological relevance since they offer high productivity and a low percentage of substrate consumption for anabolic processes. Nevertheless, the range of compounds that can be produced as sole end product of a fermentative process is limited, since the average oxidation state of substrate and products has to be identical in the absence of an external electron acceptor. This limitation could be overcome by the transfer of the surplus of electrons to a poised electrode surface, which of note is the only known anaerobic electron acceptor that cannot be depleted. In the first genetic engineering step, deletion mutants were developed that are devoid of either one, two, or all three prophages in their genome with the aim to construct a more stable chassis strain for microbe-electrode interaction, due to less prophage induced cell lysis (Gödeke et al., 2011). Current production in a bioelectrochemical system together with the analysis of cells on the anode surface were used as surrogate for the stability assessment. The λ-prophage deletion mutant produced overall 1.34fold more current (6.7 μA cm ) than the wild type and all other constructed strains and showed with 1.1 × 10 cells the highest cell density on the anode surface (2.3fold more than the wild type). The strain was further modified to contain codon optimized versions of acetolactate synthase and acetolactate decarboxylase derived from Bacillus subtilis. This allowed for the production of a mixture of acetoin and acetate from lactate in an almost 0.4:1 ratio. Further process improvement was reached by the deletion of the acetate kinase and phosphotransacetylase genes ackA/pta. The acetoin yield increased in this mutant from 40 to 86% of the theoretical maximum and acetoin was the only detectable end product. Biotechnol. Bioeng. 2017;114: 1283-1289. © 2017 Wiley Periodicals, Inc.
本研究描述了利用变形杆菌奥奈达希瓦氏菌实现缺氧产3-羟基丁酮的过程。发酵过程具有很高的生物技术相关性，因为它们具有高生产率，且用于合成代谢过程的底物消耗百分比低。然而，作为发酵过程唯一最终产物能够产生的化合物范围有限，因为在没有外部电子受体的情况下，底物和产物的平均氧化态必须相同。通过将多余的电子转移到稳定的电极表面可以克服这一限制，值得注意的是，这是唯一已知的不会耗尽的厌氧电子受体。在第一步基因工程中，构建了基因组中缺失一个、两个或所有三个原噬菌体的缺失突变体，目的是构建一个更稳定的底盘菌株用于微生物与电极的相互作用，因为原噬菌体诱导的细胞裂解较少（戈德克等人，2011年）。生物电化学系统中的电流产生以及阳极表面细胞的分析被用作稳定性评估的替代指标。λ-原噬菌体缺失突变体产生的总电流（6.7 μA/cm）比野生型和所有其他构建菌株高出1.34倍，并且在阳极表面显示出1.1×10个细胞的最高细胞密度（比野生型高2.3倍）。该菌株进一步经过改造，含有源自枯草芽孢杆菌的密码子优化版乙酰乳酸合酶和乙酰乳酸脱羧酶。这使得能够从乳酸中以几乎0.4:1的比例生产3-羟基丁酮和乙酸的混合物。通过缺失乙酸激酶和磷酸转乙酰酶基因ackA/pta实现了进一步的工艺改进。在该突变体中，3-羟基丁酮产量从理论最大值的40%提高到86%，并且3-羟基丁酮是唯一可检测到的最终产物。《生物技术与生物工程》2017年；114: 1283 - 1289。© 2017威利期刊公司
