Methylotrophic Yeast Pichia Pastoris Research Articles

Boosting Expression of a Specifically Targeted Antimicrobial Peptide K in Pichia pastoris by Employing a 2A Self-Cleaving Peptide-Based Expression System.

Background/Objectives: The current epidemic of drug-resistance bacterial strains is one of the most urgent threats to human health. Antimicrobial peptides (AMPs) are known for their good activity against multidrug resistance bacteria. Specifically targeted AMPs (STAMPs) are a fraction of AMPs that target specific bacteria and maintain the balance of the healthy microbiota of a host. We reported a STAMP Peptide K (former name: peptide 13) for E. coli. The aim of this study was to effectively produce peptide K using methylotrophic yeast Pichia pastoris. Methods: Three inserts (sequence of peptide K (K), two copies of peptide K fused with 2A sequence (KTK), and two copies of peptide K fused with 2A and an extra α mating factor (KTAK)) were designed to investigate the effect of the number of repeats and the trafficking of peptide on the yield. Results: The yield from KTK was the highest-more than two-fold higher compared with K-implying the role of the 2A sequence in heterologous peptide expression apart from the co-translation. Then, the fermentation condition for KTK was optimized. The optimized yield of KTK was 6.67 mg/mL, suggesting the efficiency of the expression system. Selectivity, antibacterial activity, biocompatibility, and the stability of the fermentation product were equivalent to the chemically synthesized peptide. The actional mechanism of the fermentation product included membrane permeabilization and ROS induction. Conclusions: Together, our work provided a new perspective to augment the yield of the antimicrobial peptide in the microbial system, building a technological foundation for their large-scale production and expanding the market application of AMPs.

Continuous cell recycling in methylotrophic yeast Pichia pastoris to enhance product yields: a case study with Yarrowia lipolytica lipase Lip2

Continuous cell recycling in methylotrophic yeast Pichia pastoris to enhance product yields: a case study with Yarrowia lipolytica lipase Lip2

Improvement of the recombinant phytase expression by intermittent feeding of glucose during the induction phase of methylotrophic yeast Pichia pastoris.

For growth of methylotrophic yeast, glycerol is usually used as a carbon source. Glucose is used in some cases, but not widely consumed due to strong repressive effect on AOX1 promoter. However, glucose is still considered as a carbon source of choice since it has low production cost and guarantees growth rate comparable to glycerol. In flask cultivation of the recombinant yeast, Pichia pastoris GS115(pPIC9K-appA38M), while methanol induction point(OD600) and methanol concentration significantly affected the phytase expression, glucose addition in induction phase could enhance phytase expression. The optimal flask cultivation conditions illustrated by Response Surface Methodology were 10.37 OD600 induction point, 2.02h before methanol feeding, 1.16% methanol concentration and 40.36μL glucose feeding amount(for 20mL culture volume) in which the expressed phytase activity was 613.4 ± 10.2U/mL, the highest activity in flask cultivation. In bioreactor fermentation, the intermittent glucose feeding showed several advantageous results such as 68h longer activity increment, 149.2% higher cell density and 200.1% higher activity compared to the sole methanol feeding method. These results implied that remaining glucose at induction point might exhibit a positive effect on the phytase expression. Glucose intermittent feeding could be exploited for economic phytase production and the other recombinant protein expression by P. pastoris GS115.

Establishment of a steroid binding assay for goldfish membrane progesterone receptor (mPR) by coupling with graphene quantum dots (GQDs).

A homogeneous assay was developed to evaluate ligands that target the membrane progesterone receptor alpha (mPRα) of goldfish. This was achieved by employing graphene quantum dots (GQDs), a type of semiconductor nanoparticle conjugated to the goldfish mPRα. When progesterone-BSA-fluorescein isothiocyanate (P4-BSA-FITC) was combined with the other agents, fluorescence was observed through Förster resonance energy transfer (FRET). However, this fluorescence was quenched by binding between the ligand and receptor. This established method demonstrated the ligand selectivity of the mPRα protein. Then, the methylotrophic yeast Pichia pastoris was used to express the goldfish mPRα (GmPRα) protein. The recombinant purified GmPRα protein was coupled with graphene quantum dots (GQDs) to generate GQD-conjugated goldfish mPRα (GQD-GmPRα). Fluorescence at a wavelength of 520nm was observed through FRET upon the combination of P4-BSA-FITC and subsequent activation by ultraviolet (UV) light. Adding free P4 to the reaction mixture resulted in a decrease in fluorescence intensity at a wavelength of 520nm. The fluorescence was reduced by the administration of GmPRα ligands but not by steroids that do not interact with GmPRα. The findings indicated that the interaction between the ligand and receptor led to the formation of a complex involving GQD-GmPRα and P4-BSA-FITC. The interaction between the compounds and GQD-GmPRα was additionally validated by a binding experiment that employed the radiolabeled natural ligand [3H]-17α,20β-dihydroxy-4-pregnen-3-one. We established a ligand-binding assay for the fish membrane progesterone receptor that is applicable for screening compounds.

