Improved production of human hemoglobin in yeast by engineering hemoglobin degradation

Olena P Ishchuk,August T Frost,Facundo Muñiz-Paredes,Saki Matsumoto,Nathalie Laforge,Nélida Leiva Eriksson,José L Martínez,Dina Petranovic

doi:10.1016/j.ymben.2021.05.002

Abstract

With the increasing demand for blood transfusions, the production of human hemoglobin (Hb) from sustainable sources is increasingly studied. Microbial production is an attractive option, as it may provide a cheap, safe, and reliable source of this protein. To increase the production of human hemoglobin by the yeast Saccharomyces cerevisiae, the degradation of Hb was reduced through several approaches. The deletion of the genes HMX1 (encoding heme oxygenase), VPS10 (encoding receptor for vacuolar proteases), PEP4 (encoding vacuolar proteinase A), ROX1 (encoding heme-dependent repressor of hypoxic genes) and the overexpression of the HEM3 (encoding porphobilinogen deaminase) and the AHSP (encoding human alpha-hemoglobin-stabilizing protein) genes — these changes reduced heme and Hb degradation and improved heme and Hb production. The reduced hemoglobin degradation was validated by a bilirubin biosensor. During glucose fermentation, the engineered strains produced 18% of intracellular Hb relative to the total yeast protein, which is the highest production of human hemoglobin reported in yeast. This increased hemoglobin production was accompanied with an increased oxygen consumption rate and an increased glycerol yield, which (we speculate) is the yeast's response to rebalance its NADH levels under conditions of oxygen limitation and increased protein-production.

Highlights

Hemoglobin (Hb) is a major blood-protein, whose main function is oxygen transport
HEM13 gene by Rox1; iii) deleted the HMX1 gene responsible for specific heme cleavage; iv) overexpressed human AHSP gene for increased stability of the α-globin; v) deleted the VPS10 and PEP4 genes for reducing targeting of hemoglobin to the vacuoles for protein degradation. Among these 6 mutations we introduced, the modification of expression the genes ROX1, HMX1, VPS10 and AHSP were not used before for hemoglobin production in S. cerevisiae
Improving heme biosynthesis and reducing heme and hemoglobin degradation increased the yield of human hemoglobin

Summary

Introduction

In humans, during erythropoiesis in the bone marrow, megakaryocyte/erythroid progenitor cells (MEP) produce Hb that is carried by differentiated erythrocytes (red blood cells, RBCs) in blood circulation (Xavier-Ferrucio and Krause, 2018). Hb is the cofactor-containing tetrameric protein that is composed of two α- and two β-globin subunits (α2β2) in adults. Each subunit carries one heme b (protoporphyrin IX) group with a ferrous iron atom ligated by the four nitrogen atoms at the center of the porphyrin ring (Bruice, 2004). While the iron atom acts as an active site for oxygen binding, the organic component of Hb contributes to the regulation of the Hb activity. The organic component ensures the reversibility of the oxygen binding to Hb (Nagai et al, 1985). Human Hb is needed for blood transfusions and for artificial blood, but in recent years donated blood has had shortages, because of decreasing rates of donation

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Conclusion