Recently, Trends in Biotechnology published an online review of Recent advances in the microbial synthesis of hemoglobin by the team of Academician Chen Jian from Jiangnan University’s Future Food Science Center and School of Bioengineering. Associate researcher Zhao Xinrui is the first author, Professor Zhou Jingwen is the corresponding author, and the authors of the paper also include Professor Chen Jian and Professor Du Guocheng.
Hemoglobin is a type of iron-containing metalloprotein that exists in prokaryotic and eukaryotic cells with heme as a prosthetic group. It has many important functions in the organism, such as transporting and storing oxygen, regulating intracellular pH, and regulating physiological metabolism. . In recent years, hemoglobin has been used in emergency medicine (as a cell-free oxygen carrier), healthcare (as an iron supplement), food processing (food-grade coloring and flavoring) and other fields. However, the acquisition of hemoglobin still needs to rely on extraction from blood or plant tissues. The extraction method is not only time-consuming and inefficient, but also the chemical reagents used can easily cause environmental pollution. Therefore, the use of microbial cell factories to synthesize hemoglobin from different sources has become a research hotspot in recent years.
There are more than 141110 hemoglobin gene coding sequences in NCBI’s GeneBank database, more than 84,424 hemoglobin amino acid sequences in the EMBL protein database, and 725 hemoglobin three-dimensional structures in the PDB protein crystal database. Data, but at this stage only 15 types of hemoglobin from different sources (human, soybean, crocodile, etc.) can be synthesized by 9 limited microorganisms (E. coli, Saccharomyces cerevisiae, Bacillus subtilis, etc.). According to the phylogenetic tree analysis of the amino acid sequence of the existing hemoglobin, it is found that hemoglobin and its subunits from different sources are divided into several categories:animal-derived hemoglobin alpha subunit, beta subunit and other types of subunits, plant-derived single-subunit hemoglobin, Hemoglobin of microbial origin (Figure 1). Therefore, it is possible to comprehensively apply the currently increasingly mature metabolic engineering and synthetic biology strategies to develop an effective and stable microbial synthesis method of hemoglobin to meet the demand for large-scale applications of hemoglobin of different species such as human, soybean, and Vitreoscilla .
Hemoglobin is a prosthetic group necessary for hemoglobin to perform its physiological functions. In order to synthesize hemoglobin efficiently, it is first necessary to increase the supply of intracellular hemoglobin. In nature, the synthesis of heme is mainly through the C4 and C5 pathways (Figure 2). By strengthening the C4 pathway and adding glycine and succinate as substrates or strengthening the C5 pathway against feedback inhibition, the intracellular heme pro-heme can be increased. Body 5-aminolevulinic acid (ALA) content. On this basis, continue to strengthen and modularize the downstream pathway of heme synthesis, and the synthesis of 115.5±2.3mg/L heme can be achieved without adding a substrate in E. coli. In addition, because the excessively high content of heme in the cell can significantly inhibit the growth of bacteria, the use of heme transporter can realize the secretion and synthesis of more than 60%of heme to reduce the toxic effect of heme on cells.
On the basis of increasing the level of hemoglobin supply, the use of E. coli, yeast and other microbial hosts to synthesize hemoglobin from different sources has been successful. Due to the mature metabolic modification and low cost of culture, more than 70%of hemoglobin is currently synthesized by E. coli. First, in order to enhance the solubility of hemoglobin in E. coli, codon optimization, promoter and vector adaptor combinations, and protein solubilization tags have been successfully applied to avoid the formation of inclusion bodies; secondly, to enhance the hemoglobin alpha subunit of animal origin The expression level, the tandem expression of genes encoding α subunits, and the co-expression of stable cofactor AHSP have also been used to improve the stable type of α subunits; in addition, due to the weak synthesis and transport capacity of heme in the E. coli host, it can be Introduce the heme transport system of Pseudomonas shiga and add high concentration of heme outside the cell to increase the intracellular heme level (Figure 3). On the basis of preliminary acquisition of hemoglobin, it can be further extended by combining human hemoglobin α subunit and bovine hemoglobin β subunit to develop a self-polymerized human bovine heterogeneous hemoglobin (180-500kDa polymer) The half-life of commercial cell-free oxygen carrier products in the blood; co-expression of methionine aminopeptidase in the host to accelerate the correct excision of the N-terminal methionine residue of hemoglobin; optimization of inducer concentration, induction temperature and other fermentation conditions to improve hemoglobin Yield.
In addition to E. coli, yeast strains such as Saccharomyces cerevisiae and Pichia pastoris are also efficient platforms for the synthesis of hemoglobin. However, in previous studies, the production of hemoglobin synthesized by Saccharomyces cerevisiae was low. In recent years, the oxygen sensing pathway has been modified by optimizing the expression level of hemoglobin α and β subunits, strengthening the heme synthesis pathway, and knocking out the transcription factor Hap1p ( Figure 3) The highest content of human hemoglobin can reach 7%of the total soluble protein in the cell. Pichia pastoris is currently an efficient host for the production of commercial soy hemoglobin. The obtained soy hemoglobin can be added to new artificial meat products to simulate the color and flavor of real meat. The Pichia strain developed by Impossible Foods, which can synthesize soybean hemoglobin commercially, has obtained FDA approval and has applied for patents in many countries. In this strain, the eight enzymes required for heme synthesis are divided into three modules, and the methanol-induced AOX1 promoter is used for enhanced expression; in addition, two copies of the soybean hemoglobin gene and the transcription activator Mxr1p are also Integrated into the genome; finally, by optimizing high-density fermentation conditions, large-scale industrial production of soybean hemoglobin was achieved (Figure 3).
Although the use of microorganisms to synthesize hemoglobin has been successful, the yield of most hemoglobin is still low, which poses a major challenge to the application of recombinant hemoglobin in production. In future research, new strategies can be used to strengthen the synthesis ability of strains (Figure 4):First, deep learning methods can be used to discover unknown hemoglobin genes with special functions or characteristics, such as clover that is resistant to discoloration at high temperatures. Hemoglobin, etc.; secondly, we should continue to look for the rate-limiting steps in the heme synthesis pathway, and apply strategies such as protein scaffolds to relieve the spatial isolation between these steps; once again, the by-products and heme oxygenation in the intracellular heme synthesis pathway should be inhibited Enzymes degrade heme; finally, because heme has a toxic effect on cells, and the overexpression of enzymes in the heme synthesis pathway will increase the metabolic burden of the host, the heme sensor is used to achieve the difference between heme synthesis and hemoglobin expression The metabolic balance is the key to breaking through the bottleneck of hemoglobin synthesis.
This review was funded by the National Key Research and Development Program (2019YFA0906400), the National Natural Science Foundation of China (31900067), and the “Light Industry Technology and Engineering 1” National First-Class Discipline Project (LITE2018-08).”synthesis of hemoglobin” was published in the journal Trendsin Biotechnology (Trendsin Biotechnology, 2021, 39(3), 286-297, https://www.sciencedirect.com/science/article/abs/pii/S0167779920302262).