Ge Fanglan, Li Xiaokun, Ge Qingrong, Zhu Di, Li Wei, Shi Fenghui, Chen Hongjin
College of Life Sciences, Sichuan Normal University, Chengdu, 610068, People's Republic of China.
Key Laboratory for Utilization and Conservation of Bio-Resources of Education of Education Department of Sichuan Province, Chengdu, People's Republic of China.
AMB Express. 2021 Dec 27;11(1):179. doi: 10.1186/s13568-021-01335-0.
5-aminolevulinic acid (ALA) has broad potential applications in the medical, agricultural and food industries. Several strategies have been implemented successfully to try to improve ALA synthesis. Nonetheless, the low yield has got in the way of large-scale bio-manufacture of 5-ALA. In this study, we explored strain engineering strategies for high-level 5-ALA production in Corynebacterium glutamicum F343 using the C4 pathway. Initially, the glutamate dehydrogenase-encoding gene gdhA was deleted to reduce glutamate yield. Then the C4 pathway was introduced in the gdhA mutant strain F2-A (∆gdhA + hemA), resulting in a 5-ALA yield of up to 3.2 g/L. Furthermore, the accumulations of downstream metabolites such as heme, porphobilinogen, and protoporphyrin IX, were decreased. After evaluating the mechanisms of this synthetic pathway by RNA-Seq, the results showed that genes involved in both the C5 pathway and heme pathways were down-regulated in strain F2-A (∆gdhA + hemA). Interestingly, upstream genes of succinyl-CoA in the tricarboxylic acid (TCA) cycle, such as icd, lpdA, were up-regulated, while its downstream genes, including sucC, sucD, sdhB, sdhA, sdhCD, were down-regulated. These changes amplify the sources of succinyl-CoA and reduce its expenditure, before pulling the carbon flux to produce 5-ALA. Furthermore, the down-regulation of most genes of the heme pathway could reduce the drainage of 5-ALA, which further enhance its accumulation. To alleviate competition between glyoxylate and the TCA cycle, the isocitrate dehydrogenase-encoding gene aceA was also knocked out, resulting in 3.86 g/L of 5-ALA. Finally, the fermentation conditions were optimized, resulting in a maximum 5-ALA yield of 5.6 g/L. Overall, the blocking of the glutamate synthesis pathway could be a powerful strategy to re-allocate the carbon flux to produce 5-ALA. It could also enable the efficient synthesis of other TCA derivatives in C. glutamicum.
5-氨基乙酰丙酸（ALA）在医学、农业和食品工业中具有广泛的潜在应用。人们已经成功实施了几种策略来尝试提高ALA的合成。尽管如此，低产量阻碍了5-ALA的大规模生物制造。在本研究中，我们探索了利用C4途径在谷氨酸棒杆菌F343中高水平生产5-ALA的菌株工程策略。最初，删除了编码谷氨酸脱氢酶的基因gdhA以降低谷氨酸产量。然后在gdhA突变菌株F2-A（∆gdhA + hemA）中引入C4途径，导致5-ALA产量高达3.2 g/L。此外，血红素、胆色素原和原卟啉IX等下游代谢物的积累减少。通过RNA-Seq评估该合成途径的机制后，结果表明，在菌株F2-A（∆gdhA + hemA）中，参与C5途径和血红素途径的基因均下调。有趣的是，三羧酸（TCA）循环中琥珀酰辅酶A的上游基因，如icd、lpdA，上调，而其下游基因，包括sucC、sucD、sdhB、sdhA、sdhCD，下调。这些变化扩大了琥珀酰辅酶A的来源并减少了其消耗，然后拉动碳通量以产生5-ALA。此外，血红素途径中大多数基因的下调可以减少5-ALA的消耗，这进一步增强了其积累。为了缓解乙醛酸和TCA循环之间的竞争，还敲除了编码异柠檬酸脱氢酶的基因aceA，产生了3.86 g/L的5-ALA。最后，对发酵条件进行了优化，5-ALA的最大产量达到5.6 g/L。总体而言，阻断谷氨酸合成途径可能是重新分配碳通量以产生5-ALA的有力策略。它还可以使谷氨酸棒杆菌中其他TCA衍生物的高效合成成为可能。
