Systems metabolic engineering of Escherichia coli for hyper-production of 5‑aminolevulinic acid

Abstract

Background 5-Aminolevulinic acid (5-ALA) is a promising biostimulant, feed nutrient, and photodynamic drug with wide applications in modern agriculture and therapy. Although microbial production of 5-ALA has been improved realized by using metabolic engineering strategies during the past few years, there is still a gap between the present production level and the requirement of industrialization. Results In this study, pathway, protein, and cellular engineering strategies were systematically employed to construct an industrially competitive 5-ALA producing Escherichia coli. Pathways involved in precursor supply and product degradation were regulated by gene overexpression and synthetic sRNA-based repression to channel metabolic flux to 5-ALA biosynthesis. 5-ALA synthase was rationally engineered to release the inhibition of heme and improve the catalytic activity. 5-ALA transport and antioxidant defense systems were targeted to enhance cellular tolerance to intra- and extra-cellular 5-ALA. The final engineered strain produced 30.7 g/L of 5-ALA in bioreactors with a productivity of 1.02 g/L/h and a yield of 0.532 mol/mol glucose, represent a new record of 5-ALA bioproduction. Conclusions An industrially competitive 5-ALA producing E. coli strain was constructed with the metabolic engineering strategies at multiple layers (protein, pathway, and cellular engineering), and the strategies here can be useful for developing industrial-strength strains for biomanufacturing.

Design of a Genetically Encoded Biosensor for High-Throughput Screening and Engineering 5-Aminolevulinic Acid Hyper-Producing Escherichia coli

“…Then, the optimized biosensor was used for subsequent HTS to achieve improved production of ALA. Based on this, combined with traditional metabolic engineering strategies, the final titer of ALA reached 58.54 g/L, with a productivity of 1.58 g/L/h. It exceeded the reported highest titer (30.7 g/L) and productivity (1.02 g/L/h) of ALA by microbial production . It shows the advantages of irrational design combined with HTS and significantly improves the production of ALA through E.…”

“…Subsequently, the ppc (encoding phosphoenolpyruvate carboxylase), coaA (encoding pantothenate kinase), and grxA (encoding reduced glutaredoxin 1) genes were overexpressed on the plasmid of hemA*, generating the PCG-hemA* plasmid. The overexpression of the ppc gene enhances the supply of oxaloacetate and introduces more carbon flux into the TCA cycle; the coaA gene catalyzes the initiation of pantothenic acid phosphorylation, and the increase of its expression can promote the biosynthesis from VB 5 to CoA; and the overexpression of the grxA gene is involved in the oxidative damage repair process of strains, and its overexpression can enhance the tolerance of strains to ALA. 9 The DM16 strain harboring PCG-hemA* was named PCG, and its ALA production increased by 13.41% compared to the DM16-hemA* strain, reaching 10.51 ± 0.29 g/L (Figure 6b). To enhance the efflux of ALA and weaken the ALA downstream pathway, the gene rhtA and the hemB antisense RNA (asRNA-hemB) were expressed in the PCG strain, generating PCG-rhtA and PCG-ashemB strains.…”

“…The expression levels of antioxidative-stress-related genes ( yfcF , sodA , katE , htpG , groL ) were also upregulated in DM16 (Figure c). According to previous studies, strengthening the ability of strains to withstand antioxidative stress is conducive to increasing the tolerance of strains to ALA, which will improve the growth of strains and increase the ALA production. , Then, the intracellular ROS levels of DM16 were determined during fermentation. It was found that DM16 had lower intracellular ROS levels than DH5α throughout the ALA fermentation (Figure b).…”

“…It exceeded the reported highest titer (30.7 g/L) and productivity (1.02 g/L/h) of ALA by microbial production. 9 It shows the advantages of irrational design combined with HTS and significantly improves the production of ALA through E. coli.…”

“…So far, many studies have been devoted to the modification of microorganisms through traditional metabolic engineering strategies to improve their ALA production, such as screening of ALA synthase, optimization of metabolic pathways, strengthening the tolerance of strains, etc. − However, further rational strategies are confronted with challenges for efficiently achieving higher ALA production. Evolutionary engineering is a highly efficient strategy for strain development and pathway gene optimization through the creation of various mutant libraries. , While the large number of diversified libraries highly desire the development of effective high-throughput screening (HTS) technology to enable rapid screening of superior performers.…”