Non-conventional Yeast Pichia Research Articles

Advances in the Application of the Non-Conventional Yeast Pichia kudriavzevii in Food and Biotechnology Industries.

Pichia kudriavzevii is an emerging non-conventional yeast which has attracted increased attention for its application in food and biotechnology areas. It is widespread in various habitats and often occurs in the spontaneous fermentation process of traditional fermented foods and beverages. The contributions of P. kudriavzevii in degrading organic acid, releasing various hydrolase and flavor compounds, and displaying probiotic properties make it a promising starter culture in the food and feed industry. Moreover, its inherent characteristics, including high tolerance to extreme pH, high temperature, hyperosmotic stress and fermentation inhibitors, allow it the potential to address technical challenges in industrial applications. With the development of advanced genetic engineering tools and system biology techniques, P. kudriavzevii is becoming one of the most promising non-conventional yeasts. This paper systematically reviews the recent progress in the application of P. kudriavzevii to food fermentation, the feed industry, chemical biosynthesis, biocontrol and environmental engineering. In addition, safety issues and current challenges to its use are discussed.

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.

Artificial Transcription Factors for Tuneable Gene Expression in Pichia pastoris.

The non-conventional yeast Pichia pastoris (syn. Komagataella phaffii) has become a powerful eukaryotic expression platform for biopharmaceutical and biotechnological applications on both laboratory and industrial scales. Despite the fundamental role that artificial transcription factors (ATFs) play in the orthogonal control of gene expression in synthetic biology, a limited number of ATFs are available for P. pastoris. To establish orthogonal regulators for use in P. pastoris, we characterized ATFs derived from Arabidopsis TFs. The plant-derived ATFs contain the binding domain of TFs from the plant Arabidopsis thaliana, in combination with the activation domains of yeast GAL4 and plant EDLL and a synthetic promoter harboring the cognate cis-regulatory motifs. Chromosomally integrated ATFs and their binding sites (ATF/BSs) resulted in a wide spectrum of inducible transcriptional outputs in P. pastoris, ranging from as low as 1- to as high as ∼63-fold induction with only small growth defects. We demonstrated the application of ATF/BSs by generating P. pastoris cells that produce β-carotene. Notably, the productivity of β-carotene in P. pastoris was ∼4.8-fold higher than that in S. cerevisiae, reaching ∼59% of the β-carotene productivity obtained in a S. cerevisiae strain optimized for the production of the β–carotene precursor, farnesyl diphosphate, by rewiring the endogenous metabolic pathways using plant-derived ATF/BSs. Our data suggest that plant-derived regulators have a high degree of transferability from S. cerevisiae to P. pastoris. The plant-derived ATFs, together with their cognate binding sites, powerfully increase the repertoire of transcriptional regulatory modules for the tuning of protein expression levels required in metabolic engineering or synthetic biology in P. pastoris.

Structural insights into the desymmetrization of bulky 1,2-dicarbonyls through enzymatic monoreduction

Benzil reductases are dehydrogenases preferentially active on aromatic 1,2-diketones, but the reasons for this peculiar substrate recognition have not yet been clarified. The benzil reductase (KRED1-Pglu) from the non-conventional yeast Pichia glucozyma showed excellent activity and stereoselectivity in the monoreduction of space-demanding aromatic 1,2-dicarbonyls, making this enzyme attractive as biocatalyst in organic chemistry. Structural insights into the stereoselective monoreduction of 1,2-diketones catalyzed by KRED1-Pglu were investigated starting from its 1.77 Å resolution crystal structure, followed by QM and classical calculations; this study allowed for the identification and characterization of the KRED1-Pglu reactive site. Once identified the recognition elements involved in the stereoselective desymmetrization of bulky 1,2-dicarbonyls mediated by KRED1-Pglu, a mechanism was proposed together with an in silico prediction of substrates reactivity.

