Yeast Pichia Pastoris Research Articles

Metabolic engineering of Pichia pastoris for myo-inositol production by dynamic regulation of central metabolism

BackgroundThe methylotrophic budding yeast Pichia pastoris GS115 is a powerful expression system and hundreds of heterologous proteins have been successfully expressed in this strain. Recently, P. pastoris has also been exploited as an attractive cell factory for the production of high-value biochemicals due to Generally Recognized as Safe (GRAS) status and high growth rate of this yeast strain. However, appropriate regulation of metabolic flux distribution between cell growth and product biosynthesis is still a cumbersome task for achieving efficient biochemical production.ResultsIn this study, P. pastoris was exploited for high inositol production using an effective dynamic regulation strategy. Through enhancing native inositol biosynthesis pathway, knocking out inositol transporters, and slowing down carbon flux of glycolysis, an inositol-producing mutant was successfully developed and low inositol production of 0.71 g/L was obtained. The inositol production was further improved by 12.7% through introduction of heterologous inositol-3-phosphate synthase (IPS) and inositol monophosphatase (IMP) which catalyzed the rate-limiting steps for inositol biosynthesis. To control metabolic flux distribution between cell growth and inositol production, the promoters of glucose-6-phosphate dehydrogenase (ZWF), glucose-6-phosphate isomerase (PGI) and 6-phosphofructokinase (PFK1) genes were replaced with a glycerol inducible promoter. Consequently, the mutant strain could be switched from growth mode to production mode by supplementing glycerol and glucose sequentially, leading to an increase of about 4.9-fold in inositol formation. Ultimately, the dissolved oxygen condition in high-cell-density fermentation was optimized, resulting in a high production of 30.71 g/L inositol (~ 40-fold higher than the baseline strain).ConclusionsThe GRAS P. pastoris was engineered as an efficient inositol producer for the first time. Dynamic regulation of cell growth and inositol production was achieved via substrate-dependent modulation of glycolysis and pentose phosphate pathways and the highest inositol titer reported to date by a yeast cell factory was obtained. Results from this study provide valuable guidance for engineering of P. pastoris for the production of other high-value bioproducts.

Heterologous Production and Evaluation of the Biological Activity of Cystatin-B From the Red Piranha Pygocentrus nattereri.

Cystatin proteins are known to form a superfamily of cysteine protease inhibitors, which play a key role in protein degradation and are related to different physiological processes, such as development and immunity. Currently, numerous immunoregulatory proteins, such as cystatins, are being used in the control and prevention of diseases in aquaculture. Thus, the objective of this study was to produce recombinant cystatin (rCYST-B) from the red piranha Pygocentrus nattereri and to evaluate its effect on bacterial growth. The gene that encodes cystatin-B was isolated from the spleen of P. nattereri and cloned in an expression system. The protein was produced via a heterologous system involving the yeast Pichia pastoris X-33. The inhibitory activity of purified cystatin-B was evaluated on papain using different concentrations (0–80.0 μg/μL) of rCYST-B. The bacteriostatic action of the protein was evaluated using the Kirby-Bauer method on the growth of Escherichia coli and Bacillus subtilis. rCYST-B showed 100% inhibition at a concentration of 60 μg/μL. Moreover, the bacteriostatic activity of E. coli and B. subtilis showed inhibition of 40.36 and 49.08% compared to the negative control (phosphate buffer), respectively. These results suggest that recombinant CYST-B has biotechnological potential for use in aquaculture.

Avoiding entry into intracellular protein degradation pathways by signal mutations increases protein secretion in Pichia pastoris.

SummaryIn our previous study, we serendipitously discovered that protein secretion in the methylotrophic yeast Pichia pastoris is enhanced by a mutation (V50A) in the mating factor alpha (MFα) prepro‐leader signal derived from Saccharomyces cerevisiae. In the present study, we investigated 20 single‐amino‐acid substitutions, including V50A, located within the MFα signal peptide, indicating that V50A and several single mutations alone provided significant increase in production of the secreted proteins. In addition to hydrophobicity index analysis, both an unfolded protein response (UPR) biosensor analysis and a microscopic observation showed a clear difference on the levels of UPR induction and mis‐sorting of secretory protein into vacuoles among the wild‐type and mutated MFα signal peptides. This work demonstrates the importance of avoiding entry of secretory proteins into the intracellular protein degradation pathways, an observation that is expected to contribute to the engineering of strains with increased production of recombinant secreted proteins.

