Overexpression of the genes of glycerol catabolism and glycerol facilitator improves glycerol conversion to ethanol in the methylotrophic thermotolerant yeast Ogataea polymorpha

Production of fuel ethanol is one of the possible ways to utilize crude glycerol, substantial amounts of which are produced by biodiesel industry. Earlier, we have described construction of the recombinant strains of methylotrophic thermotolerant yeast Ogataea polymorpha with simultaneous overexpression of the genes PDC1 and ADH1, which produced increased amounts of ethanol from glycerol. In this work, we have further improved these strains by overexpression of genes involved either in oxidative (through dihydroxyacetone) or phosphorylative (through glycerol-3-phosphate) pathway of glycerol catabolism, as well as heterologous gene coding for glycerol transporter FPS1 from Komagataella phaffii (formerly, Pichia pastoris). Obtained recombinant strains produced up to 10.7 g/L of ethanol (with ethanol productivity 30 mg/g of biomass/hr and yield 132 mg/g of consumed glycerol) from pure glycerol and up to 3.55 g/L of ethanol (with ethanol productivity 11.6 mg/g of biomass/hr and yield 72.3 mg/g of consumed glycerol) from crude glycerol as a carbon source, which is approximately 15 times more relative to that of the O. polymorpha wild-type strain and 2.2 more relative to the earlier constructed strain.     

Fed‐batch methanol feeding strategy for recombinant protein production by Pichia pastoris in the presence of co‐substrate sorbitol

Batch‐wise sorbitol addition as a co‐substrate at the induction phase of methanol fed‐batch fermentation by Pichia pastoris (Mut⁺) was proposed as a beneficial recombinant protein production strategy and the metabolic responses to methanol feeding rate in the presence of sorbitol was systematically investigated. Adding sorbitol batch‐wise to the medium provided the following advantages over growth on methanol alone: (a) eliminating the long lag‐phase for the cells and reaching ‘high cell density production’ at t = 24 h of the process (C_X = 70 g CDW/l); (b) achieving 1.8‐fold higher recombinant human erythropoietin (rHuEPO) (at t = 18 h); (c) reducing specific protease production 1.2‐fold; (d) eliminating the lactic acid build‐up period; (e) lowering the oxygen uptake rate two‐fold; and (f) obtaining 1.4‐fold higher overall yield coefficients. The maximum specific alcohol oxidase activity was not affected in the presence of sorbitol, and it was observed that sorbitol and methanol were utilized simultaneously. Thus, in the presence of sorbitol, 130 mg/l rHuEPO was produced at t = 24 h, compared to 80 mg/l rHuEPO (t = 24 h) on methanol alone. This work demonstrates not only the ease and efficiency of incorporating sorbitol to fermentations by Mut⁺ strains of P. pastoris for the production of any bio‐product, but also provides new insights into the metabolism of the methylotrophic yeast P. pastoris. Copyright © 2009 John Wiley & Sons, Ltd.     

Expression of GFP using Pichia pastoris vectors with zeocin or G‐418 sulphate as the primary selectable marker

Pichia pastoris is a popular host organism for expressing heterologous proteins, and various expression vectors for this yeast are currently available. Recently, vectors containing novel dominant antibiotic resistance markers have become a strong and developing field of research for this methylotropic yeast strain. We have developed new P. pastoris expression vectors, the pPICKanMX6 and pPICKanMX6α series. These vectors were constructed by replacing the zeocin resistance gene of the pPICZA, B, C and pPICZαA, B and C vectors with the Tn903 kan^R marker from pFA6a KanMX6, which confers G‐418 sulphate resistance in P. pastoris. The limits of antibiotic resistance in two transformant yeast strains were investigated, and the selection marker was shown to be stably retained. To demonstrate their usefulness, a gene encoding hexa‐histidine‐tagged green fluorescent protein (GFPH6) was cloned into one of the new vectors and GFP expression examined in P. pastoris cells. The protein expression levels using the pPICKanMX6B vector were comparable with that using the original plasmid, based on zeocin resistance as seen by yeast cell fluorescence. Moreover, GFPH6 was able to be isolated by immobilized metal ion affinity chromatography (IMAC) from lysates of both yeast strains. A model reporter construct has been used to demonstrate successful recombinant protein expression and its subsequent purification using these new vectors. Corresponding vectors can now also be engineered with foreign gene expression under the control of various different promoters, to increase the flexibility of P. pastoris as a cellular factory for heterologous protein production. Copyright © 2009 John Wiley & Sons, Ltd.     

