Metabolic manipulation through CRISPRi and gene deletion to enhance cadaverine production in Escherichia coli

Abstract

Due to the limiting natural resources, greenhouse effect and global warming crisis, the bio-based chemicals which are environmentally friendly materials have gradually become urgent and important. Cadaverine, a 1,5-diaminopentane (DAP), is widely used as block chemicals for synthesis of biopolymer, which can be produced from lysine by lysine decarboxylase (EC 4.1.1.18) in Escherichia coli. However, the DAP will be further utilized into by-products through downstream genes of speE, puuA, speG and ygjG, which decrease the amount of product. In this study, two approaches including Lambda-Red system for gene knockout, and clustered regularly interspaced short palindromic repeats interference (CRISPRi) for gene knockdown; are explored to manipulate the metabolic flux among 26 genetic E. coli. As a result, CadA driven by inducible T7 promoter accumulated more DAP from CRISPRi targeted on single-gene repressive strains such as BT7AiE, BT7AiP, BT7AiG and BT7AiY. The highest DAP titer and productivity was obtained to 38 g/L and 2.67 g/L/h in BT7AiY (repression of ygjG). We also investigated the co-factor pyridoxal 5'-phosphate (PLP) effect on lysine consumption and DAP production from different E. coli derivatives. In contrast to CRISPRi-mediated strains, 4 genes knockout strain (BT7AdEPGY) deal with 98% lysine consumption and achieved 37.45 g/L DAP and 3.17 g/L/h DAP productivity. The metabolic regulation by CRISPRi is a simple strategy and the results are consistent with gene knockout to manipulate the pathway for DAP production.

Keywords: CRISPRi; Cadaverine; Escherichia coli; Gene knockout; Metabolic pathway.