Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.
Department of Biological Sciences, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea; Institute for the BioCentury, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.
Metab Eng. 2023 Mar;76:63-74. doi: 10.1016/j.ymben.2023.01.006. Epub 2023 Jan 10.
Cinnamaldehyde (CAD) derived from cinnamon bark has received much attention for its potential as a nematicide and food additive. Previously, we have succeeded in developing an Escherichia coli strain (YHP05) capable of synthesizing cinnamaldehyde; however, the production titer (75 mg/L) was not sufficient for commercialization. Herein, to develop an economical and sustainable production bioprocess, we further engineered the YHP05 strain for non-auxotrophic, antibiotic-free, inducer-free hyperproduction of CAD using systematic metabolic engineering. First, the conversion of trans-cinnamic acid (t-CA) to CAD was improved by the co-expression of carboxylic acid reductase and phosphopantetheinyl transferase (PPTase) genes. Second, to prevent the spontaneous conversion of CAD to cinnamyl alcohol, 10 endogenous reductase and dehydrogenase genes were deleted. Third, all expression cassettes were integrated into the chromosomal DNA using an auto-inducible system for antibiotic- and inducer-free production. Subsequently, to facilitate CAD production, available pools of cofactors (NADPH, CoA, and ATP) were increased, and acetate pathways were deleted. With the final antibiotic-, plasmid-, and inducer-free strain (H-11MPmR), fed-batch cultivations combined with in situ product recovery (ISPR) were performed, and the production titer of CAD as high as 3.8 g/L could be achieved with 49.1 mg/L/h productivity, which is the highest CAD titer ever reported.
肉桂醛（CAD）来源于肉桂树皮，因其作为杀线虫剂和食品添加剂的潜力而备受关注。此前，我们已经成功开发出一种能够合成肉桂醛的大肠杆菌菌株（YHP05）；然而，产量（75mg/L）不足以商业化。在此，为了开发经济可持续的生产工艺，我们进一步通过系统代谢工程对 YHP05 菌株进行非营养缺陷型、无抗生素、无诱导剂的 CAD 超生产工程改造。首先，通过共表达羧酸还原酶和磷酸泛酰巯基乙胺转移酶（PPTase）基因，提高了反式肉桂酸（t-CA）向 CAD 的转化率。其次，为了防止 CAD 自发转化为肉桂醇，删除了 10 个内源还原酶和脱氢酶基因。第三，所有表达盒均使用自动诱导系统整合到染色体 DNA 中，实现无抗生素和诱导剂生产。随后，为了促进 CAD 生产，增加了可用的辅酶（NADPH、CoA 和 ATP）池，并删除了乙酸盐途径。最终获得无抗生素、质粒和诱导剂的菌株（H-11MPmR），并进行分批补料培养和原位产物回收（ISPR），CAD 的产量高达 3.8g/L，生产强度为 49.1mg/L/h，这是迄今为止报道的最高 CAD 产量。