Pichia pastoris protease-deficient and auxotrophic strains generated by a novel,user-friendly vector toolbox for gene deletion

Targeted gene knockouts play an important role in the study of gene function. For the generation of knockouts in the industrially important yeast Pichia pastoris, several protocols have been published to date. Nevertheless, creating a targeted knockout in P. pastoris still is a time-consuming process, as the existing protocols are labour intensive and/or prone to accumulate nucleotide mutations. In this study, we introduce a novel, user-friendly vector-based system for the generation of targeted knockouts in P. pastoris. Upon confirming the successful knockout, respective selection markers can easily be recycled. Excision of the marker is mediated by Flippase (Flp) recombinase and occurs at high frequency (≥95%). We validated our knockout system by deleting 20 (confirmed and putative) protease genes and five genes involved in biosynthetic pathways. For the first time, we describe gene deletions of PRO3 and PHA2 in P. pastoris, genes involved in proline, and phenylalanine biosynthesis, respectively. Unexpectedly, knockout strains of PHA2 did not display the anticipated auxotrophy for phenylalanine but rather showed a bradytroph phenotype on minimal medium hinting at an alternative but less efficient pathway for production of phenylalanine exists in P. pastoris. Overall, all knockout vectors can easily be adapted to the gene of interest and strain background by efficient exchange of target homology regions and selection markers in single cloning steps. Average knockout efficiencies for all 25 genes were shown to be 40%, which is comparably high.     

Simplified protocol for faster transformation of (a large number of) Pichia pastoris strains

During the last couple of decades, the methylotrophic yeast, Pichia pastoris, has emerged as an important yeast species owing to its increasing importance both in industry and in basic research. The presently available methods for P. pastoris transformation necessitate the preparation of competent cells, which requires lots of resource, space, time, and efforts. This limits the number of transformations that can be performed by an individual in a given time. This paper is reporting a modification in the available protocols, which makes P. pastoris transformation hassle-free. In the present, modified procedure, cells were grown in patches on YPD plate(s), and the rest of the steps were carried out in small Eppendorf tubes. This modified protocol does not require a big centrifuge and shaker. This modified procedure of P. pastoris transformation with its unique way of competent cells preparation will be helpful for those working with this yeast species.     

Construction of recombinant industrial <Emphasis Type="Italic">S. cerevisiae</Emphasis> strain with barley lipid-transfer protein 1 secretion capability and lower PrA activity

Beer barley LTP1 in beer is an important component of beer foam, and it participates in the formation of beer foam. The digestion of beer barley LTP1 by proteinase A from brewing yeast leads to the decline of beer foam stability, especially for the unpasteurized beer. The objective of this study was to construct an industrial brewing yeast strain to secrete recombinant barley LTP1 into fermenting wort during beer fermentation for the foam stability improvement. We constructed barley LTP1 expression cassette and transformed into the host industrial yeast cells to replace partial PEP4 alleles using homologous recombination method. The expression of b-LTP1 was under control of the constitutive yeast ADH1 promoter, and the concentration of recombinant barley LTP1 secreted by recombinants reached 26.23 mg/L after incubation in YEPD medium for 120 h. The PrA activity of the recombinant strain declined compared with the host strain. The head retention of beer brewed with the recombinant industrial strain (326 ± 12 s) was improved when the host strain WZ65 (238 ± 7 s) and the constructed strain S.c-P-1 (273 ± 10 s) with partial PEP4 gene deficiency were used as control. The present study may provide reference for brewing industries and researches on beer foam stability.     

Comparing cytosolic expression to peroxisomal targeting of the chimeric L1/L2 (ChiΔH-L2) gene from human papillomavirus type 16 in the methylotrophic yeasts Pichia pastoris and Hansenula polymorpha

The chimeric ChiΔH‐L2 gene from human papillomavirus type 16, consisting of structural proteins L1 and L2, was successfully expressed in the cytosol of both Pichia pastoris and Hansenula polymorpha during methanol induction. In addition, a novel approach was employed whereby ChiΔH‐L2 was targeted to the peroxisome using peroxisomal targeting sequence 1 (PTS1) to compare ChiΔH‐L2 yields in the peroxisome vs the cytosol. The ChiΔH‐L2 gene was yeast‐optimized and cloned into plasmids aimed at genomic integration. Levels of intracellular ChiΔH‐L2 accumulation in the cytosol were highest in P. pastoris KM71 strain KMChiΔH‐L2 (1.43 mg/l), compared to the maximum production level of 0.72 mg/l obtained with H. polymorpha. ChiΔH‐L2 targeting to the peroxisome was successful; however, it appeared to negatively affect ChiΔH‐L2 production in both P. pastoris and H. polymorpha. Copyright © 2012 John Wiley & Sons, Ltd.     