A Cost-Effective Pichia pastoris Cell-Free System Driven by Glycolytic Intermediates Enables the Production of Complex Eukaryotic Proteins.

Cell-free systems are particularly attractive for screening applications and the production of difficult-to-express proteins. However, the production of cell lysates is difficult to implement on a larger scale due to large time requirements, cultivation costs, and the supplementation of cell-free reactions with energy regeneration systems. Consequently, the methylotrophic yeast Pichia pastoris, which is widely used in recombinant protein production, was utilized in the present study to realize cell-free synthesis in a cost-effective manner. Sensitive disruption conditions were evaluated, and appropriate signal sequences for translocation into ER vesicles were identified. An alternative energy regeneration system based on fructose-1,6-bisphosphate was developed and a ~2-fold increase in protein production was observed. Using a statistical experiment design, the optimal composition of the cell-free reaction milieu was determined. Moreover, functional ion channels could be produced, and a G-protein-coupled receptor was site-specifically modified using the novel cell-free system. Finally, the established P. pastoris cell-free protein production system can economically produce complex proteins for biotechnological applications in a short time.

Directed evolution of Baeyer-Villiger monooxygenase for highly secretory expressed in Pichia pastoris and efficient preparation of chiral pyrazole sulfoxide.

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is a highly distinguished expression platform for the excellent synthesis of various heterologous proteins in recent years. With the advantages of high-density fermentation, P. pastoris can produce gram amounts of recombinant proteins. While not every protein of interest can be expressed to such high titers, such as Baeyer-Villiger monooxygenase (BVMO) (AcPSMO) which is responsible for pyrazole sulfide asymmetric oxidation. In this work, an excellent yeast expression system was established to facilitate efficient AcPSMO expression, which exhibited 9.5-fold enhanced secretion. Subsequently, an ultrahigh throughput screening method based on fluorescence-activated cell sorting by fusing super folder green fluorescent protein (sfGFP) in the C-terminal of AcPSMO was developed, and directed evolution was performed. The protein expression level of the superior mutant AcPSMOP1 (S58T/T252P/E336N/H456D) reached 84.6 mg/L at 100 mL shaking flask, which was 4.7 times higher than the levels obtained with the wild-type. Finally, the optimized chassis cells were used for high-density fermentation on a 5-L scale, and AcPSMOP1 protein yield of 3.4 g/L was achieved, representing approximately 85% of the total protein secreted. By directly employing the pH-adjusted supernatant as a biocatalyst, 20 g/L pyrmetazole sulfide was completely transformed into the corresponding (S)-sulfoxide, with a 78.8% isolated yield. This work confers dramatic benefits for efficient secretion of other BVMOs in P. pastoris.

Characterization of Prolyl-4-Hydroxylase Substrate Specificity Using Pichia pastoris as an Efficient Eukaryotic Expression System.

The use of eukaryotic expression systems facilitates the heterologous expression of complex eukaryotic proteins in their post-translationally modified and biologically active state, as a prerequisite for subsequent biochemical characterization and functional analysis. Here we describe the complete workflow for the expression of Arabidopsis thaliana prolyl-4-hydroxylases (P4Hs) in the methylotrophic yeast Pichia pastoris (renamed as Komagataella phaffii), for the extraction of the recombinant enzymes, purification by affinity chromatography, and characterization of P4H activity and specificity toward oligopeptide substrates by mass spectrometry. We expressed eight of the 13 Arabidopsis P4Hs and show that they are all active against proline-rich extensin-derived peptides. However, three of them differed in substrate specificity and were also able to hydroxylate the CLEL9 signaling peptide, featuring a single proline within its mature peptide sequence.

