Redirection of metabolic flux in Shewanella oneidensis MR-1 by CRISPRi and modular design for 5-aminolevulinic acid production

Abstract

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.

Keywords

Shewanella oneidensis MR-1 / CRISPRi / 5-Aminolevulinic acid / Metabolic flux / Heme synthesis / C4 pathway / C5 pathway

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Funding

Ministry of Science and Technology, Taiwan(MOST 108-2221-E-006-004-MY3)