Pichia pastoris Metabolic Engineering Services

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Pichia pastoris metabolic engineering services provide specialized solutions for optimizing the metabolic pathways of this widely used yeast species to enhance the production of valuable biochemicals, biofuels, pharmaceuticals, and other industrially relevant products. Our comprehensive services offer support from initial project design to final strain optimization, ensuring precise and efficient metabolic modifications tailored to your specific research and biotechnological needs.

Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation(P Cai, et al.,2021)

Overview Service Process Examples and Solutions Case Study Frequently Asked Questions

Overview

Pichia pastoris, a methylotrophic yeast, is a popular system for heterologous protein expression due to its ability to grow to high cell densities and perform post-translational modifications. Our metabolic engineering services leverage advanced genetic engineering techniques, such as CRISPR/Cas9, homologous recombination, and synthetic biology approaches, to optimize the metabolic pathways of Pichia pastoris for improved production of target compounds.

Service Process

The process of Pichia pastoris metabolic engineering involves several critical and interrelated steps:

Project Consultation: Collaborating with researchers to define specific metabolic engineering goals, including target compounds, desired metabolic modifications, and intended applications.
Pathway Analysis and Design: Analyzing existing metabolic pathways and designing modifications to optimize the production of target compounds. This includes pathway reconstruction and flux balance analysis.
Vector Design and Construction: Designing and constructing expression vectors or CRISPR/Cas9 systems tailored to the specific genetic modifications needed for the metabolic pathway.
Yeast Transformation: Introducing the genetic material into Pichia pastoris cells using techniques such as electroporation or lithium acetate transformation.
Selection and Screening: Selecting successfully transformed yeast cells using selectable markers and screening for desired metabolic modifications using assays such as HPLC, GC-MS, and enzymatic assays.
Strain Optimization: Optimizing the engineered strains through iterative rounds of modification and selection to enhance the production of target compounds. This may include optimizing growth conditions and media composition.
Characterization and Validation: Characterizing the engineered yeast strains to confirm the presence and functionality of the metabolic modifications. This includes growth assays, metabolic profiling, and functional assays.
Scale-Up and Production: Scaling up the engineered strains for large-scale production and further applications in research or industry.
Reporting and Consultation: Providing a detailed report of the findings and offering further consultation to interpret the results and plan subsequent research steps.

For more information about our Pichia pastoris Metabolic Engineering Services or to discuss your specific needs, please contact us. Our team of experts is available to provide guidance and support for your research and biotechnological projects, ensuring you achieve your scientific and industrial goals.

Examples and Solutions

The following table provides an overview of various case studies in Pichia pastoris metabolic engineering and the solutions we offer to support your research and biotechnological endeavors:

Case Study	Description	Solutions We Offer
Ethanol Production Optimization	Engineering yeast strains to enhance ethanol production from biomass.	Pathway design, genetic modification, strain optimization, and scale-up.
Antibiotic Production Enhancement	Modifying metabolic pathways to increase the yield of antibiotics.	CRISPR/Cas9 gene editing, pathway optimization, and production scaling.
Organic Acid Production	Developing strains for the efficient production of organic acids for industrial use.	Metabolic pathway reconstruction, strain engineering, and yield optimization.
Industrial Enzyme Production	Engineering yeast to produce high levels of industrial enzymes.	Pathway design, strain development, and production optimization.
Biofuel Production Strains	Optimizing metabolic pathways for the efficient production of biofuels.	Gene pathway engineering, strain development, and production optimization.
Synthetic Pathway Construction	Constructing synthetic pathways in yeast for the production of novel biochemicals.	Synthetic biology, pathway integration, and functional assays.