Microbial synthesis of long-chain α-alkenes from methanol by engineering Pichia pastoris

α-Alkenes (terminal alkenes) are important fuel and platform chemicals that are mainly produced from petroleum. Microbial synthesis might provide a sustainable approach for α-alkenes. In this work, we engineered the methylotrophic yeast Pichia pastoris to produce long-chain (C15:1, C17:1 and C17:2) α-alkenes via a decarboxylation of fatty acids. Combinatorial engineering, including enzyme selection, expression optimization and peroxisomal compartmentalization, enabled the production of 1.6 mg/L α-alkenes from sole methanol. This study represents the first case of α-alkene biosynthesis from methanol and also provides a reference for the construction of methanol microbial cell factories of other high-value chemicals.Graphical

Identification of Saccharum CaM gene family and functioncharacterization of ScCaM1 during cold and oxidant exposure in Pichia pastoris.

Calmodulin (CaM) plays an essential role in binding calcium ions and mediating the interpretation of Ca2+signals in plants under various stresses. However, the evolutionary relationship of CaM family proteins inSaccharumhas not been elucidated. To deduce and explore the evolution and function ofSaccharumCaM family. A total of 104 typical CaMs were obtained fromSaccharum spontaneumand other 18 plant species. The molecular characteristics and evolution of those CaM proteins were analyzed. A typicalCaMgene,ScCaM1, was subsequently cloned from sugarcane (Saccharumspp. hybrid). Its expression patterns in different tissues and under various abiotic stresses were assessed by quantitative real-time PCR. Then the green fluorescent protein was used to determine the subcellular localization of ScCaM1. Finally, the function ofScCaM1was evaluated via heterologous yeast expression systems. Three typical CaM members (SsCaM1, SsCaM2, and SsCaM3) were identified from theS. spontaneumgenome database.CaMs were originated from the two last common ancestors before the origin of angiosperms. The number of CaM family members did not correlate to the genome size but correlated with allopolyploidization events. TheScCaM1was more highly expressed in buds and roots than inother tissues. The expression patterns ofScCaM1suggested that it was involved in responses to various abiotic stresses in sugarcane via different hormonal signaling pathways. Noteworthily, its expression levels appeared relatively stable during the cold exposure in the cold-tolerant variety but significantly suppressed in the cold-susceptible variety. Moreover,therecombinant yeast (Pichia pastoris) overexpressingScCaM1grew better than the wild-type yeast strain under cold and oxidative stresses. It was revealed that theScCaM1played a positive role in reactive oxygen species scavenging and conferred enhanced cold and oxidative stress tolerance to cells. This study provided comprehensive information on the CaM gene family inSaccharumand would facilitate further investigation of their functional characterization.

Low specific growth rate and temperature in fed-batch cultures of a beta-propeller phytase producing Pichia pastoris strain under GAP promoter trigger increased KAR2 and PSA1-1 gene expression yielding enhanced extracellular productivity

Previously, we showed that the methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii) could produce and secrete the beta-propeller phytase FTEII in an active form under the control of the AOX1 promoter and methanol as the inductor. In this work, we engineered P. pastoris strains to construct a constitutive P. pastoris expression system (GAP promoter) and extracellularly produce the phytase FTEII. We optimized the culture conditions to increase the extracellular volumetric phytase productivity (Qp) and evaluated the impact of the optimization process on the physiological response of the host. Moreover, we analyzed the expression levels of the FTEII gene and endogenous genes for P. pastoris cells in cultures with the lowest and highest Qp to understand which processes (from heterologous gene expression to protein secretion) might be responsible for the increase in Qp. The results indicate that a low specific growth rate and temperature in the fed-batch phase increases the Qp, which was correlated with an upregulation of the KAR2 and PSA1–1/MPG1 genes rather than increased heterologous gene transcription.