Sustainable Second-Generation Bioethanol Production from Enzymatically Hydrolyzed Domestic Food Waste Using Pichia anomala as Biocatalyst

In the current study, a domestic food waste containing more than 50% of carbohydrates was assessed as feedstock to produce second-generation bioethanol. Aiming to the maximum exploitation of the carbohydrate fraction of the waste, its hydrolysis via cellulolytic and amylolytic enzymatic blends was investigated and the saccharification efficiency was assessed in each case. Fermentation experiments were performed using the non-conventional yeast Pichia anomala (Wickerhamomyces anomalus) under both separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) modes to evaluate the conversion efficiencies and ethanol yields for different enzymatic loadings. It was shown that the fermentation efficiency of the yeast was not affected by the fermentation mode and was high for all handlings, reaching 83%, whereas the enzymatic blend containing the highest amount of both cellulolytic and amylolytic enzymes led to almost complete liquefaction of the waste, resulting also in ethanol yields reaching 141.06 ± 6.81 g ethanol/kg waste (0.40 ± 0.03 g ethanol/g consumed carbohydrates). In the sequel, a scale-up fermentation experiment was performed with the highest loading of enzymes in SHF mode, from which the maximum specific growth rate, μmax, and the biomass yield, Yx/s, of the yeast from the hydrolyzed waste were estimated. The ethanol yields that were achieved were similar to those of the respective small scale experiments reaching 138.67 ± 5.69 g ethanol/kg waste (0.40 ± 0.01 g ethanol/g consumed carbohydrates).

Exchange of endogenous and heterogeneous yeast terminators in Pichia pastoris to tune mRNA stability and gene expression.

In the yeast Saccharomyces cerevisiae, terminator sequences not only terminate transcription but also affect expression levels of the protein-encoded upstream of the terminator. The non-conventional yeast Pichia pastoris (syn. Komagataella phaffii) has frequently been used as a platform for metabolic engineering but knowledge regarding P. pastoris terminators is limited. To explore terminator sequences available to tune protein expression levels in P. pastoris, we created a ‘terminator catalog’ by testing 72 sequences, including terminators from S. cerevisiae or P. pastoris and synthetic terminators. Altogether, we found that the terminators have a tunable range of 17-fold. We also found that S. cerevisiae terminator sequences maintain function when transferred to P. pastoris. Successful tuning of protein expression levels was shown not only for the reporter gene used to define the catalog but also using betaxanthin production as an example application in pathway flux regulation. Moreover, we found experimental evidence that protein expression levels result from mRNA abundance and in silico evidence that levels reflect the stability of mRNA 3′-UTR secondary structure. In combination with promoter selection, the novel terminator catalog constitutes a basic toolbox for tuning protein expression levels in metabolic engineering and synthetic biology in P. pastoris.

Transcriptional profiling reveals molecular basis and the role of arginine in response to low-pH stress in Pichia kudriavzevii

The non-conventional yeast Pichia kudriavzevii is considered to be a promising biotechnological host for the production of organic acids under low-pH conditions. However, little is known about the low-pH stress response in P.kudriavzevii, which significantly restricts its future development. In this study, P.kudriavzevii N-X showed great tolerance to low-pH stress, but the cell aggregation upon extremely acidic conditions might be unfavorable for low-pH fermentation. We therefore conducted RNA-Seq to compare global gene expression of P.kudriavzevii N-X in response to different pH stresses. Totally 434 genes were identified to be differentially expressed genes (DEGs), and annotation and enrichment analysis suggested that multiple genes associated with regulation of membrane lipid composition, filamentous growth and arginine metabolism were differentially expressed. The increased specific activity of arginase and intracellular ammonia concentration of P.kudriavzevii cultured at pH 2.0 further implied potential roles of arginine in response to extreme low-pH conditions. Extracellular supplementation of 5mM arginine resulted in increased pHi and cell growth at pH 2.0, meanwhile the cell aggregation was partially suppressed. Additionally, overexpression of ARG J involving in arginine synthesis can also enhance the cell growth and reduce the aggregation effect. These results suggested that increasing arginine flux might be an alternative approach in the developing of P.kudriavzevii as a platform host for production of organic acids under low-pH conditions.

