Luna-Flores Carlos H, Stowers Chris C, Cox Brad M, Nielsen Lars K, Marcellin Esteban
1Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane, QLD 4072 Australia.
3BioEngineering and Bioprocessing R&D, Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268 USA.
Biotechnol Biofuels. 2018 Aug 13;11:224. doi: 10.1186/s13068-018-1222-9. eCollection 2018.
Propionic acid (PA) is used as a food preservative and increasingly, as a precursor for the synthesis of monomers. PA is produced mainly through hydrocarboxylation of ethylene, also known as the 'oxo-process'; however, species are promising biological PA producers natively producing PA as their main fermentation product. However, for fermentation to be competitive, a PA yield of at least 0.6 g/g is required.
A new strain able to reach the required yield was obtained using genome shuffling. To gain insight into the changes leading to the improved phenotype, the genome of the new strain was sequenced, and metabolomics and transcriptomics data were obtained. In combination, the data revealed three key mutations: (i) a mutation in the promoter region of a sugar transporter which enables an increase in the uptake rate of sucrose; (ii) a mutation in a polar amino acid transporter which improves consumption of amino acids and acid tolerance; and (iii) a mutation in a gene annotated as a cytochrome C biogenesis gene, which is likely responsible for the coupling of the Wood-Werkman cycle to ATP production were responsible for the phenotype. The bioprocess was further enhanced with a feeding strategy that achieved 70 g/L of product. The proposed bioprocess is expected to outperform the economics of the current 'oxo-process' by 2020.
In this study, using genome shuffling, we obtained a strain capable of producing PA exceeding the commercial needs. The multiomics comparison between the novel strain and the wild type revealed overexpression of amino acid pathways, changes in sucrose transporters and an increased activity in the methylglyoxal and the glucuronate interconversion pathways. The analysis also suggests that a mutation in the cytochrome C biogenesis gene, coupled with ATP production through the Wood-Werkman cycle, may be responsible for the increased PA production.
丙酸（PA）用作食品防腐剂，并且越来越多地用作单体合成的前体。PA主要通过乙烯的氢羧化反应生产，也称为“羰基合成法”；然而，一些物种是有前景的生物PA生产者，它们天然地将PA作为主要发酵产物生产。然而，为了使发酵具有竞争力，需要至少0.6 g/g 的PA产量。
使用基因组改组获得了一种能够达到所需产量的新菌株。为了深入了解导致表型改善的变化，对新菌株的基因组进行了测序，并获得了代谢组学和转录组学数据。综合这些数据揭示了三个关键突变：（i）糖转运蛋白启动子区域的一个突变，其能够提高蔗糖的摄取速率；（ii）极性氨基酸转运蛋白的一个突变，其改善了氨基酸的消耗和耐酸性；（iii）一个注释为细胞色素C生物合成基因的基因中的一个突变，其可能负责伍德-韦克曼循环与ATP产生的偶联，这些突变导致了该表型。通过一种补料策略进一步提高了生物过程水平，该策略实现了70 g/L的产物产量。预计到2020年，所提出的生物过程在经济性上将优于当前的“羰基合成法”。
在本研究中，使用基因组改组，我们获得了一种能够生产超过商业需求的PA的菌株。新菌株与野生型之间的多组学比较揭示了氨基酸途径的过表达、蔗糖转运蛋白的变化以及甲基乙二醛和葡萄糖醛酸相互转化途径中活性的增加。分析还表明，细胞色素C生物合成基因中的一个突变，与通过伍德-韦克曼循环产生ATP相结合，可能是PA产量增加的原因。
