High-level secretory production of leghemoglobin in Pichia pastoris through enhanced globin expression and heme biosynthesis

Shao, Youran; Xue, Changlu; Liu, Wenqian; Zuo, Siqi; Wei, Peilian; Huang, Lei; Lian, Jiazhang; Xu, Zhinan

doi:10.1016/j.biortech.2022.127884

Cited by 44 publications

(50 citation statements)

References 31 publications

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“…K. phaffii has been widely employed as a powerful platform for the expression of thousands of recombinant proteins, including various heme proteins [ 22 ]. In order to achieve high production of heme proteins, the heme supply should be sufficient.…”

Section: Resultsmentioning

confidence: 99%

“…K. phaffii harbors a native synthetic pathway for heme production. However, the biosynthesis of heme in K. phaffii is tightly regulated, resulting in extremely low level of intracellular heme which would be difficult to meet the requirement for high production of heme proteins [ 22 ]. Therefore, firstly we sought to construct a high heme-producing K. phaffii strain.…”

Section: Resultsmentioning

confidence: 99%

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Engineering Escherichia coli for efficient assembly of heme proteins

Wang

Bai

et al. 2023

Microb Cell Fact

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Background Heme proteins, such as hemoglobin, horseradish peroxidase and cytochrome P450 (CYP) enzyme, are highly versatile and have widespread applications in the fields of food, healthcare, medical and biological analysis. As a cofactor, heme availability plays a pivotal role in proper folding and function of heme proteins. However, the functional production of heme proteins is usually challenging mainly due to the insufficient supply of intracellular heme. Results Here, a versatile high-heme-producing Escherichia coli chassis was constructed for the efficient production of various high-value heme proteins. Initially, a heme-producing Komagataella phaffii strain was developed by reinforcing the C4 pathway-based heme synthetic route. Nevertheless, the analytical results revealed that most of the red compounds generated by the engineered K. phaffii strain were intermediates of heme synthesis which were unable to activate heme proteins. Subsequently, E. coli strain was selected as the host to develop heme-producing chassis. To fine-tune the C5 pathway-based heme synthetic route in E. coli, fifty-two recombinant strains harboring different combinations of heme synthesis genes were constructed. A high-heme-producing mutant Ec-M13 was obtained with negligible accumulation of intermediates. Then, the functional expression of three types of heme proteins including one dye-decolorizing peroxidase (Dyp), six oxygen-transport proteins (hemoglobin, myoglobin and leghemoglobin) and three CYP153A subfamily CYP enzymes was evaluated in Ec-M13. As expected, the assembly efficiencies of heme-bound Dyp and oxygen-transport proteins expressed in Ec-M13 were increased by 42.3–107.0% compared to those expressed in wild-type strain. The activities of Dyp and CYP enzymes were also significantly improved when expressed in Ec-M13. Finally, the whole-cell biocatalysts harboring three CYP enzymes were employed for nonanedioic acid production. High supply of intracellular heme could enhance the nonanedioic acid production by 1.8- to 6.5-fold. Conclusion High intracellular heme production was achieved in engineered E. coli without significant accumulation of heme synthesis intermediates. Functional expression of Dyp, hemoglobin, myoglobin, leghemoglobin and CYP enzymes was confirmed. Enhanced assembly efficiencies and activities of these heme proteins were observed. This work provides valuable guidance for constructing high-heme-producing cell factories. The developed mutant Ec-M13 could be employed as a versatile platform for the functional production of difficult-to-express heme proteins.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Engineering Escherichia coli for efficient assembly of heme proteins

Wang

Bai

et al. 2023

Microb Cell Fact

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“…[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%

“…Additionally, the result of SDS‐PAGE showed that the secreted S‐Hb was severely degraded during the process of fed‐batch fermentation (≈40%). [ 20 ] Thus, the proteases that result in the degradation of hemoproteins should be inhibited. Furthermore, a previous study found that the expression level of V‐Hb was inversely proportional to the ethanol production in E. coli .…”

Section: Introductionmentioning

confidence: 99%

“…[27,28] Notably, a previous study found that among four commonly used microbial hosts (E. coli, S. cerevisiae, P. pastoris, and Yarrowia lipolytica), P. pastoris exhibited the highest catalytic activity and stability for human cytochrome P450 2D6. [29] Although three other hemoproteins (P-Mb, S-Hb, and V-Hb) have been expressed successfully in P. pastoris, [9,20,25] except for P450-BM3, there are still three bottlenecks that hinder the efficient synthesis of these high-active hemoproteins. The first is the low expressional level of the globin component in multitudinous hemoproteins.…”

Section: Introductionmentioning

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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Characterization and engineering of peroxisome targeting sequences for compartmentalization engineering in Pichia pastoris

Ye,

Hong,

Gao

et al. 2024

Biotech & Bioengineering

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Peroxisomal compartmentalization has emerged as a highly promising strategy for reconstituting intricate metabolic pathways. In recent years, significant progress has been made in the peroxisomes through harnessing precursor pools, circumventing metabolic crosstalk, and minimizing the cytotoxicity of exogenous pathways. However, it is important to note that in methylotrophic yeasts (e.g. Pichia pastoris), the abundance and protein composition of peroxisomes are highly variable, particularly when peroxisome proliferation is induced by specific carbon sources. The intricate subcellular localization of native proteins, the variability of peroxisomal metabolic pathways, and the lack of systematic characterization of peroxisome targeting signals have limited the applications of peroxisomal compartmentalization in P. pastoris. Accordingly, this study established a high‐throughput screening method based on β‐carotene biosynthetic pathway to evaluate the targeting efficiency of PTS1s (Peroxisome Targeting Signal Type 1) in P. pastoris. First, 25 putative endogenous PTS1s were characterized and 3 PTS1s with high targeting efficiency were identified. Then, directed evolution of PTS1s was performed by constructing two PTS1 mutant libraries, and a total of 51 PTS1s (29 classical and 22 noncanonical PTS1s) with presumably higher peroxisomal targeting efficiency were identified, part of which were further characterized via confocal microscope. Finally, the newly identified PTS1s were employed for peroxisomal compartmentalization of the geraniol biosynthetic pathway, resulting in more than 30% increase in the titer of monoterpene compared with when the pathway was localized to the cytosol. The present study expands the synthetic biology toolkit and lays a solid foundation for peroxisomal compartmentalization in P. pastoris.

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High-level secretory production of leghemoglobin in Pichia pastoris through enhanced globin expression and heme biosynthesis

Cited by 44 publications

References 31 publications

Engineering Escherichia coli for efficient assembly of heme proteins

Engineering Escherichia coli for efficient assembly of heme proteins

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

Characterization and engineering of peroxisome targeting sequences for compartmentalization engineering in Pichia pastoris

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