Efficient gene disruption by posttransformational directed internal homologous recombination in Pichia pastoris

In this study, we designed an improved method to construct gene-deficient strain in non-conventional yeast Pichia pastoris. This method achieved high efficiency of gene deletion without disruption of NHEJ-related genes. The disruption efficiency of Och1 reached about 80%. This simple method will be a new useful molecular tool for P. pastoris.

Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing.

The non-conventional yeast Pichia pastoris is a popular host for recombinant protein production in scientific research and industry. Typically, the expression cassette is integrated into the genome via homologous recombination. Due to unknown integration events, a large clonal variability is often encountered consisting of clones with different productivities as well as aberrant morphological or growth characteristics. In this study, we analysed several clones with abnormal colony morphology and discovered unpredicted integration events via whole genome sequencing. These include (i) the relocation of the locus targeted for replacement to another chromosome (ii) co-integration of DNA from the E. coli plasmid host and (iii) the disruption of untargeted genes affecting colony morphology. Most of these events have not been reported so far in literature and present challenges for genetic engineering approaches in this yeast. Especially, the presence and independent activity of E. coli DNA elements in P. pastoris is of concern. In our study, we provide a deeper insight into these events and their potential origins. Steps preventing or reducing the risk for these phenomena are proposed and will help scientists working on genetic engineering of P. pastoris or similar non-conventional yeast to better understand and control clonal variability.

Stereoselective reduction of aromatic ketones by a new ketoreductase from Pichia glucozyma

A new NADPH-dependent benzil reductase (KRED1-Pglu) was identified from the genome of the non-conventional yeast Pichia glucozyma CBS 5766 and overexpressed in E. coli. The new protein was characterised and reaction parameters were optimised for the enantioselective reduction of benzil to (S)-benzoin. A thorough study of the substrate range of KRED1-Pglu was conducted; in contrast to most other known ketoreductases, KRED1-Pglu prefers space-demanding substrates, which are often converted with high stereoselectivity. A molecular modelling study was carried out for understanding the structural determinants involved in the stereorecognition experimentally observed and unpredictable on the basis of steric properties of the substrates. As a result, a new useful catalyst was identified, enabling the enantioselective preparation of different aromatic alcohols and hydroxyketones.

Genome shuffling of the nonconventional yeast Pichia anomala for improved sugar alcohol production.

BackgroundSugar alcohols have been widely applied in the fields of food and medicine owing to their unique properties. Compared to chemical production, microbial production of sugar alcohols has become attractive because of its environmentally friendly and sustainable characteristics. Our previous study identified the nonconventional yeast Pichia anomala TIB-x229 as a potential producer of sugar alcohols from glucose. To further improve strain performance, we combined genome shuffling with optimized high throughput screening methods for the directed improvement of nonconventional yeast and complex phenotypes.ResultsTo accelerate strain improvement, a practical genome shuffling procedure was developed and successfully applied in the nonconventional yeast P. anomala to increase sugar alcohol production. Through two rounds of genome shuffling, an improved P. anomala isolate GS2-3 could produce 47.1 g/L total sugar alcohols from 100 g/L glucose, which was 32.3% higher than the original strain. In this process, a simple and accurate colorimetric assay was optimized and used for high throughput screening of sugar alcohol-producing strains. Moreover, a fluorescence-activated cell sorting method was developed to efficiently screen protoplast fusions for genome shuffling of nonconventional yeast.ConclusionAn efficient genome shuffling procedure was developed and applied to enhance the sugar alcohol production of the nonconventional yeast P. anomala. Our results provide a general platform for strain improvement of polyol-producing microorganisms or nonconventional microorganisms in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0303-8) contains supplementary material, which is available to authorized users.

Impact of multi-metals (Cd, Pb and Zn) exposure on the physiology of the yeast Pichia kudriavzevii.

