Fine-Tuning of hemB Using CRISPRi for Increasing 5-Aminolevulinic Acid Production in Escherichia coli.

Tianyuan Su,Qi Guo,Yi Zheng,Qian Wang,Qingsheng Qi,Quanfeng Liang

doi:10.3389/fmicb.2019.01731

Abstract

5-aminolevulinic acid (5-ALA) is an important metabolic intermediate in the biosynthesis of heme and has been broadly applied in medicine, agriculture, and organic synthesis. Compared to the chemical synthesis methods, microbial fermentation of ALA has significant economic and environmental advantages. However, the heme biosynthesis pathway downstream of ALA is essential for cell survival, so it cannot be completely blocked. In this work, we fine-tuned the expression of HemB, the key enzyme involved in heme biosynthesis, using CRISPR interference (CRISPRi), and investigated its effect on promoting ALA accumulation. The activity of HemB was down-regulated by 15, 19, 33, 36, 71, and 80% respectively, with six CRISPRi sites targeting various regions of hemB. ALA accumulation in these hemB weakened strains varied from 90.2 to 493.1% compared to that of the original strain. This work provided new insights into fine-tuning of heme biosynthesis pathway for promoting ALA production.

Highlights

In Escherichia coli, ALA is natively synthesized via the C5 pathway
We found that ALA production is related to the HemB activity, which makes the higher inhibition of hemB, the higher the ALA accumulation (Figures 4, 5)
CRISPR interference (CRISPRi) was applied to fine tune the expression of hemB for reducing the metabolic flux from ALA to heme and promoting the accumulation of ALA

Summary

Introduction

In Escherichia coli, ALA is natively synthesized via the C5 pathway. ALA biosynthesis through this pathway is tightly regulated by the downstream metabolite heme (Figure 1; Woodard and Dailey, 1995). This is consistent with the report that gltX and hemA are subjected to tightly feedback inhibition by their end product heme (Li et al, 2014). Combinatorial inhibition using 27 arrays containing sgRNAs with different repression capacities targeting the three genes obtained the most efficient strain, BNX122, which produced 20.5 ± 0.85 g/L of GlcNAc with a yield of 0.46 ± 0.010 g/g glucose and xylose in shake flask culture These works demonstrated that CRISPRi is efficient for the fine-tuning of single or multiple genes to a suitable level in the metabolic pathway and promotes the production of valueadded compounds

Methods

Results

Conclusion