Deciphering cell wall sensors enabling the construction of robust P. pastoris for single-cell protein production

Single-cell protein (SCP) production in the methylotrophic yeast Pichia pastoris has the potential to achieve a sustainable protein supply. However, improving the methanol fermentation efficiency and reducing carbon loss has been a long-standing challenge with far-reaching scientific and practical implications. Here, comparative transcriptomics revealed that PAS_0305, a gene directly associated with cell wall thickness under methanol stress, can be used as a target for unlocking cell wall sensors. Intracellular trehalose accumulation confirmed that cell wall sensors were activated after knocking out PAS_0305, which resulted in increased cell wall permeability. Genome-wide signal perturbations were transduced through the HOG module and the CWI pathway, which was confirmed to connected by Pbs2-Mkk. As a consequence of CWI pathway activation, ΔPAS_0305 elicited a rescue response of cell wall remodeling by increasing the β-1,3-glucan content and decreasing the chitin/mannose content. Remarkably, perturbations in global stress signals led to a fine-tuning of the metabolic network of ΔPAS_0305, resulting in a superior phenotype with highest crude protein and methanol conversion rate of 67.21% and 0.46 gDCW/g. Further genome-scale metabolic models were constructed to validate the experimental results, confirming that unlocking cell wall sensors resulted in maximized flux from methanol towards SCP and effectively addressing the issue of carbon loss in methanol fermentation. This work sheds new light on the potential of manipulating cellular signaling pathways to optimize metabolic networks and achieve exceptional phenotypic characteristics, providing new strategies for constructing versatile cell factories in P. pastoris.

Enhanced production of human interferon α2b in glycoengineered Pichia pastoris by robust control of methanol feeding and implications of various control strategies

Interferon α2b (IFNα2b), a prominent cytokine molecule, is bestowed with immunomodulatory, antiproliferative, and antiviral properties. Like any other recombinant protein produced in methylotrophic yeast Pichia pastoris, Human interferon α2b (huIFN α2b) production in P. pastoris depends on methanol utilization during the induction phase. This study investigates the impact of varying residual methanol concentrations on huIFNα2b productivity and assesses control strategies towards the tight control of residual methanol concentration in fed-batch experiments carried out in the fermentation calorimeter. Real-time monitoring of P. pastoris metabolism was facilitated by metabolic heat rate measurements and exhaust gas analyser. Optimal methanol concentration screening performed at different concentrations viz., 1.5, 3, 5, and 7 g/L, revealed the highest huIFNα2b productivity at 3 g/L (0.21 mg/g h). Methanol sensor provided input data for three different control strategies (on/off, proportional integral (PI), and model-based adaptive PI) to maintain optimal methanol levels (3 g/L). Model-based adaptive control resulted in the highest huIFNα2b yield, productivity (244.34 mg/L, 0.31 mg/g h), surpassing on/off (188.7 mg/L, 0.16 mg/g h) and PI (202.3 mg/L, 0.28 mg/g h) control strategies. The model-based adaptive PI control framework developed in this study could be employed for other heterologous protein production in P. pastoris.

Deglycosylated RBD produced in Pichia pastoris as a low-cost sera COVID-19 diagnosis tool and a vaccine candidate.

During the COVID-19 outbreak, numerous tools including protein-based vaccines have been developed. The methylotrophic yeast Pichia pastoris (synonymous to Komagataella phaffii) is an eukaryotic cost-effective and scalable system for recombinant protein production, with the advantages of an efficient secretion system and the protein folding assistance of the secretory pathway of eukaryotic cells. In a previous work, we compared the expression of SARS-CoV-2 Spike Receptor Binding Domain in P. pastoris with that in human cells. Although the size and glycosylation pattern was different between them, their protein structural and conformational features were indistinguishable. Nevertheless, since high mannose glycan extensions in proteins expressed by yeast may be the cause of a nonspecific immune recognition, we deglycosylated RBD in native conditions. This resulted in a highly pure, homogenous, properly folded and monomeric stable protein. This was confirmed by circular dichroism and tryptophan fluorescence spectra and by SEC-HPLC, which were similar to those of RBD proteins produced in yeast or human cells. Deglycosylated RBD was obtained at high yields in a single step, and it was efficient in distinguishing between SARS-CoV-2-negative and positive sera from patients. Moreover, when the deglycosylated variant was used as an immunogen, it elicited a humoral immune response ten times greater than the glycosylated form, producing antibodies with enhanced neutralizing power and eliciting a more robust cellular response. The proposed approach may be used to produce at a low cost, many antigens that require glycosylation to fold and express, but do not require glycans for recognition purposes.

