Heme biosensor-guided in vivo pathway optimization and directed evolution for efficient biosynthesis of heme

Abstract

Background Heme has attracted much attention because of its wide applications in medicine and food. The products of genes hemBCDEFY convert 5-aminolevulinic acid to protoporphyrin IX (PPIX; the immediate precursor of heme); protoporphyrin ferrochelatase (FECH) inserts Fe2+ into PPIX to generate heme. Biosynthesis of heme is limited by the need for optimized expression levels of multiple genes, complex regulatory mechanisms, and low enzymatic activity; these problems need to be overcome in metabolic engineering to improve heme synthesis. Results We report a heme biosensor-guided screening strategy using the heme-responsive protein HrtR to regulate tcR expression in Escherichia coli, providing a quantifiable link between the intracellular heme concentration and cell survival in selective conditions (i.e., the presence of tetracycline). This system was used for rapid enrichment screening of heme-producing strains from a library with random ribosome binding site (RBS) variants and from a FECH mutant library. Through up to four rounds of iterative evolution, strains with optimal RBS intensities for the combination of hemBCDEFY were screened; we obtained a PPIX titer of 160.8 mg/L, the highest yield yet reported in shaken-flask fermentation. A high-activity FECH variant was obtained from the saturation mutagenesis library. Fed-batch fermentation of strain SH20C, harboring the optimized hemBCDEFY and the FECH mutant, produced 127.6 mg/L of heme. Conclusion We sequentially improved the multigene biosynthesis pathway of PPIX and performed in vivo directed evolution of FECH, based on a heme biosensor, which demonstrated the effectiveness of the heme biosensor-based pathway optimization strategy and broadens our understanding of the mechanism of heme synthesis.

Transcriptome Profiling Provides Insights into the Early Development of Tiller Buds in High- and Low-Tillering Orchardgrass Genotypes

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Development of a growth-coupled selection platform for directed evolution of heme biosynthetic enzymes in Corynebacterium glutamicum

“…By using the tetracycline efflux protein TcR as a reporter, the intracellular heme concentration was coupled with the cellular resistance to tetracycline, which decided the cell growth rate under appropriate tetracycline stress. This biosensing system was also successfully applied to directed evolution of ferrochelatase from B. subtilis , and 14 mutants with increased cellular resistance to tetracycline as well as a high-activity variant with 2.04-fold higher heme production were obtained from a saturation mutagenesis library ( Zhang et al, 2023 ).…”