Case Study

: The engineered strain overexpressing the keto-acid degradation pathway was able to produce 284 mg/L of isobutanol when supplemented with 2-ketoisovalerate. To improve the production of isobutanol and eliminate the need to supplement the production media with the expensive 2-ketoisovalerate intermediate, we overexpressed a portion of the amino acid l-valine biosynthetic pathway in the engineered strain. While heterologous expression of the pathway genes from the yeast Saccharomyces cerevisiae did not lead to improvement in isobutanol production in the engineered P. pastoris, overexpression of the endogenous l-valine biosynthetic pathway genes led to a strain that is able to produce 0.89 g/L of isobutanol. Fine-tuning the expression of bottleneck enzymes by employing an episomal plasmid-based expression system further improved the production titer of isobutanol to 2.22 g/L, a 43-fold improvement from the levels observed in the original strain. Finally, heterologous expression of a broad-substraterange alcohol-O-acyltransferase led to the production of isobutyl acetate ester and isopentyl acetate ester at 51 and 24 mg/L, respectively

Efects of diferent carbon sources on isobutanol production in engineered P. pastoris(W Siripong, et al.,2018)

Efects of diferent carbon sources on isobutanol production in engineered P. pastoris. Total isobutanol production in minimum yeast media with either 2% glycerol (a) or 2% glucose (b) as the sole carbon source. Specifc isobutanol production in minimum yeast media with either 2% glycerol (c) or 2% glucose (d) as the sole carbon source. Engineered strains (PP100, PP200, PP300) were pre-cultured in 5-mL aliquots in MGYH (2% glycerol) minimal medium overnight and used to inoculate either 5 mL fresh MGYH (2% glycerol, a, c) or 5 mL fresh MGYH_glu (2% glucose, b, d) to achieve an initial optical density of 0.05 at 600 nm (OD600). The cultures were grown at 30 °C and 250 rpm in an orbital shaking incubator. Samples were taken at 24 and 48 h time points and the supernatants were analyzed on HPLC to quantify the isobutanol content. Values are the mean of three biological replicates ± standard deviation (n = 3)

RT-PCR analysis of isobutanol biosynthetic pathway genes including PpIlv5 (a), PpIlv3 (b) PpIlv6 (c), PpIlv2 (d) in engineered yeast.(W Siripong, et al.,2018)

The engineered strains PP100, PP200, and PP300 were pre-cultured in 5-mL aliquots in MGYH (2% glycerol) minimal medium overnight and used to inoculate either 10 mL fresh MGYH (2% glycerol) to achieve an initial optical density of 0.05 at 600 nm (OD600). The cultures were grown at 30 °C and 250 rpm in an orbital shaking incubator. Samples were taken at 48 h time points for real time RT-PCR analysis

Frequently Asked Questions

Q: What is Pichia pastoris metabolic engineering?

A: Pichia pastoris metabolic engineering involves the genetic modification of yeast strains to optimize their metabolic pathways for the production of target compounds. This can include introducing, deleting, or modifying specific genes to redirect metabolic fluxes and increase the yield of desired products.

Q: How is Pichia pastoris metabolic engineering performed?

A: Pichia pastoris metabolic engineering is performed through a series of steps including project consultation, pathway analysis and design, vector design and construction, yeast transformation, selection and screening, strain optimization, characterization and validation, scale-up and production, and reporting. Each step ensures precise and efficient metabolic modifications.

Q: What are the applications of Pichia pastoris metabolic engineering?

A: Applications include biofuel production, pharmaceutical production, industrial enzyme production, biochemicals production, synthetic biology, and agricultural biotechnology. Engineered yeast strains are used to produce valuable bioproducts and address various industrial and environmental challenges.

Q: What are the key steps in the Pichia pastoris metabolic engineering process?

A: Key steps include project consultation, pathway analysis and design, vector design and construction, yeast transformation, selection and screening, strain optimization, characterization and validation, scale-up and production, and reporting. These steps ensure comprehensive and accurate development of engineered yeast strains.

Q: Why is Pichia pastoris metabolic engineering important?

A: Pichia pastoris metabolic engineering is important for advancing research, developing new bioproducts, optimizing industrial processes, and addressing environmental challenges. Engineered yeast strains provide valuable tools for enhancing production yields and creating novel compounds.

Please note that all services are for research use only. Not intended for any clinical use.

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