Hybrid-architectured double-promoter expression systems enhance and upregulate-deregulated gene expressions in Pichia pastoris in methanol-free media

Demir, İrem; Çalı́k, Pınar

doi:10.1007/s00253-020-10796-5

Cited by 11 publications

(3 citation statements)

References 54 publications

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“…Thus, the constitutive expressions of gut1 and gut2 were achieved under the control of the bidirectional P HHX1 promoter (Vogl et al, 2018), generating mutant Z1. Then, based on the strain Z1, the gt1 gene was further overexpressed by replacing its promoter with a strong constitutive promoter P GAP (Demir & Calık, 2020), creating mutant Z2. To evaluate the carbon source utilization capacity of the recombinant strains, Z1 and Z2 were cultured in MG medium.…”

Section: Resultsmentioning

confidence: 99%

Metabolic engineering of Komagataella phaffii for synergetic utilization of glucose and glycerol

Wang

Zhao

Luo

et al. 2022

Yeast

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Komagataella phaffii GS115 is a proven heterologous expression system and has recently been exploited for the production of value‐added biochemicals from glucose through metabolic engineering. A major challenge for high‐level biochemical production is the appropriate distribution of carbon flux between cell growth and product biosynthesis. In this study, we report the development of a synergetic glucose and glycerol coutilization strategy for K. phaffii, potentially enabling this strain to consume glycerol for growth while conserving more glucose for product formation. First, several potential genes encoding mediator proteins and transcriptional factors that were considered to be associated with carbon catabolite repression in K. phaffii were screened, and deletion of gss1, a glucose sensor, appeared to be able to eliminate the glucose‐induced repression of glycerol utilization in a mixed glucose–glycerol medium. Transcriptome comparisons between the parent strain and the Δgss1 mutant were then performed, and the glycerol‐metabolism genes that were subjected to glucose regulation were identified. Second, coutilization of glucose and glycerol in K. phaffii was achieved by overexpressing genes relevant to glycerol metabolism, namely, gt1, gut1, and gut2. Furthermore, knockout or knockdown of pfk and zwf genes resulted in a reduction of carbon flux from glucose towards glycolysis and the pentose phosphate pathway. With these efforts, the cell metabolism of the final strain was divided into growth and production modules. This study describes a promising strategy to address the challenge of carbon flux distribution in K. phaffii, and would be valuable in engineering this strain as a versatile fermentation platform for biochemical production.

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Section: Resultsmentioning

confidence: 99%

Metabolic engineering of Komagataella phaffii for synergetic utilization of glucose and glycerol

Wang

Zhao

Luo

et al. 2022

Yeast

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“…Using de novo synthetic sequence to replace native cis -acting DNA assists in achieving the architecture of the hybrid promoter ( Ergün et al, 2020 ). The hybrid-architectured promoter design refers to collecting monodirectional double-promoter expression system (DPESs) with hybrid architecture composed of engineered promoter variants P ADH2 -Cat 8 -L 2 and Pm AOX1 and the natural promoter P GAP for enhancing and upregulating deregulated gene expressions in P. pastoris in media free of methanol ( Demir and Calik, 2020 ). Biofunctional DPESs exhibited higher transcription and expression upregulation power relative to twin DPESs (two-copy expression systems).…”

Section: Promoter Engineeringmentioning

confidence: 99%

Current advances of Pichia pastoris as cell factories for production of recombinant proteins

Pan

Yang²,

Wu³

et al. 2022

Front. Microbiol.

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Pichia pastoris (syn. Komagataella spp.) has attracted extensive attention as an efficient platform for recombinant protein (RP) production. For obtaining a higher protein titer, many researchers have put lots of effort into different areas and made some progress. Here, we summarized the most recent advances of the last 5 years to get a better understanding of its future direction of development. The appearance of innovative genetic tools and methodologies like the CRISPR/Cas9 gene-editing system eases the manipulation of gene expression systems and greatly improves the efficiency of exploring gene functions. The integration of novel pathways in microorganisms has raised more ideas of metabolic engineering for enhancing RP production. In addition, some new opportunities for the manufacture of proteins have been created by the application of novel mathematical models coupled with high-throughput screening to have a better overview of bottlenecks in the biosynthetic process.

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“…This species is widely applied as a heterologous protein production host, and its utilization has been widely reported in the literature [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 ]. The main advantages of this organism are the possibility to run high-density fermentation according to established protocols, fast-paced and automation-friendly genetic engineering [ 10 ], eukaryotic post-translational modifications [ 11 , 12 ], high secretory efficiency and biomass yields [ 13 , 14 ], stable genetic constructs [ 15 ], and an increasing collection of publicly available tools [ 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ].…”

Section: Introductionmentioning

confidence: 99%

Industrial Production of Proteins with Pichia pastoris—Komagataella phaffii

Barone

Emmerstorfer-Augustin

Biundo

et al. 2023

Biomolecules

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Since the mid-1960s, methylotrophic yeast Komagataella phaffii (previously described as Pichia pastoris) has received increasing scientific attention. The interest for the industrial production of proteins for different applications (e.g., feed, food additives, detergent, waste treatment processes, and textile) is a well-consolidated scientific topic, and the importance for this approach is rising in the current era of environmental transition in human societies. This review aims to summarize fundamental and specific information in this scientific field. Additionally, an updated description of the relevant products produced with K. phaffii at industrial levels by a variety of companies—describing how the industry has leveraged its key features, from products for the ingredients of meat-free burgers (e.g., IMPOSSIBLE™ FOODS, USA) to diabetes therapeutics (e.g., Biocon, India)—is provided. Furthermore, active patents and the typical workflow for industrial protein production with this strain are reported.

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Hybrid-architectured double-promoter expression systems enhance and upregulate-deregulated gene expressions in Pichia pastoris in methanol-free media

Cited by 11 publications

References 54 publications

Metabolic engineering of Komagataella phaffii for synergetic utilization of glucose and glycerol

Metabolic engineering of Komagataella phaffii for synergetic utilization of glucose and glycerol

Current advances of Pichia pastoris as cell factories for production of recombinant proteins

Industrial Production of Proteins with Pichia pastoris—Komagataella phaffii

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