Metabolic Engineering Of Saccharomyces Cerevisiae For Efficient Synthesis Of Hemoglobins And Myoglobins

Both hemoglobin（Hb）and myoglobin（Mb）are heme-binding proteins composed of heme and globin,which are closely associated with the formation of meat color and the metallic taste of meat,and can be used as color additives in plant-bassed and cell-based atifical meat alternative recently.In higher mammals,Hb is a tetramer metallic protein consisting of two pairs ofαand twoβsubunits,each globin containing a heme as a prosthetic group.In plants,Hb exists as a monomer.In contrast,Mb exists mainly as monomers in different muscle tissues.With the focus on artificial meat products,the global demand for Hb and Mb continues to increase.At present,Hb and Mb are mainly extracted from blood by chromatography methods.However,traditional extraction methods are limited by the problems of complex process,high extraction cost makes,or low raw materials in nature.Comparatively,microbial fermentation producing Hb and Mb from cheap substrates,with the advantages of short growth cycle,low production cost,and green environment protection,is an important way to meet market demand in the furture.Herein,we enginnered GRAS Saccharomyces cerevisiae CEN.PK2-1C strain as a chassis for the synthesis of hemoglobin and myoglobin by reconstructing the heme synthetic pathway.The engineered strain is optimized via metabolic engineering strategies,such as subcellular localization,relieving rate-limiting steps,inhibiting degradation and protein secretion engineering.The main research results are as follows:（1）The food-grade Saccharomyces cerevisiae chassis for the intracellular high-efficiency expression of hemoglobin and myoglobin were contructed by using expression elements to establish an inducible expression strategy,screening the most suitable inducible GAL system,co-expressing the hemoglobinαsubunit stabilizing protein（AHSP）to promote the stability of protein translation and folding.In shake flask of fermentation conditions,the titer of soybean hemoglobin was 124.3 mg/L,the titer of clover hemoglobin was 20.7 mg/L,the titer of porcine myoglobin was 100.4 mg/L,the titer of bovine myoglobin was 78.9 mg/L,the titer of porcine hemoglobin was 15.2 mg/L,and the titer of bovine hemoglobin was 14.5 mg/L.（2）The spatial barrier between mitochondria and cytoplasm in the heme synthesis pathway was removed by subcellular colocalization in Saccharomyces cerevisiae.It was determined that Hem2p,Hem3p,Hem4p,Hem12p,and Hem13p were located in the cytoplasm,and Hem1p,Hem14p,and Hem15p were located in the mitochondria,and the mitochondrial localization signal MLS_Hem1,MLS_Hem14,and MLS_Hem15 were verified by truncating,laying the foundation for the spatial reconstruction of the heme synthesis pathway from mitochondria to cytoplasm.（3）The intracellular supply of heme can be improved by modularizing the engineering strategy of regulating endogenous heme synthesis.The best C4 pathway for synthesizing heme precursor ALA was screened,and multi-copy integration of HEM1 gene in the genome stably to provide ALA.The mitochondrial localization signal of Hem14p and Hem15p were removed to localize in cytoplasm with Hem13p for multi-enzyme cascade reaction.The combined metabolic engineering was used to achieve 0.3 mg/L intracellular heme,and promotes thet expression of higher active hemoglobin and myoglobin.（4）By screening secretory signal peptides,α-factor signal peptides can be used to achieve the extracellular expression of monosubunit hemoprotein.According to the mechanism of N-glycosylation modification in Saccharomyces cerevisiae,the glycosylation modification in the secretion process was resolved by knockouting out glycosyltransferase.The unmodified soybean hemoglobin was obtained after knocking out ALG12,and the titer of s Hb was 8.1 mg/L increased by about 24.9%.This result suggested that the ALG12（α-1,6-mannose-transferase）is a key factor limiting soybean hemoglobin production in the secretion process.