Construction of a genome-engineered stable 5-aminolevulinic acid producing Corynebacterium glutamicum by increasing succinyl-CoA supply

Abstract

5-Aminolevulinic acid (5-ALA), a versatile precursor for tetrapyrrole derivatives (such as heme, chlorophyll, and cobalamin), drives advancing microbial cell factories to meet growing biomedical and industrial demands. However, there remain two challenges that limit yield and scalability: the limitations of conventional plasmid-based gene expression systems and the lack of fine regulation of succinyl-CoA. In this study, to address these limitations, we integrated multiple copies of hemA ^C132A of the heterologous C4 pathway on the genome. For fine regulating the supply of succinyl-CoA, the genes related to the tricarboxylic acid cycle (TCA cycle) oxidation branch pathway were combinatorially screened. The optimal combination of icd and lpd was confirmed by ribosome binding site (RBS) engineering, which was integrated on the genome with optimized expression intensity. Succinyl-CoA supply was further increased by genome integration and expression optimization of key CoA biosynthetic gene coaA, pantothenic acid synthesis-related gene panB-panC, and β-alanine synthesis-related gene panD. The optimized genomically stable chassis achieved a high 5-ALA production of 6.38 ± 0.16 g/L, which was 8.63-fold higher than the single hemA ^C132A copy strain A1 (0.74 ± 0.07 g/L). From these findings, a stable and high-yield 5-ALA synthetic strain was successfully constructed, providing a new strategy for production of biochemicals derived from succinyl-CoA in C. glutamicum.

Keywords: 5-Aminolevulinic acid; C4 pathway; Corynebacterium glutamicum; Succinyl-CoA.

Fig. 1

Development of genome-integrated C4 ALA production system in C. glutamicum and evaluation of glycine supplementation concentrations. (A) The 5-ALA production in strains with different hemA^C132A copy numbers. (B) The growth of strains with different hemA^C132A copies numbers. (C) Effect of different concentrations of glycine on the synthesis of 5-ALA. The data represent the averages of three replicates with the associated standard deviations. ANOVA of the 5-ALA titer was used for statistical analysis by SPSSAU (https://spssau.com/About_spssau.html) (∗p < 0.05, ∗∗p < 0.01).

Fig. 2

Schematic representation of 5-ALA production from glucose in C. glutamicum via the C4 pathway. Arrows in blue represent overexpressed. The × in red represent gene was knockout. Error bars represent standard error of the mean (SEM) from three independent replicates.

Fig. 3

Co-expression of icd and lpd increases 5-ALA production. (A) Rational combination of icd and lpd genetic elements for optimized C4 metabolic flux. (B) Effects of overexpression of icd and lpd in different combinations on 5-ALA synthesis, A12 was obtained by introducing plasmid pXMJ19 into A4. (C) Effects of different promoters in the genome manipulating icd-lpd operon expression on 5-ALA synthesis. (D) Strategies for enhancing icd-lpd operons in the genome. (E) Growth of strains in which different promoters in the genome manipulate icd-lpd operon expression. The data represent the averages of three replicates with the associated standard deviations. Student's t-test of the growth rate was used for statistical analysis by SPSSAU (https://spssau.com/About_spssau.html) (∗p < 0.05, ∗∗p < 0.01).

Fig. 4

Effects of the combinatorial optimization of CoA pathway genes. (A) Effects of single overexpression of CoA synthesis-related genes and combination of overexpression of CoA synthesis-related genes on 5-ALA production. (B) In the C4 pathway for the synthesis of 5-ALA, CoA is recycled. The data represent the averages of three replicates with the associated standard deviations. Student's t-test of the growth rate was used for statistical analysis by SPSSAU (https://spssau.com/About_spssau.html) (∗p < 0.05, ∗∗p < 0.01).