Institute of Biochemical Engineering, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615, Bielefeld, Germany.
Metab Eng. 2019 Sep;55:220-230. doi: 10.1016/j.ymben.2019.07.006. Epub 2019 Jul 15.
Gasification is a suitable technology to generate energy-rich synthesis gas (syngas) from biomass or waste streams, which can be utilized in bacterial fermentation processes for the production of chemicals and fuels. Established microbial processes currently rely on acetogenic bacteria which perform an energetically inefficient anaerobic CO oxidation and acetogenesis potentially hampering the biosynthesis of complex and ATP-intensive products. Since aerobic oxidation of CO is energetically more favorable, we exploit in this study the Gram-negative β-proteobacterium Hydrogenophaga pseudoflava DSM1084 as novel host for the production of chemicals from syngas. We sequenced and annotated the genome of H. pseudoflava and established a genetic engineering toolbox, which allows markerless chromosomal modification via the pk19mobsacB system and heterologous gene expression on pBBRMCS2-based plasmids. The toolbox was extended by identifying strong endogenous promotors such as P which proved to yield high expression under heterotrophic and autotrophic conditions. H. pseudoflava showed relatively fast heterotrophic growth in complex and minimal medium with sugars and organic acids which allows convenient handling in lab routines. In autotrophic bioreactor cultivations with syngas, H. pseudoflava exhibited a growth rate of 0.06 h and biomass specific uptakes rates of 14.2 ± 0.3 mmol H g h, 73.9 ± 1.8 mmol CO g h, and 31.4 ± 0.3 mmol O g h. As proof of concept, we engineered the carboxydotrophic bacterium for the aerobic production of the C sesquiterpene (E)-α-bisabolene from the C carbon source syngas by heterologous expression of the (E)-α-bisabolene synthase gene agBIS. The resulting strain H. pseudoflava (pOCEx1:agBIS) produced 59 ± 8 μg (E)-α-bisabolene L with a volumetric productivity Q of 1.2 ± 0.2 μg L h and a biomass-specific productivity q of 13.1 ± 0.6 μg g h. The intrinsic properties and the genetic repertoire of H. pseudoflava make this carboxydotrophic bacterium a promising candidate for future aerobic production processes to synthesize more complex or ATP-intensive chemicals from syngas.
气化是一种从生物质或废物流中产生富含能量的合成气（syngas）的合适技术，可用于细菌发酵过程以生产化学品和燃料。现有的微生物工艺目前依赖于产乙酸菌，该菌进行能量效率低下的厌氧 CO 氧化和产乙酸作用，可能会阻碍复杂和需要大量 ATP 的产物的生物合成。由于 CO 的有氧氧化在能量上更为有利，因此我们在本研究中利用革兰氏阴性β变形菌 Hydrogenophaga pseudoflava DSM1084 作为新型宿主，从合成气中生产化学品。我们对 H. pseudoflava 的基因组进行了测序和注释，并建立了遗传工程工具包，该工具包允许通过 pk19mobsacB 系统进行无标记染色体修饰，并在基于 pBBRMCS2 的质粒上进行异源基因表达。该工具包通过鉴定强内源性启动子（如 P）得到了扩展，证明在异养和自养条件下，P 可以产生高表达。H. pseudoflava 在含有糖和有机酸的复杂和最小培养基中表现出相对较快的异养生长，这使其在实验室常规操作中便于处理。在使用合成气的自养生物反应器培养中，H. pseudoflava 的生长速率为 0.06 h，生物质比吸收速率分别为 14.2 ± 0.3 mmol H g h、73.9 ± 1.8 mmol CO g h 和 31.4 ± 0.3 mmol O g h。作为概念验证，我们通过异源表达（E）-α-法尼烯合酶基因 agBIS，对产羧基细菌进行工程改造，使其能够从 C 碳源合成气中有氧生产 C 倍半萜（E）-α-毕澄茄烯。所得菌株 H. pseudoflava（pOCEx1：agBIS）产生了 59 ± 8 μg（E）-α-毕澄茄烯 L，比生产率 Q 为 1.2 ± 0.2 μg L h，生物质比生产率 q 为 13.1 ± 0.6 μg g h。H. pseudoflava 的固有特性和遗传组成使其成为未来有氧生产工艺的有前途的候选者，可用于从合成气中合成更复杂或需要大量 ATP 的化学品。
