Sustainable and high-level microbial production of plant hemoglobin in Corynebacterium glutamicum

Abstract

Background Plant hemoglobin shows great potential as a food additive to circumvent the controversy of using animal materials. Microbial fermentation with engineered microorganisms is considered as a promising strategy for sustainable production of hemoglobin. As an endotoxin-free and GRAS (generally regarded as safe) bacterium, Corynebacterium glutamicum is an attractive host for hemoglobin biosynthesis. Results Herein, C. glutamicum was engineered to efficiently produce plant hemoglobin. Hemoglobin genes from different sources including soybean and maize were selected and subjected to codon optimization. Interestingly, some candidates optimized for the codon usage bias of Escherichia coli outperformed those for C. glutamicum regarding the heterologous expression in C. glutamicum. Then, saturated synonymous mutation of the N-terminal coding sequences of hemoglobin genes and fluorescence-based high-throughput screening produced variants with 1.66- to 3.45-fold increase in hemoglobin expression level. To avoid the use of toxic inducers, such as isopropyl-β-d-thiogalactopyranoside, two native inducible expression systems based on food additives propionate and gluconate were developed. Promoter engineering improved the hemoglobin expression level by 2.2- to 12.2-fold. Combination of these strategies and plasmid copy number modification allowed intracellular production of hemoglobin up to approximately 20% of total protein. Transcriptome and proteome analyses of the hemoglobin-producing strain revealed the cellular response to excess hemoglobin accumulation. Several genes were identified as potential targets for further enhancing hemoglobin production. Conclusions In this study, production of plant hemoglobin in C. glutamicum was systematically engineered by combining codon optimization, promoter engineering, plasmid copy number modification, and multi-omics-guided novel target discovery. This study offers useful design principles to genetically engineer C. glutamicum for the production of hemoglobin and other recombinant proteins.

High-level expression of leghemoglobin in Kluyveromyces marxianus by remodeling the heme metabolism pathway

“…Previous reports showed that bovine hemoglobin, deer mouse hemoglobin, and cowpea leghemoglobin II exhibited similar faster mobilities in SDS-PAGE ( Arredondo-Peter et al, 1997 ; Natarajan et al, 2011 ). A recent study observed LBA expressed intracellularly in C. glutamicum exhibiting a migration pattern on SDS-PAGE also below 15 kDa ( Wang et al, 2023 ). The results indicate that LBA exhibits a faster mobility in SDS-PAGE, which might be related to the conformation of LBA.…”

Automated characterization and analysis of expression compatibility between regulatory sequences and metabolic genes in Escherichia coli

“…Due to the challenge of directly measuring intracellular expression levels of target proteins, many studies fused the coding sequence (CDS) of the target gene with a fluorescent protein CDS [ 6 , 22 , 37 ]. This approach allows the fluorescence values of the fusion protein to serve as an indirect reflection of the target protein's expression levels.…”

Biosynthesis of High‐Active Hemoproteins by the Efficient Heme‐SupplyPichia PastorisChassis

“…In the latest study, S‐Hb, maize‐Hb, rice‐Hb, and S. cerevisiae ‐Hb were successfully synthesized in Corynebacterium glutamicum . [ 62 ] Among them, the highest expression of S‐Hb, comprising 20% of the total protein, was achieved using a high‐throughput screening method based on the fusion of Hb with a green fluorescent protein. This method optimized the N‐terminal coding sequence of Hb genes, native inducible promoters, and plasmid copy number.…”

“…glutamicum , could reach 28% by adding 1 g L −1 ALA to the fermentation medium. [ 62 ] However, this heme‐binding ratio is still low, and this approach would increase the culture cost by over 60%. [ 16 ] Therefore, in this study, the rate‐limiting steps catalyzed by Hem2p, Hem3p, and Hem4p were eliminated using protein scaffolds, promoting the transport of their substrates (ALA, PBG, and HMB) and improving the heme titer (90.24%, Figure 4a ).…”