Improved production of human hemoglobin in yeast by engineering hemoglobin degradation

Ishchuk, Olena P.; Frost, August T.; Muñiz-Paredes, Facundo; Matsumoto, Saki; Laforge, Nathalie; Eriksson, Nélida Leiva; Martínez, José Luis; Petranović, Dina

doi:10.1016/j.ymben.2021.05.002

Cited by 33 publications

(20 citation statements)

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“…Note that hemoglobin folds with heme as a prosthetic group, which requires balancing of heme biosynthesis and its recombinant protein production (Fig. 4a ) 35 . We generated eight specific models to simulate the maximum recombinant protein secretion under various growth rates.…”

Section: Resultsmentioning

confidence: 99%

Improving recombinant protein production by yeast through genome-scale modeling using proteome constraints

et al. 2022

Nat Commun

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Eukaryotic cells are used as cell factories to produce and secrete multitudes of recombinant pharmaceutical proteins, including several of the current top-selling drugs. Due to the essential role and complexity of the secretory pathway, improvement for recombinant protein production through metabolic engineering has traditionally been relatively ad-hoc; and a more systematic approach is required to generate novel design principles. Here, we present the proteome-constrained genome-scale protein secretory model of yeast Saccharomyces cerevisiae (pcSecYeast), which enables us to simulate and explain phenotypes caused by limited secretory capacity. We further apply the pcSecYeast model to predict overexpression targets for the production of several recombinant proteins. We experimentally validate many of the predicted targets for α-amylase production to demonstrate pcSecYeast application as a computational tool in guiding yeast engineering and improving recombinant protein production.

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Section: Resultsmentioning

confidence: 99%

Improving recombinant protein production by yeast through genome-scale modeling using proteome constraints

et al. 2022

Nat Commun

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“…The combination of deleting proteinase A and proteinase B for improved recombinant protein production has been reported for several microbial hosts, including S. cerevisiae [25,38,39]. Removal of solely PEP4 has however also proven effective for production of several proteins [25,41,42].…”

Section: Discussionmentioning

confidence: 99%

Engineering Saccharomyces cerevisiae for the production and secretion of Affibody molecules

et al. 2022

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Background Affibody molecules are synthetic peptides with a variety of therapeutic and diagnostic applications. To date, Affibody molecules have mainly been produced by the bacterial production host Escherichia coli. There is an interest in exploring alternative production hosts to identify potential improvements in terms of yield, ease of production and purification advantages. In this study, we evaluated the feasibility of Saccharomyces cerevisiae as a production chassis for this group of proteins. Results We examined the production of three different Affibody molecules in S. cerevisiae and found that these Affibody molecules were partially degraded. An albumin-binding domain, which may be attached to the Affibody molecules to increase their half-life, was identified to be a substrate for several S. cerevisiae proteases. We tested the removal of three vacuolar proteases, proteinase A, proteinase B and carboxypeptidase Y. Removal of one of these, proteinase A, resulted in intact secretion of one of the targeted Affibody molecules. Removal of either or both of the two additional proteases, carboxypeptidase Y and proteinase B, resulted in intact secretion of the two remaining Affibody molecules. The produced Affibody molecules were verified to bind their target, human HER3, as potently as the corresponding molecules produced in E. coli in an in vitro surface-plasmon resonance binding assay. Finally, we performed a fed-batch fermentation with one of the engineered protease-deficient S. cerevisiae strains and achieved a protein titer of 530 mg Affibody molecule/L. Conclusion This study shows that engineered S. cerevisiae has a great potential as a production host for recombinant Affibody molecules, reaching a high titer, and for proteins where endotoxin removal could be challenging, the use of S. cerevisiae obviates the need for endotoxin removal from protein produced in E. coli.

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“…Nowadays, the biosynthesis of hemoglobin has been developed as a feasible option by the advancements in synthetic biology [21]. Although mammalian hemoglobins have been successfully synthesized in Escherichia coli [22,23] and Saccharomyces cerevisiae [24][25][26], the challenges of low expressional level and complex process of purification still limited the large-scale production. Engineering the degradation pathway of heterologous proteins in S. cerevisiae increased the production of human hemoglobin to about 18% of the total cellular protein.…”

Section: Introductionmentioning

confidence: 99%

“…Engineering the degradation pathway of heterologous proteins in S. cerevisiae increased the production of human hemoglobin to about 18% of the total cellular protein. However, when fused with α-Factor signal peptide, only trace amounts of human hemoglobin were detected by Western blot in the 360-fold concentrated fermentation broth [25]. P. pastoris, recognized for its robust capabilities of protein expression and post-translational modification [27], has emerged as an efficient system for the synthesis of hemoglobin [28,29].…”

Section: Introductionmentioning

confidence: 99%

Efficient Secretory Expression for Mammalian Hemoglobins in Pichia pastoris

Li,

Zhang,

Luo

et al. 2024

Fermentation

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Mammalian hemoglobins (HB) are a kind of heme-binding proteins that play crucial physiological roles in various organisms. The traditional techniques employed for the extraction of HB are expensive and time-consuming, while the yields of mammalian HB in previous reports were quite low. The industrial Pichia pastoris is a highly effective platform for the secretory expression of heterologous proteins. To achieve efficient secretory expression of HB in P. pastoris, multiple strategies were applied, including the selection of a suitable host, the screening of optimal endogenous signal peptides, the knockout of VPS10, VTH1, and PEP5, and the co-expression of Alpha-Hemoglobin Stabilizing Protein (AHSP). In addition, the conditions for producing HB were optimized at shaking-flask level (BMMY medium with 100 mg/L of hemin, 2% methanol, and 24 °C). Based on these conditions, the higher titers of bovine hemoglobin (bHB, 376.9 ± 13.3 mg/L), porcine hemoglobin (pHB, 119.2 ± 7.3 mg/L), and human hemoglobin (hHB, 101.1 ± 6.7 mg/L) were achieved at fermenter level. The engineered P. pastoris strain and comprehensive strategies can also be applied to facilitate the synthesis of other high-value-added hemoproteins or hemoenzymes.

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Improved production of human hemoglobin in yeast by engineering hemoglobin degradation

Cited by 33 publications

References 50 publications

Improving recombinant protein production by yeast through genome-scale modeling using proteome constraints

Improving recombinant protein production by yeast through genome-scale modeling using proteome constraints

Engineering Saccharomyces cerevisiae for the production and secretion of Affibody molecules

Efficient Secretory Expression for Mammalian Hemoglobins in Pichia pastoris

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