Metal contamination of the environment is frequently associated to the presence of two or more metals. This work aimed to study the impact of a mixture of metals (Cd, Pb and Zn) on the physiology of the non-conventional yeast Pichia kudriavzevii. The incubation of yeast cells with 5 mg/l Cd, 10 mg/l Pb and 5 mg/l Zn, for 6 h, induced a loss of metabolic activity (assessed by FUN-1 staining) and proliferation capacity (evaluated by a clonogenic assay), with a small loss of membrane integrity (measured by trypan blue exclusion assay). The staining of yeast cells with calcofluor white revealed that no modification of chitin deposition pattern occurred during the exposure to metal mixture. Extending for 24 h, the exposure of yeast cells to metal mixture provoked a loss of membrane integrity, which was accompanied by the leakage of intracellular components. A marked loss of the metabolic activity and the loss of proliferation capacity were also observed. The analysis of the impact of a single metal has shown that, under the conditions studied, Pb was the metal responsible for the toxic effect observed in the metal mixture. Intracellular accumulation of Pb seems to be correlated with the metals' toxic effects observed.

Biotransformation of aromatic ketones and ketoesters with the non-conventional yeast Pichia glucozyma

The non-conventional yeast Pichia glucozyma CBS 5766 has been used for the biotransformation of different aromatic ketones and ketoesters. The growth and biotransformation conditions were optimised for the reduction of acetophenone and under optimised conditions, propiophenone, butyrophenone and valerophenone were reduced to the corresponding (S)-alcohols with high yields and enantioselectivity. Ketoreductase(s) of Pichia glucozyma showed high catalytic activities also towards aromatic β- and γ-ketoesters, being often competitive with esterase(s). These concurrent activities allowed for the preparation of hydroxyesters, hydroxyacids and lactones often in a very selective manner.

Low pH d-xylonate production with Pichia kudriavzevii

d-Xylonic acid is one of the top 30 most desirable chemicals to be derived from biomass sugars identified by the US Department of Energy, being applicable as a non-food substitute for d-gluconic acid and as a platform chemical. We engineered the non-conventional yeast Pichia kudriavzevii VTT C-79090T to express a d-xylose dehydrogenase coding gene from Caulobacter crescentus. With this single modification the recombinant P. kudriavzevii strain produced up to 171gL−1 of d-xylonate from 171gL−1d-xylose at a rate of 1.4gL−1h−1 and yield of 1.0g [g substrate consumed]−1, which was comparable with d-xylonate production by Gluconobacter oxydans or Pseudomonas sp. The productivity of the strain was also remarkable at low pH, producing 146gL−1d-xylonate at 1.2gL−1h−1 at pH 3.0. This is the best low pH production reported for d-xylonate. These results encourage further development towards industrial scale production.

Metabolic engineering of the non-conventional yeast Pichia ciferrii for production of rare sphingoid bases

The study describes the identification of sphingolipid biosynthesis genes in the non-conventional yeast Pichia ciferrii, the development of tools for its genetic modification as well as their application for metabolic engineering of P. ciferrii with the goal to generate strains capable of producing the rare sphingoid bases sphinganine and sphingosine. Several canonical genes encoding ceramide synthase (encoded by PcLAG1 and PcLAF1), alkaline ceramidase (PcYXC1) and sphingolipid C-4-hydroxylase(PcSYR2), as well as structural genes for dihydroceramide Δ4-desaturase (PcDES1) and sphingolipid Δ8-desaturase (PcSLD1) were identified, indicating that P. ciferrii would be capable of synthesizing desaturated sphingoid bases, a property not ubiquitously found in yeasts.In order to convert the phytosphingosine-producing P. ciferrii wildtype into a strain capable of producing predominantly sphinganine, Syringomycin E-resistant mutants were isolated. A stable mutant almost exclusively producing high levels of acetylated sphinganine was obtained and used as the base strain for further metabolic engineering. A metabolic pathway required for the three-step conversion of sphinganine to sphingosine was implemented in the sphinganine producing P. ciferrii strain and subsequently enhanced by screening for the appropriate heterologous enzymes, improvement of gene expression and codon optimization. These combined efforts led to a strain capable of producing 240mgL−1 triacetyl sphingosine in shake flask, with tri- and diacetyl sphinganine being the main by-products. Lab-scale fermentation of this strain resulted in production of up to 890mgkg−1 triacetyl sphingosine. A third by-product was unequivocally identified as triacetyl sphingadienine. It could be shown that inactivation of the SLD1 gene in P. ciferrii efficiently suppresses triacetyl sphingadienine formation. Further improvement of the described P. ciferrii strains will enable a biotechnological route to produce sphinganine and sphingosine for cosmetic and pharmaceutical applications.