Cloning and Overexpressing Membrane Proteins Using Pichia pastoris (Komagataella phaffii)

AbstractUnderstanding the structure and function of key proteins located within biological membranes is essential for fundamental knowledge and therapeutic applications. Robust cell systems allowing their actual overexpression are required, among which stands the methylotrophic yeast Pichia pastoris. This system proves highly efficient in producing many eukaryotic membrane proteins of various functions and structures at levels and quality compatible with their subsequent isolation and molecular investigation. This article describes a set of basic guidelines and directions to clone and select recombinant P. pastoris clones overexpressing eukaryotic membrane proteins. Illustrative results obtained for a panel of mammalian membrane proteins are presented, and hints are given on a series of experimental parameters that may substantially improve the amount and/or the functionality of the expressed proteins. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Designing and cloning a P. pastoris expression vectorBasic Protocol 2: Integrative transformation of P. pastoris and selection of recombinant clonesBasic Protocol 3: Culturing transformed P. pastoris for membrane protein expressionBasic Protocol 4: Yeast cell lysis and membrane preparationBasic Protocol 5: Immunodetection of expressed membrane proteins: western blotAlternate Protocol 1: Immunodetection of expressed membrane proteins: dot blotAlternate Protocol 2: Immunodetection of expressed membrane proteins: yeastern blotBasic Protocol 6: Activity assay: ligand‐binding analysis of an expressed GPCR

Development of a continuous fermentation process for the production of recombinant uricase enzyme by Pichia pastoris.

Recent studies in the biopharmaceutical industry have shown an increase in the productivity and production efficiency of recombinant proteins by continuous culture. In this research, a new upstream fermentation process was developed for the production of recombinant uricase in the methylotrophic yeast Pichia pastoris. Expression of recombinant protein in this system is under the control of the AOX1 promoter and therefore requires methanol as an inducing agent and carbon/energy source. Considering the biphasic growth characteristics of conventional fed-batch fermentation, physical separation of the growth and induction stages for better control of the continuous fermentation process resulted in higher dry-cell weight (DCW) and enhanced recombinant urate oxidase activity. The DCW and recombinant uricase activity enzyme for fed-batch fermentation were 79g/L and 6.8u/mL. During the continuous process, in the growth fermenter at a constant dilution rate of 0.025h-1 , DCW increased to 88.39g/L. In the induction fermenter, at methanol feeding rates of 30, 60, and 80mL/h, a recombinant uricase activity was 4.13, 7.2, and 0u/mL, respectively. The optimum methanol feeding regime in continuous fermentation resulted in a 4.5-fold improvement in productivity compared with fed-batch fermentation from 0.04u/mL/h (0.0017mg/mL/h) to 0.18u/mL/h (0.0078mg/mL/h).

Economical production of Pichia pastoris single cell protein from methanol at industrial pilot scale

BackgroundMethanol, synthesized from CO2, is a potentially sustainable one-carbon (C1) resource for biomanufacturing. The use of methanol as a feedstock to produce single cell protein (SCP) has been investigated for decades as an alternative to alleviate the high global demand for animal-derived proteins. The methylotrophic yeast Pichia pastoris is an ideal host for methanol-based SCP synthesis due to its natural methanol assimilation ability. However, improving methanol utilization, tolerance to higher temperature, and the protein content of P. pastoris are also current challenges, which are of great significance to the economical industrial application using methanol as a feedstock for SCP production.ResultsIn the present work, adaptive laboratory evolution (ALE) has been employed to overcome the low methanol utilization efficiency and intolerance to a higher temperature of 33 °C in P. pastoris, associated with reduced carbon loss due to the lessened detoxification of intracellular formaldehyde through the dissimilation pathway and cell wall rearrangement to temperature stress resistance following long-term evolution as revealed by transcriptomic and phenotypic analysis. By strengthening nitrogen metabolism and impairing cell wall synthesis, metabolic engineering further increased protein content. Finally, the engineered strain via multi-strategy produced high levels of SCP from methanol in a pilot-scale fed-batch culture at 33 °C with a biomass of 63.37 g DCW/L, methanol conversion rate of 0.43 g DCW/g, and protein content of 0.506 g/g DCW. SCP obtained from P. pastoris contains a higher percentage of protein compared to conventional foods like soy, fish, meat, whole milk, and is a source of essential amino acids, including methionine, lysine, and branched-chain amino acids (BCAAs: valine, isoleucine, leucine).ConclusionsThis study clarified the unique mechanism of P. pastoris for efficient methanol utilization, higher temperature resistance, and high protein synthesis, providing a P. pastoris cell factory for SCP production with environmental, economic, and nutritional benefits.

