Ishchuk Olena P, Frost August T, Muñiz-Paredes Facundo, Matsumoto Saki, Laforge Nathalie, Eriksson Nélida Leiva, Martínez José L, Petranovic Dina
Department of Biology and Biological Engineering, Systems and Synthetic Biology, Chalmers University of Technology, SE41296, Gothenburg, Sweden.
Department of Biotechnology and Biomedicine, Section for Synthetic Biology, Technical University of Denmark, DK-2800, Kgs. Lyngby, Denmark.
Metab Eng. 2021 Jul;66:259-267. doi: 10.1016/j.ymben.2021.05.002. Epub 2021 May 10.
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.
随着输血需求的不断增加，人们越来越多地研究从可持续来源生产人血红蛋白（Hb）。微生物生产是一个有吸引力的选择，因为它可能提供这种蛋白质的廉价、安全和可靠来源。为了提高酿酒酵母生产人血红蛋白的产量，通过几种方法减少了Hb的降解。删除HMX1基因（编码血红素加氧酶）、VPS10基因（编码液泡蛋白酶受体）、PEP4基因（编码液泡蛋白酶A）、ROX1基因（编码缺氧基因的血红素依赖性阻遏物），并过表达HEM3基因（编码胆色素原脱氨酶）和AHSP基因（编码人α-血红蛋白稳定蛋白）——这些变化减少了血红素和Hb的降解，提高了血红素和Hb的产量。胆红素生物传感器验证了血红蛋白降解的减少。在葡萄糖发酵过程中，工程菌株产生的细胞内Hb占酵母总蛋白的18%，这是酵母中报道的人血红蛋白的最高产量。血红蛋白产量的增加伴随着耗氧率的增加和甘油产量的增加，我们推测这是酵母在氧气限制和蛋白质产量增加的条件下为重新平衡其NADH水平而做出的反应。