High-level production of tetraacetyl phytosphingosine (TAPS) by combined genetic engineering of sphingoid base biosynthesis and L-serine availability in the non-conventional yeast Pichia ciferrii

The non-conventional yeast Pichia ciferrii is known to secrete the sphingoid long-chain base phytosphingosine in a tetraacetylated form (TAPS). Sphingolipids are important ingredients in cosmetic applications as they play important roles in human skin. Our work aimed to improve TAPS production by genetic engineering of P. ciferrii. In the first step we improved precursor availability by blocking degradation of L-serine, which is condensed with palmitoyl-CoA by serine palmitoyltransferase in the first committed step of sphingolipid biosynthesis. Successive deletion of two genes, SHM1 and SHM2, encoding L-serine hydroxymethyltransferases, and of CHA1 encoding L-serine deaminase, resulted in a strain producing 65mg(TAPS)g−1(cdw), which is a threefold increase in comparison with the parental strain. Attempts to increase the metabolic flux into and through the L-serine biosynthesis pathway did not improve TAPS production. However, genetic engineering of the sphingolipid pathway further increased secretion of TAPS. Blocking of sphingoid long-chain base phosphorylation by deletion of the LCB kinase gene PcLCB4 resulted in a further increase in TAPS production by 78% and significant secretion of the direct precursor of phytosphingosine, sphinganin, in a triacetylated form (TriASa). Overproduction of two serine palmitoyltransferase subunits, Lcb1 and Lcb2, together with a deletion of the gene ORM12 encoding a putative negative regulator of sphingolipid synthesis resulted in a strain producing 178mg(TAPS)g−1(cdw). Additional overproduction of the C4-hydroxylase Syr2 converting sphinganine to phytosphingosine reduced TriASa production and further improved TAPS production. The final recombinant P. ciferrii strain produced up to 199mg(TAPS)g−1(cdw) with a maximal production rate of 8.42mg×OD600nm−1h−1 and a titer of about 2gL−1, and should be applicable for industrial TAPS production.

Enantioselective reduction and deracemisation using the non-conventional yeast Pichia glucozyma in water/organic solvent biphasic systems: preparation of ( S)-1,2-diaryl-2-hydroxyethanones (benzoins)

Water/organic solvent two-liquid-phase systems have been successfully applied in the synthesis of enantiomerically pure ( S)-benzoin through two different methodologies catalysed by whole cells from the non-conventional yeast Pichia glucozyma: the stereoselective monoreduction of benzil and the deracemisation of benzoin. The presence of the organic solvent influences the redox systems implied in the reactions, avoiding the formation of the corresponding diols, increasing the enantioselectivity and allowing the easy isolation of the products in high yields and excellent enantiomeric excesses. The use of both strategies has been extended to the preparation of different chiral benzoin derivatives.

Chromate-resistant mutants of the yeast Pichia guilliermondii: Selection and properties

Chromate-resistant mutants of the non-conventional yeast Pichia guilliermondii L2 were selected by different methods. The isolated mutants were capable of better growth and higher biomass yield at toxic (1.8–2.4 mM) chromate concentrations than the parent strain. The capacity of the mutants for extracellular chromate reduction and chelation of Cr(III) in the culture liquid was demonstrated. The effectiveness of these processes was shown to correlate with the resistance of P. guilliermondii strains to chromate. Extracellular metabolites of the yeast cells cultivated without chromate were shown to be capable of reducing chromate and forming stable soluble Cr(III)-biocomplexes.