Heterologous synthesis of N and M fragments of capsid protein VP2 of avian infectious bursal disease virus in yeast Pichia pastoris

BACKGROUND: Infectious bursal disease is one of the most common and economically important viral diseases of birds. Vaccination is currently the most effective way to control IBD. Subunit vaccines contain only the immunogenic protein of the pathogen or its fragments, but do not contain other proteins, lipopolysaccharides, toxins, which avoids vaccination side effects.
 AIM: The aim of the work was to obtain yeast Pichia pastoris strains that synthesize and secrete the fragments of major coat protein VP2 of the infectious bursal disease virus.
 MATERIALS AND METHODS: The DNA sequences encoding the N and M fragments of VP2 protein, were cloned under the control of the AOX1 gene promoter and integrated into the genome of P. pastoris strains X-33 (mut+) and GS115 (his4).
 RESULTS: The analysis of proteins secreted by the obtained strains revealed the presence of additional proteins with a molecular weights corresponding to the target proteins.
 CONCLUSIONS: Thus, the obtained strains of P. pastoris producers of N and M fragments of VP2 protein can be used for antigen production to create a subunit vaccine against avian IBD.

Construction of an l-Tyrosine Chassis in Pichia pastoris Enhances Aromatic Secondary Metabolite Production from Glycerol.

Bioactive plant-based secondary metabolites such as stilbenoids, flavonoids, and benzylisoquinoline alkaloids (BIAs) are produced from l-tyrosine (l-Tyr) and have a wide variety of commercial applications. Therefore, building a microorganism with high l-Tyr productivity (l-Tyr chassis) is of immense value for large-scale production of various aromatic compounds. The aim of this study was to develop an l-Tyr chassis in the nonconventional yeast Pichia pastoris (Komagataella phaffii) to produce various aromatic secondary metabolites (resveratrol, naringenin, norcoclaurine, and reticuline). Overexpression of feedback-inhibition insensitive variants of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (ARO4K229L) and chorismate mutase (ARO7G141S) enhanced l-Tyr titer from glycerol in P. pastoris. These engineered P. pastoris strains increased the titer of resveratrol, naringenin, and norcoclaurine by 258, 244, and 3400%, respectively, after expressing the corresponding heterologous pathways. The titer of resveratrol and naringenin further increased by 305 and 249%, resulting in yields of 1825 and 1067 mg/L, respectively, in fed-batch fermentation, which is the highest titer from glycerol reported to date. Furthermore, the resveratrol-producing strain accumulated intermediates in the shikimate pathway. l-Tyr-derived aromatic compounds were produced using crude glycerol byproducts from biodiesel fuel (BDF) production. Constructing an l-Tyr chassis is a promising strategy to increase the titer of various aromatic secondary metabolites and P. pastoris is an attractive host for high-yield production of l-Tyr-derived aromatic compounds from glycerol.

Effect of an intermolecular disulfide bond introduced into the first loop of CH1 domain of Adalimumab Fab on thermal stability and antigen-binding activity

The introduction of intermolecular disulfide bonds by amino acid mutations is an effective method for stabilizing dimeric proteins. X-ray crystal structure of Fab of a therapeutic antibody, adalimumab, revealed the first loop of the CH1 domain to be partially unsolved at position 135-141. To find new sites for the introduction of intermolecular disulfide bonds in adalimumab Fab, Fab mutants targeting the unsolved region were predicted using molecular simulation software. Four Fab mutants, H:K137C-L:I117C, H:K137C-L:F209C, H:S138C-L:F116C and H:S140C-L:S114C, were expressed in the methylotrophic yeast Pichia pastoris. SDS-PAGE analysis of these mutants indicated that H:K137C-L:F209C, H:S138C-L:F116C and H:S140C-L:S114C mutants mostly formed intermolecular disulfide bonds, whereas some H:K137C-L:I117C mutants formed intermolecular disulfide bonds and some did not. Differential scanning calorimetry measurements showed increased thermal stability in all Fab mutants with engineered disulfide bonds. The bio-layer interferometry measurements, for binding of the antigen tumor necrotic factor α, indicated that Fab mutants had less antigen-binding activity than wild-type Fab. In particular, the KD value of H:K137C-L:F209C was ~17 times higher than that of wild-type Fab. Thus, we successfully introduced intermolecular disulfide bonds between the first loop region of the CH1 and CL domains and observed that it increases the thermostability of Fab and affects the antigen-binding activity.