Recombinant Production of Food Allergens in Yeast Pichia pastoris.

The methylotrophic yeast Pichia pastoris (P. pastoris) is one of the expression systems widely used to produce recombinant heterologous proteins. In this chapter, the methodology to produce recombinant food allergens in P. pastoris is described. The methodology begins with the preparation of competent P. pastoris cells followed by the transformation of the competent cells by electroporation as well as the preparation of plasmid DNA for transformation. Moreover, the screening of yeast transformants by direct PCR to ensure integration of allergen DNA followed by small-scale expression of recombinant allergen in yeast cells is also described.

Development of an enzyme-linked immunosorbent assay for determining FSH plasma concentrations in green sea turtle (Chelonia mydas), using recombinant gonadotropins

Follicle-stimulating hormone (FSH) is involved in the regulation of essential reproductive processes such as gametogenesis and follicular growth. There are presently no immunoassays for measuring FSH in turtles. Recently we produced green sea turtle (Chelonia mydas) recombinant (r) FSH as a single-chain polypeptide using the methylotrophic yeast Pichia pastoris expression system, and polyclonal antibodies for the recombinant FSH. In this work we developed a competitive enzyme-linked immunosorbent assay (ELISA) for the measurement of FSH concentrations in plasma samples from green sea turtles. We used the rFSHβα for standard, rFSHβ for coating and a cmFSHβ antibody. The sensitivity of the assay was 0.13 ng/ml and the intra-assay and inter-assay coefficients of variation were 5.54% and 13.52% respectively. Parallelism was observed between the linearized FSH standard curves and the corresponding serial dilutions of green sea turtle plasma samples. We also observed parallelism between the linearized standard and serial dilutions of plasma samples from the loggerhead sea turtle (Caretta caretta), hawksbill sea turtle (Eretmochelys imbricate), and African softshell turtle (Trionyx triunguis). The ELISA was used to study the FSH plasma concentrations during the reproductive cycles and was compared to hormonal steroid concentrations (Testosterone, Estradiol and Progesterone). This revealed a positive correlation between FSH and estradiol concentrations in females; estradiol concentrations were increased immediately after FSH elevation. In addition, nested females presented an increase in FSH concentrations prior to progesterone elevation in January to April, slightly before egg laying. This ELISA will increase our understanding of gonadotropin functions, and their effects on reproduction in the green sea turtle.

Optimization of a Purification Method for the Recombinant Platelet-Derived Growth Factor rhPDGF-BB Expressed in the Methylotrophic Yeast Pichia Pastoris

Optimization of a Purification Method for the Recombinant Platelet-Derived Growth Factor rhPDGF-BB Expressed in the Methylotrophic Yeast Pichia Pastoris

Expression and purification of human interferon alpha 2a (IFNα2a) in the methylotrophic yeast Pichia pastoris

Human interferon alpha 2a (IFNα2a) is a secreted glycoprotein that exerts a wide spectrum of biological effects, such as triggering of antiviral, antitumor and immunosuppressive responses. IFNα2a is used as pharmaceutical polypeptide in chronic hepatitis C virus (HCV) infection, chronic myelogenous leukemia, advanced renal cell carcinoma, and metastatic malignant melanoma. So far, the pharmaceutical polypeptide of this cytokine is produced in prokaryotic expression systems (E. coli). Here we report the expression and purification of recombinant human IFNα2a in the methylotrophic yeast Pichia pastoris. The cDNA encoding for human IFNα2a, modified to bear the P. pastoris codon bias, was cloned into the pPinkα-HC vector in order to be expressed as a secreted protein upon induction. Proper expression and secretion of recombinant human IFNα2a (approximately 19 kDa) was confirmed by PCR-sequencing, SDS-PAGE and Western blot analysis following methanol-induced expression in a number of individual transformed strains. Purification of the recombinant protein was performed by affinity chromatography, achieving a robust yield of purified active form. The purified recombinant protein showed an impressive stability to thermal denaturation as observed by Differential Scanning Fluorimetry. The biological activity of the P. pastoris-produced IFNα2a was confirmed in A549 and HT29 cells by monitoring transcriptional up-regulation of a panel of known interferon-stimulated genes (ISGs). Our results document that the P. pastoris expression system is a suitable system for producing biologically functional IFNα2a in a secreted form.

