Institute of Synthetic Biology, Biomedical Center, Guangdong Province Key Laboratory of Improved Variety Reproduction in Aquatic Economic Animals and South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, People's Republic of China.
Biotechnology Research Centre, School of Life Science, Sun Yat-Sen University, Guangzhou, 510275, People's Republic of China.
J Ind Microbiol Biotechnol. 2018 Feb;45(2):123-139. doi: 10.1007/s10295-018-2003-y. Epub 2018 Jan 17.
Putrescine is widely used in the industrial production of bioplastics, pharmaceuticals, agrochemicals, and surfactants. Because the highest titer of putrescine is much lower than that of its precursor L-ornithine reported in microorganisms to date, further work is needed to increase putrescine production in Corynebacterium glutamicum. We first compared 7 ornithine decarboxylase genes and found that the Enterobacter cloacae ornithine decarboxylase gene speC1 was most suitable for putrescine production in C. glutamicum. Increasing NADPH availability and blocking putrescine oxidation and acetylation were chosen as targets for metabolic engineering. The putrescine producer C. glutamicum PUT4 was first constructed by deleting puo, butA and snaA genes, and replacing the fabG gene with E. cloacae speC1. After adaptive evolution with C. glutamicum PUT4, the evolved strain C. glutamicum PUT-ALE, which produced an 96% higher amount of putrescine compared to the parent strain, was obtained. The whole genome resequencing indicates that the SNPs located in the odhA coding region may be associated with putrescine production. The comparative proteomic analysis reveals that the pentose phosphate and anaplerotic pathway, the glyoxylate cycle, and the ornithine biosynthetic pathway were upregulated in the evolved strain C. glutamicum PUT-ALE. The aspartate family, aromatic, and branched chain amino acid and fatty acid biosynthetic pathways were also observed to be downregulated in C. glutamicum PUT-ALE. Reducing OdhA activity by replacing the odhA native start codon GTG with TTG and overexpression of cgmA or pyc458 further improved putrescine production. Repressing the carB, ilvH, ilvB and aroE expression via CRISPRi also increased putrescine production by 5, 9, 16 and 19%, respectively.
腐胺广泛应用于生物塑料、制药、农用化学品和表面活性剂的工业生产。由于迄今为止在微生物中报道的腐胺的最高效价远低于其前体 L-鸟氨酸，因此需要进一步工作来提高谷氨酸棒杆菌中的腐胺产量。我们首先比较了 7 种鸟氨酸脱羧酶基因，发现肠杆菌科克雷伯氏菌的鸟氨酸脱羧酶基因 speC1 最适合在谷氨酸棒杆菌中生产腐胺。增加 NADPH 的可用性并阻断腐胺的氧化和乙酰化被选为代谢工程的目标。首先通过删除 puo、butA 和 snaA 基因，并将 fabG 基因替换为 E. cloacae speC1 构建腐胺生产菌 C. glutamicum PUT4。在 C. glutamicum PUT4 进行适应性进化后，获得了与亲本菌株相比产腐胺量提高了 96%的进化菌株 C. glutamicum PUT-ALE。全基因组重测序表明，位于 odhA 编码区的 SNPs 可能与腐胺的产生有关。比较蛋白质组学分析表明，戊糖磷酸和氨甲酰磷酸途径、乙醛酸循环和鸟氨酸生物合成途径在进化菌株 C. glutamicum PUT-ALE 中上调。天冬氨酸家族、芳香族、支链氨基酸和脂肪酸生物合成途径在 C. glutamicum PUT-ALE 中也观察到下调。通过将 odhA 天然起始密码子 GTG 替换为 TTG 来降低 OdhA 活性，并过表达 cgmA 或 pyc458 进一步提高腐胺产量。通过 CRISPRi 抑制 carB、ilvH、ilvB 和 aroE 的表达也分别使腐胺产量提高了 5%、9%、16%和 19%。