Cloning of the Penicillium minioluteum gene encoding dextranase and its expression in Pichia pastoris

The DEX gene encoding an extracellular dextranase was isolated from the genomic DNA library of Penicillium minioluteum by hybridization using the dextranase cDNA as a probe. Comparison of the gene and cDNA sequences revealed that the DEX gene does not contain introns. Amino acid sequences comparison of P. minioluteum dextranase with other reported dextranases reveals a significant homology (29% identity) with a dextranase from Arthrobacter sp. CB-8. The DEX gene fragment encoding a mature protein of 574 amino acids was expressed in the methylotrophic yeast Pichia pastoris by using the SUC2 gene signal sequence from Saccharomyces cerevisiae under control of the alcohol oxidase-1 (AOX1) promoter. Over 3·2g/l of enzymatically active dextranase was secreted into the medium after induction by methanol. The yeast product was indistinguishable from the native enzyme in specific activity and the N-terminus of both proteins were identical.     

Performance of indigenous yeasts in the processing of Chinese strong‐flavoured liquor during spontaneous mixed solid‐state or submerged fermentation

To explore the in situ metabolic characteristics of yeasts involved in the spontaneous fermentation process of Chinese strong‐flavoured liquor, a comparison was conducted between solid‐state fermentation (SSF) and submerged fermentation (SmF) when supplemented with 24 indigenous yeast strains, with a focus on the production of ethanol and a broad range of volatile compounds responsible for the characteristics of Chinese strong‐flavoured liquor. Under the various experimental conditions, the 24 indigenous yeast strains showed different influences on the mixed fermentation system. The fluctuations caused by different yeast strains in the mixed system were less than those caused by the different fermentation modes relative to the formation of flavour compounds. SSF was found to be more suitable for the production of ethanol, methanol and ethyl lactate, whereas SmF was more suitable for the production of 10 higher alcohols, four esters and four acids. This study revealed the relationships amongst the indigenous yeasts, SSF, and the distinctive flavour profiles of Chinese strong‐flavoured liquor. This work provides evidence of the existence of internal stability in spontaneous SSF, thereby facilitating a better understanding of the fermentative mechanism in the SSF process for Chinese strong‐flavoured liquor production Copyright © 2015 The Institute of Brewing & Distilling     

Glucose metabolism and ethanol production in adh multiple and null mutants of Kluyveromyces lactis

Four genes coding for alcohol dehydrogenase (ADH) activities were identified in Kluyveromyces lactis. Due to the presence in this yeast of multiple ADH isozymes, mutants in the individual genes constructed by gene replacement yielded no clear phenotype. We crossed these mutants and developed a screening procedure which allowed us to identify strains lacking several ADH activities. The analysis of the adh triple mutants revealed that each activity confers to the cell the ability to grow on ethanol as the sole carbon source. On the contrary, adh null strains failed to grow on this substrate, indicating that no other important ADH activities are present in K. lactis cells. In the adh null mutants we also found a residual production of ethanol, as has been reported to be the case in Saccharomyces cerevisiae. This production showed a ten-fold increase when the K1ADHI activity was reintroduced in the null mutant and cells were cultivated under oxygen-limiting conditions. Differently from S. cerevisiae, glycerol is poorly accumulated in K. lactis adh null mutants.     

Cloning of levansucrase from Leuconostoc mesenteroides and its expression in Pichia pastoris

A gene (m1ft) encoding levansucrase from Leuconostoc mesenteroides has been previously cloned in Escherichia coli. Presently, m1ft was cloned and secretively expressed in Pichia pastoris. Methanol induction of recombinant M1FT resulted in the production of active levansucrase (PM1FT). PM1FT-5 was expressed as an active form, but the protein accumulated mainly inside the cells, representing about 5% of total cell proteins. M1FT was secreted into the culture supernatant with a maximum yield of 14,400 U/L using fed-batch fermentations. P. pastoris-derived M1FT displayed catalytic activities comparable to those of the E. coli-derived M1FT. PM1FT was glycosylated at its 2 potential N-glycosylation sites.     

