Yi Ying-Chen, Ng I-Son
Department of Chemical Engineering, National Cheng Kung University, Tainan, 70101, Taiwan.
Bioresour Bioprocess. 2021 Feb 7;8(1):13. doi: 10.1186/s40643-021-00366-6.
Programming non-canonical organisms is more attractive due to the prospect of high-value chemical production. Among all, Shewanella oneidensis MR-1 possesses outstanding heme synthesis ability and is well-known for electron transfer, thus has high potential in microbial fuel cell and bioremediation. However, heme, as the final product of C4 and C5 pathways, is regulated by heme cluster for the high-value 5-aminolevulinic acid (ALA) for cancer photodynamic therapy, which has never been explored in MR-1. Herein, the heme metabolism in MR-1 was firstly optimized for ALA production. We applied CRISPR interference (CRISPRi) targeted on the genes to fine-tune carbon flux in TCA cycle and redirected the carbon out-flux from heme, leading to a significant change in the amino acid profiles, while downregulation of the essential hemB showed a 2-fold increasing ALA production via the C5 pathway. In contrast, the modular design including of glucokinase, GroELS chaperone, and ALA synthase from Rhodobacter capsulatus enhanced ALA production markedly in the C4 pathway. By integrating gene cluster under dual T7 promoters, we obtained a new strain M::TRG, which significantly improved ALA production by 145-fold. We rewired the metabolic flux of MR-1 through this modular design and successfully produced the high-value ALA compound at the first time.
由于有望实现高价值化学品的生产，对非标准生物体进行编程更具吸引力。其中，嗜水气单胞菌MR-1具有出色的血红素合成能力，并以电子转移而闻名，因此在微生物燃料电池和生物修复方面具有很高的潜力。然而，血红素作为C4和C5途径的最终产物，受血红素簇调控以生成用于癌症光动力疗法的高价值5-氨基乙酰丙酸（ALA），而这在MR-1中从未被探索过。在此，首先对MR-1中的血红素代谢进行优化以生产ALA。我们应用靶向基因的CRISPR干扰（CRISPRi）来微调三羧酸循环中的碳通量，并将碳流出从血红素重新导向，导致氨基酸谱发生显著变化，而必需的hemB的下调显示通过C5途径的ALA产量增加了2倍。相比之下，包括来自荚膜红细菌的葡萄糖激酶、GroELS伴侣蛋白和ALA合酶的模块化设计在C4途径中显著提高了ALA产量。通过在双T7启动子下整合基因簇，我们获得了一个新菌株M::TRG，其将ALA产量显著提高了145倍。我们通过这种模块化设计重新调整了MR-1的代谢通量，并首次成功生产出高价值的ALA化合物。
