Stable and Efficient Biosynthesis of 5-Aminolevulinic Acid Using Plasmid-Free Escherichia coli

Abstract

5-Aminolevulinic acid (5-ALA) is a key metabolic intermediate of the heme biosynthesis pathway, which has broad application prospects in agriculture and medicine. However, segregational instability of plasmid-based expression systems and low yield have hampered large-scale manufacture of 5-ALA. In this study, two important genes of the 5-ALA C5 biosynthesis pathway, hemA and hemL, were integrated into Escherichia coli MG1655 for chemically induced chromosomal evolution (CIChE). The highest hemA and hemL copy-number, 98 per genome, was obtained in CIChE strain MG136. The 5-ALA titer of this strain reached 2724 mg/L in optimized condition. Then, after undergoing adaptative evolution and the deletion of recA, strain MG136a ΔrecA::FRT could stably produce 4550 mg/L 5-ALA from glucose, 450 times the amount produced by hemA-hemL single copy strain MG1655-hemAL. This study constructed a plasmid-free E. coli strain for 5-ALA production, which will provide the basis for further manipulation of metabolic regulation and optimization of fermentation.

Strain engineering for high‐level 5‐aminolevulinic acid production in Escherichia coli

“…As presented in this study, our strategies in engineering the TCA cycle for improved succinyl‐CoA production generated higher levels of 5‐ALA (via Shemin pathway) compared to previous strategies in which carbon flux was directed toward α‐ketoglutarate precursor for 5‐ALA biosynthesis via the C5 pathway (Noh, Lim, Park, Seo, & Jung, 2017). While similar studies generated comparable 5‐ALA titers (Cui et al, 2019; Yu, Yi, Shih, & Ng, 2019; Zhang et al, 2019), our study showed higher 5‐ALA yields particularly when no structurally related carbons were supplemented. Future strain engineering for 5‐ALA biosynthesis via the Shemin pathway could include tuning of carbon flux via the glyoxylate shunt by manipulation of aceA expression which showed physiological advantages over iclR inactivation (Noh et al, 2017).…”

Tuning the Binding Affinity of Heme-Responsive Biosensor for Precise and Dynamic Pathway Regulation

“…To address this issue, we employed multi-copy integrated hemA/hemL strains that can provide different in vivo heme concentration (Cui et al, 2019). The positive correlation between the green fluorescence intensity and the in vivo heme concentration was observed and guaranteed the characterization of the regulatory system (Figure 1B).…”

“…To evaluate the heme-responsive biosensor, the hrtO-hybrid trc promoter was placed upstream of gfp under the control of HrtR, resulting in plasmid P1. A recombinant E. coli strain containing different copy numbers of glutamyl-tRNA reductase gene (hemA) and glutamate-1-semialdehyde aminotransferase gene (hemL) on the genome was employed to achieve different intracellular heme accumulation (Cui et al, 2019). Strains S1, S20, S35, S65, and S100 represented 1, 20, 35, 65, and 100 copies of hemA/hemL integrated on the genome.…”

Metabolic engineering of microorganisms for the production of multifunctional non‐protein amino acids: γ‐aminobutyric acid and δ‐aminolevulinic acid

“…Initially, efforts for increasing the yields focused on natural producers and usually performed through random mutagenesis and optimizing fermentation conditions. Recently, remarkable efforts have been made to improve the yield of biosynthetic GABA and ALA, through natural or engineered strains (Choi et al ., 2015 ; Cui et al ., 2019 ; Zhang et al ., 2019 ; Zhang et al ., 2020 ). We screened the development in recent five years from the representative and authoritative journals, and summarized their metabolic engineering strategies in detail, which include every metabolic engineering step and its effectiveness, as well as the synthetic pathway, substrate, final titre, biomass, fermentation time, form and scale, as well as the calculated yields based on the substrate (Tables 1 and 2 ).…”