Improvement of the slide culture technique for the assessment of yeast viability

This work aimed to improve the slide culture technique (SCT) for the assessment of yeast viability. Thus, all the steps of the SCT were standardized: a sample of 20 μL containing 1 × 10⁵ cells/mL was placed in a ~ 20 × 20 mm YPD agar block and incubated for 16–24 h, at 25°C. It was proposed the use of calcofluor white (CFW) to facilitate the microscopic observation of yeast cells. The viability of cell populations in different physiological states (healthy, ethanol stressed and starved cells), assessed by SCT (without or with CFW), did not differ significantly (p  < 0.01). In addition, the viability of healthy and ethanol stressed cells determined by the SCT and the standard plate count technique (PCT) did not differ significantly (p  < 0.01). In conclusion, the improved SCT is a fast and reliable alternative to PCT for the evaluation of yeast viability in research and in the industry. Copyright © 2017 The Institute of Brewing & Distilling     

Bioethanol production from sugar beet molasses and thick juice by free and immobilised Saccharomyces cerevisiae

The aim of this study was the investigation and comparison of the potential of sugar beet molasses and thick juice as raw materials for bioethanol production, as renewable and sustainable energy sources. Ethanol fermentation of a wide range of initial sugar concentrations (100–300 g/L) was performed using either free or immobilised Saccharomyces cerevisiae in calcium alginate beads in the absence of any added nutrients. In general, immobilised cells showed better fermentative performance, enhanced ethanol productivity, stability and cell viability compared with free cells, under the same fermentation conditions. The high concentration of non‐sugar components contained in molasses affected yeast fermentation performance and viability. Maximum ethanol concentration in fermented media of 84.6 and 109.5 g/L were obtained by immobilised cells for initial sugar concentrations of 200 and 250 g/L for molasses and thick juice, respectively. However, the highest ethanol yields of 31.7 L per 100 kg of molasses and 37.6 L per 100 kg of thick juice were obtained by immobilised cells at an initial sugar concentration of 175 g/L. In the high gravity fermentation process, thick juice resulted in a higher ethanol yield per mass of raw material compared with molasses. This study shows the advantage of immobilised yeast for the efficient production of high gravity bioethanol from thick juice, which was a more favourable raw material than molasses. © 2018 The Institute of Brewing & Distilling     

Disruption of the KEX1 gene in Pichia pastoris allows expression of full‐length murine and human endostatin

Endostatin is a potent angiogenesis inhibitor. In order to isolate sufficient quantities of soluble protein for in vivo studies in mice, we expressed murine endostatin in Pichia pastoris. Analysis of the expressed protein by mass spectrometry indicated that the protein was truncated. N‐terminal sequence analysis determined that the N‐terminus was intact, suggesting that the C‐terminal lysine was missing. In Saccharomyces cerevisiae, Kex1p can cleave lysine and arginine residues from the C‐terminus of peptides and proteins. We hypothesized that the KEX1 homologue in P. pastoris is responsible for the loss of the C‐terminal lysine of endostatin. To test this hypothesis, we cloned and disrupted the P. pastoris KEX1 gene. Although the overall amino acid identity between the P. pastoris and the S. cerevisae Kex1p is only 36%, the amino acid residues involved in the catalytic activity or close to the active residues are highly conserved. Disruption of the KEX1 reading frame allowed expression of murine and human endostatin with the C‐terminal lysine. The KEX1 disruption strain may be a useful tool for the expression of other proteins with a C‐terminal basic amino acid. Addition of a lysine to the C‐terminus of recombinant proteins may protect the C‐terminus from degradation by other carboxypeptidases. 3·5 kb of the P. pastoris KEX1 gene locus have been deposited in the GeneBank database and are available under Accession No. AF095574. Copyright © 1999 John Wiley & Sons, Ltd.     

Expression of unique chimeric human papilloma virus type 16 (HPV‐16) L1‐L2 proteins in Pichia pastoris and Hansenula polymorpha

Cervical cancer is ranked the fourth most common cancer in women worldwide. Despite two prophylactic vaccines being commercially available, they are unaffordable for most women in developing countries. We compared the optimized expression of monomers of the unique HPV type 16 L1‐L2 chimeric protein (SAF) in two yeast strains of Pichia pastoris, KM71 (Mut^s) and GS115 (Mut⁺), with Hansenula polymorpha NCYC 495 to determine the preferred host in bioreactors. SAF was uniquely created by replacing the h4 helix of the HPV‐16 capsid L1 protein with an L2 peptide. Two different feeding strategies in fed‐batch cultures of P. pastoris Mut^s were evaluated: a predetermined feed rate vs. feeding based on the oxygen consumption by maintaining constant dissolved oxygen levels (DO stat). All cultures showed a significant increase in biomass when methanol was fed using the DO stat method. In P. pastoris the SAF concentrations were higher in the Mut^s strains than in the Mut⁺ strains. However, H. polymorpha produced the highest level of SAF at 132.10 mg L^?1 culture while P. pastoris Mut^s only produced 23.61 mg L^?1. H. polymorpha showed greater potential for the expression of HPV‐16 L1/L2 chimeric proteins despite the track record of P. pastoris as a high‐level producer of heterologous proteins.     