A Systematic Review of the Potential of Pichia pastoris (Komagataella phaffii) as an Alternative Host for Biologics Production.

The methylotrophic yeast Pichia pastoris is garnering interest as a chassis cell factory for the manufacture of recombinant proteins because it effectively satisfies the requirements of both laboratory and industrial set up. The optimisation of P. pastoris cultivation is still necessary due to strain- and product-specific problems such as promoter strength, methanol utilisation type, and culturing conditions to realize the high yields of heterologous protein(s) of interest. Techniques integrating genetic and process engineering have been used to overcome these problems. Insight into the Pichia as an expression system utilizing MUT pathway and the development of methanol free systems are highlighted in this systematic review. Recent developments in the improved production of proteins in P. pastoris by (i) diverse genetic engineering such as codon optimization and gene dosage; (ii) cultivating tactics including co-expression of chaperones; (iii) advances in the use of the 2A peptide system, and (iv) CRISPR/Cas technologies are widely discussed. We believe that by combining these strategies, P. pastoris will become a formidable platform for the production of high value therapeutic proteins.

Purification Method Optimization of Recombinant Platelet-Derived Growth Factor rhPDGF-BB Expressed in Methylotrophic Yeast <i>Pichia pastoris</i>

Recombinant human platelet-derived growth factor rhPDGF-BB is one of the major cytokines, which has been approved for medical use. Medical drug “becaplermin”, containing rhPDGF-BB has been approved for neuropathic ulcer and severe skin burns treatment, as well as in periodontal surgery (in combination with osteoconductive matrices). In this article, we sought to optimize purification process to obtain high purity rhPDGF-BB using methylotrophic yeast Pichia pastoris – a production host for rhPDGF-BB. A faster and simpler chromatography purification method has been suggested which allows to obtain rhPDGF-BB with purity 98% as determined by SDS-PAGE and containing host cell proteins (HCP) 33 ± 4 ng/mg, as measured by ELISA. The effective proliferative dose of rhPDGF-BB measured by WST-1 proliferative assay on 3T3 mouse fibroblast cell culture is 5.02 ± 2.64 ng/mL, which is comparable to commercially available analogues. The optimized method can be attractive for production scale use.

A novel CRISPR/Cas9 system with high genomic editing efficiency and recyclable auxotrophic selective marker for multiple-step metabolic rewriting in Pichia pastoris

The methylotrophic budding yeast Pichia pastoris has been utilized to the production of a variety of heterologous recombinant proteins owing to the strong inducible alcohol oxidase promoter (pAOX1). However, it is difficult to use P. pastoris as the chassis cell factory for high-valuable metabolite biosynthesis due to the low homologous recombination (HR) efficiency and the limitation of handy selective markers, especially in the condition of multistep biosynthetic pathways. Hence, we developed a novel CRISPR/Cas9 system with highly editing efficiencies and recyclable auxotrophic selective marker (HiEE-ReSM) to facilitate cell factory in P. pastoris. Firstly, we improved the HR rates of P. pastoris through knocking out the non-homologous-end-joining gene (Δku70) and overexpressing HR-related proteins (RAD52 and RAD59), resulting in higher positive rate compared to the basal strain, achieved 97%. Then, we used the uracil biosynthetic genes PpURA3 as the reverse screening marker, which can improve the recycling efficiency of marker. Meanwhile, the HR rate is still 100% in uracil auxotrophic yeast. Specially, we improved the growth rate of uracil auxotrophic yeast strains by overexpressing the uracil transporter (scFUR4) to increase the uptake of exogenous uracil from medium. Meanwhile, we explored the optimal concentration of uracil (90 mg/L) for strain growth. In the end, the HiEE-ReSM system has been applied for the inositol production (250 mg/L) derived from methanol in P. pastoris. The systems will contribute to P. pastoris as an attractive cell factory for the complex compound biosynthesis through multistep metabolic pathway engineering and will be a useful tool to improve one carbon (C1) bio-utilization.