Comparative proteomics analysis of Pichia pastoris cultivating in glucose and methanol

The methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii) has been extensively engineered for protein production, and is attracting attention as a chassis cell for methanol biotransformation toward production of small molecules. However, the relatively unclear methanol metabolism hampers the metabolic rewiring to improve the biosynthetic efficiency. We here performed a label-free quantitative proteomic analysis of Pichia pastoris when cultivated in minimal media containing methanol and glucose, respectively. There were 243, 158 up-regulated proteins and 244, 304 down-regulated proteins in log and stationary phase, respectively, when cultivated in methanol medium compared with that of glucose medium. Peroxisome enrichment further improved the characterization of more differentially expressed proteins (481 proteins in log phase and 524 proteins in stationary phase). We demonstrated the transaldolase isoenzyme (Tal2, Protein ID: C4R244) was highly up-regulated in methanol medium cultivation, which plays an important role in methanol utilization. Our work provides important information for understanding methanol metabolism in methyltrophic yeast and will help to engineer methanol biotransformation in P. pastoris.

92 Effects of Increasing Dose of a Novel E. coli Phytase on Total Tract Digestibility of Minerals and Energy in Pigs

Abstract The objective was to test the hypothesis that an increasing dose of a novel thermostable 6-phytase that was derived from E. coli and expressed in a Pichia Pastoris yeast (Superphy; Hanley International, LLC, Belmont, MA) increases apparent total tract digestibility (ATTD) of minerals and energy by pigs. Forty-eight barrows (initial weight: 22.61 kg) were housed in metabolism crates and fed 6 diets based on corn and soybean meal. The positive control (PC) diet contained P at the requirement. The negative control (NC) diet contained no feed phosphate, but contained 0, 250, 500, 1,000, or 2,000 phytase unit/kg. Feces were collected for 4 d after 5 d of adaptation. Dried and ground fecal samples were analyzed for dry matter, energy, Ca, P and other minerals. Data were analyzed using a model that included diet as fixed variable and contrast statements were used to compare PC and NC diets and to demonstrate linear and quadratic effects of phytase. Results indicated that ATTD of dry matter and energy and concentration of digestible energy in NC without phytase were greater (P < 0.05) than in PC, but ATTD of ash, K, Mg, Na, Cu, Fe, Mn, and Zn was not different between the 2 diets. There was no effect of phytase on ATTD of dry matter and energy or on digestible energy in diets. As dietary phytase increased, ATTD of ash and Mg increased (linear, P < 0.05) and ATTD of Ca and the standardized total tract digestibility (STTD) of P also increased (quadratic, P < 0.05), but ATTD of K, Na, Cu, Fe, Mn, and Zn was not affected by phytase. In conclusion, the novel enhanced E. Coli based phytase increased ATTD of Ca, P, and Mg and STTD of P if included in a diet containing Ca and P below requirements.

The fluorescent protein iLOV as a reporter for screening of high-yield production of antimicrobial peptides in Pichia pastoris.

