Systematic engineering of Saccharomyces cerevisiae for efficient synthesis of hemoglobins and myoglobins

Xue, Ji-ke; Zhou, Jingwen; Li, Jianghua; Du, Guocheng; Chen, Jian; Wang, Miao; Zhao, Xinrui

doi:10.1016/j.biortech.2022.128556

Cited by 18 publications

(30 citation statements)

References 38 publications

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“…The results showed that all synthesized mammalian hemoglobins displayed the characteristic spectra in the Soret band region, peaking at 410 nm. This spectral similarity is a ributed to heme moiety binding in globin components, which was consistent with the previous research (Figure 9A) [26,62]. It demonstrated that the activities of synthesized HBs were close to the native HBs, suggesting they are suitable for applications in food processing and biocatalytic processes.…”

Section: Biochemical Properties Of Various Mammalian Hemoglobins Synt...supporting

confidence: 91%

“…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%

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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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Section: Biochemical Properties Of Various Mammalian Hemoglobins Synt...supporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Efficient Secretory Expression for Mammalian Hemoglobins in Pichia pastoris

Li,

Zhang,

Luo

et al. 2024

Fermentation

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“…To facilitate the accumulation of ALA, our previous study investigated the effect of multi‐copy integrated expression of the HEM1 gene on ALA synthesis in S. cerevisiae . [ 19 ] However, the results showed no significant difference in intracellular heme levels between low‐ and high‐copy expression of HEM1 . Therefore, a single cassette of native HEM1 (P GAP ‐ HEM1 ) was incorporated into the AOXTT DOWN‐gRNA locus [ 41 ] of the X33‐ Δku70 genome, generating the HEME‐1 strain.…”

Section: Resultsmentioning

confidence: 98%

“…In addition, the low expressional level limited the application of these bacterial ALA biosynthetic enzymes to synthesize heme in P. pastoris . [ 19 ] Hence, the synthesis of ALA in the mitochondria should be maintained during the reconstruction of the heme biosynthetic pathway. To facilitate the accumulation of ALA, our previous study investigated the effect of multi‐copy integrated expression of the HEM1 gene on ALA synthesis in S. cerevisiae .…”

Section: Resultsmentioning

confidence: 99%

“…[9] Hence, enhancing intracellular heme biosynthesis is a feasible approach for producing holo-hemoproteins. [19,20] As spatial segregation is a huge obstacle to synthesizing and utilizing heme in P. pastoris, it is necessary to determine the accurate intracellular localization of all HBS and the detailed MLS of HBS located in the mitochondria at first. Eight HBS were fused with the monomeric red fluorescent protein (m-Scarlet) and integrated into the genome of the X33-Δku70 strain, respectively (Figure 3a).…”

Section: Removing the Spatial Segregation During Heme Biosynthesismentioning

confidence: 99%

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Biosynthesis of High‐Active Hemoproteins by the Efficient Heme‐SupplyPichia PastorisChassis

Yu,

Zhao,

Zhou

et al. 2023

Advanced Science

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Microbial synthesis of valuable hemoproteins has become a popular research topic, and Pichia pastoris is a versatile platform for the industrial production of recombinant proteins. However, the inadequate supply of heme limits the synthesis of high‐active hemoproteins. Here a strategy for enhancing intracellular heme biosynthesis to improve the titers and functional activities of hemoproteins is reported. After selecting a suitable expressional strategy for globins, the efficient heme‐supply P. pastoris chassis is established by removing the spatial segregation during heme biosynthesis, optimizing precursor synthesis, assembling rate‐limiting enzymes using protein scaffolds, and inhibiting heme degradation. This robust chassis produces several highly active hemoproteins, including porcine myoglobin, soy hemoglobin, Vitreoscilla hemoglobin, and P450‐BM3, which can be used in the development of artificial meat, high‐cell‐density fermentation, and whole‐cell catalytic synthesis of high‐value‐added compounds. Furthermore, the engineered chassis strain has great potential for producing and applying other hemoproteins with high activities in various fields.

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Chloroplast expression of myoglobin and functional characterization of C. reinhardtii biomass for alternative meat applications

Phadnis,

Prakash

2024

Algal Research

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Systematic engineering of Saccharomyces cerevisiae for efficient synthesis of hemoglobins and myoglobins

Cited by 18 publications

References 38 publications

Efficient Secretory Expression for Mammalian Hemoglobins in Pichia pastoris

Efficient Secretory Expression for Mammalian Hemoglobins in Pichia pastoris

Biosynthesis of High‐Active Hemoproteins by the Efficient Heme‐SupplyPichia PastorisChassis

Chloroplast expression of myoglobin and functional characterization of C. reinhardtii biomass for alternative meat applications

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