First aspects on acetate metabolism in the yeast Dekkera bruxellensis: a few keys for improving ethanol fermentation

Dekkera bruxellensis is continuously changing its status in fermentation processes, ranging from a contaminant or spoiling yeast to a microorganism with potential to produce metabolites of biotechnological interest. In spite of that, several major aspects of its physiology are still poorly understood. As an acetogenic yeast, minimal oxygen concentrations are able to drive glucose assimilation to oxidative metabolism, in order to produce biomass and acetate, with consequent low yield in ethanol. In the present study, we used disulfiram to inhibit acetaldehyde dehydrogenase activity to evaluate the influence of cytosolic acetate on cell metabolism. D. bruxellensis was more tolerant to disulfiram than Saccharomyces cerevisiae and the use of different carbon sources revealed that the former yeast might be able to export acetate (or acetyl‐CoA) from mitochondria to cytoplasm. Fermentation assays showed that acetaldehyde dehydrogenase inhibition re‐oriented yeast central metabolism to increase ethanol production and decrease biomass formation. However, glucose uptake was reduced, which ultimately represents economical loss to the fermentation process. This might be the major challenge for future metabolic engineering enterprises on this yeast.     

Optimization of conditions for secretion of dengue virus type 2 envelope domain III using Pichia pastoris

We have developed a recombinant clone of the methylotrophic yeast Pichia pastoris capable of secreting dengue virus type 2 envelope domain III (sEDIII-2). We explored various induction parameters including media composition, temperature, pH, and methanol concentration, to optimize conditions for sEDIII-2 expression in shake flask culture. Induction at 20 °C in the presence of 2% (v/v) methanol in a medium buffered to pH 5.8 resulted in highest secretion of sEDIII-2. This yield could be further enhanced up to 70% by repeated induction of the same initial biomass. Using a fed-batch cultivation strategy, we observed that shake-flask yields can be scaled up ∼ 8-fold in a bioreactor. We obtained ∼ 94% purity with > 70% recovery after purification. This study, which demonstrates for the first time the feasibility of secreting envelope domain III using the P. pastoris host, will be relevant to sub-unit approaches to dengue vaccine development.     

Substrate specificity of alcohol dehydrogenase from the yeast Hansenyls polymorpha CBS 4732 and Candida utilis CBS 621

The substrate specificity of alcohol dehydrogenase (ADH) from Hansenula polymorpha and Candida utilis has been compared with that of the classical ADH from baker's yeast. Cell-free extracts of H. polymorpha and C. utilis exhibited a much higher ratio of butanol to ethanol oxidation than baker's yeast ADH. This was also observed with the purified enzymes. The ratio of activities with ethanol and butanol was pH-dependent. With the baker's yeast enzyme the activity strongly decreased with increasing chain length, whereas the enzymes form H. polymorpha and C. utilis showed a high reactivity with long-chain alcohols. In addition, the affinity constant for ethanol was more than tenfold lower than that of the baker's yeast enzyme. The purified preparation yielded several protein bands on polyacrylamide slab gels, each of which showed activity with both ethanol and butanol.     

Downregulation by organic nitrogen of AOX1 promoter used for controlled expression of foreign genes in the yeast Pichia pastoris

Pichia pastoris is a well-established cell factory for recombinant protein synthesis. Various optimization strategies of processes based on AOX1 promoter have been investigated, including methanol co-feeding with glycerol or sorbitol during the induction stage. Compared with carbon sources, comparatively little research has been devoted to the effects of nitrogen sources. Several reports have described the benefits of adding casamino acids (CA) to the recombinant protein production medium, however, without considering its effects at the gene expression level. Using enhanced green fluorescent protein as a reporter protein, monitored using flow cytometry, CA was shown to downregulate AOX1 promoter induction. Despite higher growth rates, cultures containing CA exhibited slower transition to the induced state, whereas metabolite analysis revealed that methanol consumption was reduced in the presence of CA compared with its absence. The repressive effect of CA was further confirmed by analysing the synthesis of extracellular recombinant Candida antarctica lipase under control of the AOX1 promoter. These findings highlight nitrogen source selection as an important consideration for AOX1-based protein production.