SummaryThe methylotrophic yeast Pichia pastoris is commonly used for the production of recombinant proteins at scale. The identification of an optimally overexpressing strain following transformation can be time and reagent consuming. Fluorescent reporters like GFP have been used to assist identification of superior producers, but their relatively big size, maturation requirements and narrow temperature range restrict their applications. Here, we introduce the use of iLOV, a flavin‐based fluorescent protein, as a fluorescent marker to identify P. pastoris high‐yielding strains easily and rapidly. The use of this fluorescent protein as a fusion partner is exemplified by the production of the antimicrobial peptide NI01, a difficult target to overexpress in its native form. iLOV fluorescence correlated well with protein expression level and copy number of the chromosomally integrated gene. An easy and simple medium‐throughput plate‐based screen directly following transformation is demonstrated for low complexity screening, while a high‐throughput method using fluorescence‐activated cell sorting (FACS) allowed for comprehensive library screening. Both codon optimization of the iLOV_NI01 fusion cassettes and different integration strategies into the P. pastoris genome were tested to produce and isolate a high‐yielding strain. Checking the genetic stability, process reproducibility and following the purification of the active native peptide are eased by visualization of and efficient cleavage from the iLOV reporter. We show that this system can be used for expression and screening of several different antimicrobial peptides recombinantly produced in P. pastoris.

Fungal pretreatments of Napier grass and sugarcane leaves for high recovery of lignocellulosic enzymes and methane production

Effective biological pretreatment is a sought-after approach to solving the dilemma between environmental protection versus sustainable and economical production of bio-based products. In many countries, grasses are among the most attractive and widely cultivated crops for bioenergy. Here, Napier grass, including Alafal, Pakchong 1, and Merkeron varieties, and sugarcane were investigated for their potential applications in lignocellulosic enzyme and biomethane production. This was accomplished through biological pretreatments using a wood-rotting fungal Xylaria species under solid-state fermentation (SoSF) at ambient temperatures. Merkeron produced an enzymatic cocktail of xylanase and cellulase at activities of 103.1 ± 2.5 or 42.4 ± 1.61 U/g, respectively, on day 14. In contrast, using yeast Pichia pastoris expressing engineered Bacillus firmus xylanases, the highest xylanase activities were achieved between 58 and 61 U/g using Pakchong 1 or sugarcane leaves under submerged fermentation (SmF) after 2–4 days. Preferential fermentation mode between SoSF and SmF, utilized by fungi or yeast was observed during lignocellulosic saccharification. The Highest cellulase and xylanase productivity was achieved at 0.24–0.51 U/g/h, using the yeast-pretreatment via SmF. Alternatively, Xylaria-pretreated Napier grass mixed with cow manure effectively increased the daily biogas yield up to 212.8 ± 2.3 cm3/gVS with methane content over 80% and methane productivity of 2.31%/day, respectively. Thus, this study highlights potential uses of fungi in eco-friendly pretreatment of abundant feedstocks for optimal production of lignocellulosic enzymes and green energy biomethane.

Assessment of recombinant glutathione-S-transferase (HaGST-8) silica nano-conjugates for effective removal of pesticides.

Diverse glutathione-S-transferases (GSTs) are produced by insect pests including Helicoverpa armigera (HaGSTs) for detoxification of insecticides or xenobiotic compounds that they encounter. In an earlier study, the HaGST-8 gene was isolated from H. armigera larvae exposed to pesticide mixtures and the recombinant protein was expressed in the yeast Pichia pastoris. In this investigation, HaGST-8 was successfully immobilized on glutaraldehyde-activated APTES functionalized silica nanoparticles to obtain SiAPT-HaGST-8 nano-conjugates. Although enzyme activity associated with these conjugates was comparable to that of free HaGST-8, the specific activity of the former was found to be 1.25 times higher than the latter. In comparison with the free enzyme (that demonstrated a pH optimum of 9.0), for the nano-conjugates, the pH range was extended between pH 8.0 to 9.0. The optimum temperature for activity of both forms of the enzyme was found to be 30°C. Stability of the enzyme was improved from 20d for free HaGST-8 to 30d for SiAPT-HaGST-8 nano-conjugates. Some loss in GST activity was detected after every reuse cycle of nano-conjugates and in all, 63% reduction was observed after three cycles. When 3 kinds of pesticides (namely, chlorpyrifos, dichlorvos and cypermethrin) were reacted with SiAPT-HaGST-8, more than 80% reduction in levels were observed. On the basis of the results obtained, the use of such silica nanoparticle-based systems for stable enzyme conjugation followed by effective removal of pesticides from aqueous media is envisaged.

Production of selenomethionine labeled polyglycine hydrolases in Pichia pastoris

Producing recombinant proteins with incorporated selenomethionine (SeMet) facilitates solving X-ray crystallographic structures of novel proteins. Production of SeMet labeled proteins in the yeast Pichia pastoris (syn. Komagataella phaffii) is difficult because SeMet is mildly toxic, reducing protein expression levels. To counteract this yield loss for a novel protease, Epicoccum sorghi chitinase modifying protein (Es-cmp), a novel disease promoting protease secreted by these plant pathogenic fungi, we isolated a yeast strain that secreted more protein. By comparing the expression level of 48 strains we isolated one that produced significantly more protein. This strain was found to be gene dosed, having four copies of the expression cassette. After optimization the strain produced Es-cmp in defined media with SeMet at levels nearly equal to that of the original strain in complex media. Also, we produced SeMet labeled protein for a homologous protease from the fungus Fusarium vanettenii, Fvan-cmp, by directly selecting a gene dosed strain on agar plates with increased zeocin. Linearization of plasmid with PmeI before electroporation led to high numbers of 1 mg/mL zeocin resistant clones with significantly increased expression compared to those selected on 0.1 mg/mL. The gene dosed strains expressing Es-cmp and Fvan-cmp allowed production of 8.5 and 16.8 mg of SeMet labeled protein from 500 mL shake flask cultures. The results demonstrate that selection of P. pastoris expression strains by plating after transformation on agar with 1 mg/mL zeocin rather than the standard 0.1 mg/mL directly selects gene dosed strains that can facilitate production of selenomethionine labeled proteins.

Determination of the Optimum Annealing Temperature in the Production of CuO Nanoparticles with Antimicrobial Properties

A copper oxide (CuO) nanoparticle, a transition metal oxide with a wide variety of easy production methods, can be used as an antimicrobial agent against various types of bacteria. CuO nanoparticles were produced by the sol–gel method, annealed and structural characteristics and antimicrobial properties of these particles were investigated. Single-phase monoclinic of CuO nanoparticle formation was confirmed by X-ray powder diffraction (XRD) spectra, FTIR techniques, differential scanning calorimetry with thermal gravimetry were used to characterize. It was determined that annealing in the temperature range of 150–900∘C affects both structure and particle size and antimicrobial characteristics. CuO nanoparticle size was found to be between about 25–70[Formula: see text]nm at 150–900∘C annealing temperature, which does not have this wide temperature range in the literature. These results were supported by the TEM micrographs of the CuO nanoparticles observed at 150∘C and 900∘C. The antimicrobial activity of the synthesized nanoparticles was tested with the disc diffusion assay against Staphylococcus aureus, Escherichia coli, Agrobacterium tumefaciens, and yeast Pichia pastoris. The antimicrobial properties of the nanoparticles first increased and then decreased and disappeared as the annealing temperature increased. The antimicrobial properties of CuO nanoparticles disappeared at 750∘C and above, while the maximum antimicrobial effect was at 300∘C. The inhibition zones were examined and similar results were observed in all tested microorganisms.

Going beyond the limit: Increasing global translation activity leads to increased productivity of recombinant secreted proteins in Pichia pastoris

Yeasts are widely used cell factories for commercial heterologous protein production, however, specific productivities are usually tightly coupled to biomass formation. This greatly impacts production processes, which are commonly not run at the maximum growth rate, thereby resulting in suboptimal productivities. To tackle this issue, we evaluated transcriptomics datasets of the yeast Pichia pastoris (syn. Komagataella phaffii), which is known for its high secretory efficiency and biomass yield. These showed a clear downregulation of genes related to protein translation with decreasing growth rates, thus revealing the yeast translation machinery as cellular engineering target. By overexpressing selected differentially expressed translation factors, translation initiation was identified to be the main rate-limiting step. Specifically, overexpression of factors associated with the closed-loop conformation, a structure that increases stability and rates of translation initiation before start codon scanning is initiated, showed the strongest effects. Overexpression of closed-loop factors alone or in combination increased titers of different heterologous proteins by up to 3-fold in fed-batch processes. Furthermore, translation activity, correlating to the obtained secreted recombinant protein yields, selected transcript levels and total protein content were higher in the engineered cells. Hence, translation factor overexpression, globally affects the cell. Together with the observed impact on the transcriptome and total protein content, our results indicate that the capacity of P. pastoris for protein production is not at its limit yet.

Influence of Dissolved Oxygen Level on Chitin-Glucan Complex and Mannans Production by the Yeast Pichia pastoris.

The yeast Pichia pastoris was cultivated under different dissolved oxygen (DO) levels (5, 15, 30 and 50% of the air saturation) to evaluate its impact on the production of the cell-wall polysaccharide chitin–glucan complex (CGC) and mannans. Decreasing the DO level from 50 to 15% had no significant impact on cell growth but substrate conversion into biomass was improved. Under such conditions, a mannans content in the biomass of 22 wt% was reached, while the CGC content in the biomass was improved from 15 to 18 wt%, confirming that the DO level also impacted on P. pastoris cell-wall composition. Overall mannans and CGC volumetric productivity values of 10.69 and 8.67 g/(L. day) were reached, respectively. On the other hand, the polymers’ composition was not significantly affected by decreasing the DO level. These results demonstrated that considerable energy savings can be made in the polysaccharide production process by reducing the DO level during cultivation of P. pastoris by improving the overall polymers’ productivity without altering their composition. This has impact on the polysaccharide production costs, which is of considerable relevance for process scale-up and products’ commercialization.

Enhancing Homologous Recombination Efficiency in Pichia pastoris for Multiplex Genome Integration Using Short Homology Arms.

There is a growing interest in establishing the methylotrophic yeast Pichia pastoris as microbial cell factories for producing fuels, chemicals, and natural products, particularly with methanol as the feedstock. Although CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based genome editing technology has been established for the integration of multigene biosynthetic pathways, long (500-1000 bp) homology arms are generally required, probably due to low homologous recombination (HR) efficiency in P.pastoris. To achieve efficient genome integration of heterologous genes with short homology arms, we aimed to enhance HR efficiency by introducing the recombination machinery from Saccharomyces cerevisiae. First, we overexpressed HR related genes, including RAD52, RAD59, MRE11, and SAE2, and evaluated their effects on genome integration efficiency. Then, we constructed HR efficiency enhanced P.pastoris, which enabled single-, two-, and three-loci integration of heterologous gene expression cassettes with ∼40 bp homology arms with efficiencies as high as 100%, ∼98%, and ∼81%, respectively. Finally, we demonstrated the construction of β-carotene producing strain and the optimization of betaxanthin producing strain in a single step. The HR efficiency enhanced P.pastoris strains can be used for the construction of robust cell factories, and our machinery engineering strategy can be employed for the modification of other nonconventional yeasts.

Nitroreduction of flutamide by Cunninghamella elegans NADPH: Cytochrome P450 reductase

The microbial model of mammalian drug metabolism, Cunninghamella elegans, has three cytochrome P450 reductase genes in its genome: g1631 (CPR_A), g4301 (CPR_B), and g7609 (CPR_C). The nitroreductase activity of the encoded enzymes was investigated via expression of the genes in the yeast Pichia pastoris X33. Whole cell assays with the recombinant yeast demonstrated that the reductases converted the anticancer drug flutamide to the nitroreduced metabolite that was also produced from the same substrate when incubated with human NADPH: cytochrome P450 reductase. The nitroreductase activity extended to other substrates such as the related drug nilutamide and the environmental contaminants 1-nitronaphthalene and 1,3-dinitronaphthalene. Comparative experiments with cell lysates of recombinant yeast were conducted under aerobic and reduced oxygen conditions and demonstrated that the reductases are oxygen sensitive.