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Abstract Methanol is a promising feedstock for biomanufacturing, but the efficiency of methanol-based bioprocesses is limited by the low rate of methanol utilization pathways and methanol toxicity. Yeast diversity is an attractive biological resource to develop efficient bioprocesses since any effort with strain improvement is more deserving if applied to innate robust strains with relevant catabolic and biosynthetic potential. The present study is in line with such rational and describes the isolation and molecular identification of seven isolates of the methylotrophic species Candida boidinii from waters derived from the traditional curation of olives, in different years, and from contaminated superficial soil near fuel stations. The yeast microbiota from those habitats was also characterized. The four C. boidinii isolates obtained from the curation of olives’ water exhibited significantly higher maximum specific growth rates (range 0.15–0.19 h−1), compared with the three isolates obtained from the fuel contaminated soils (range 0.05–0.06 h−1) when grown on methanol as the sole C-source (1% (v/v), in shake flasks, at 30°C). The isolates exhibit significant robustness towards methanol toxicity that increases as the cultivation temperature decreases from 30°C to 25°C. The better methanol-based growth performance exhibited by C. boidinii isolates from olives´ soaking waters could not be essentially attributed to higher methanol tolerance. These methanol-efficient catabolizing isolates are proposed as a promising platform to develop methanol-based bioprocesses.

Methanol is a promising feedstock for biomanufacturing, but the efficiency of methanol-based bioprocesses is limited by the low rate of methanol utilization pathways and methanol toxicity. Yeast diversity is an attractive biological resource to develop efficient bioprocesses since any effort with strain improvement is more deserving if applied to innate robust strains with relevant catabolic and biosynthetic potential. The present study is in line with such rational and describes the isolation and molecular identification of seven isolates of the methylotrophic species Candida boidinii from waters derived from the traditional curation of olives, in different years, and from contaminated superficial soil near fuel stations. The yeast microbiota from those habitats was also characterized. The four C. boidinii isolates obtained from the curation of olives' water exhibited significantly higher maximum specific growth rates (range 0.15–0.19 h−1), compared with the three isolates obtained from the fuel contaminated soils (range 0.05–0.06 h−1) when grown on methanol as the sole C-source (1% (v/v), in shake flasks, at 30°C). The isolates exhibit significant robustness towards methanol toxicity that increases as the cultivation temperature decreases from 30°C to 25°C. The better methanol-based growth performance exhibited by C. boidinii isolates from olives´ soaking waters could not be essentially attributed to higher methanol tolerance. These methanol-efficient catabolizing isolates are proposed as a promising platform to develop methanol-based bioprocesses. [ABSTRACT FROM AUTHOR]

Researchers are often interested in proteins that are present in cells in small ratios compared to the total amount of proteins. These proteins include transcription factors, hormones and specific membrane proteins. However, sufficient amounts of well-purified protein preparations are required for functional and structural studies of these proteins, including the creation of artificial proteoliposomes and the growth of protein 2D and 3D crystals. This aim can be achieved by the expression of the target protein in a heterologous system. This review describes the applications of yeast heterologous expression systems in studies of plant membrane proteins. An initial brief description introduces the widely used heterologous expression systems of the baker's yeast Saccharomyces cerevisiae and the methylotrophic yeast Pichia pastoris. S. cerevisiae is further considered a convenient model system for functional studies of heterologously expressed proteins, while P. pastoris has the advantage of using these yeast cells as factories for producing large quantities of proteins of interest. The application of both expression systems is described for functional and structural studies of membrane proteins from plants, namely, K+- and Na+-transporters, various ATPases and anion transporters, and other transport proteins. [ABSTRACT FROM AUTHOR]

Methylotrophic yeasts have been widely recognized as a promising host for production of recombinant proteins and value-added chemicals. Promoters for controlled gene expression are critical for construction of efficient methylotrophic yeasts cell factories. Here, we summarized recent advances in characterizing and engineering promoters in methylotrophic yeasts, such as Komagataella phaffii and Ogataea polymorpha. Constitutive and inducible promoters controlled by methanol or other inducers/repressors were introduced to demonstrate their applications in production of proteins and chemicals. Furthermore, efforts of promoter engineering, including site-directed mutagenesis, hybrid promoter, and transcription factor regulation to expand the promoter toolbox were also summarized. This mini-review also provides useful information on promoters for the application of metabolic engineering in methylotrophic yeasts. Key Points: • The characteristics of six methylotrophic yeasts and their promoters are described. • The applications of Komagataella phaffii and Ogataea polymorpha in metabolic engineeringare expounded. • Three promoter engineering strategies are introduced in order to expand the promoter toolbox. [ABSTRACT FROM AUTHOR]

Methylotrophic yeasts have been used as the platform for expression of heterologous proteins since the 1980’s. They are highly productive and allow producing eukaryotic proteins with an acceptable glycosylation level. The first Pichia pastoris-based system for expression of recombinant protein was developed on the basis of the treeexudate-derived strain obtained in the US southwest. Being distributed free of charge for scientific purposes, this system has become popular around the world. As methylotrophic yeasts were classified in accordance with biomolecular markers, strains used for production of recombinant protein were reclassified as Komagataella phaffii. Although patent legislation suggests free access to these yeasts, they have been distributed on a contract basis. Whereas their status for commercial use is undetermined, the search for alternative stains for expression of recombinant protein continues. Strains of other species of methylotrophic yeasts have been adapted, among which the genus Ogataearepresentatives prevail. Despite the phylogenetic gap between the genus Ogataeaand the genus Komagataellarepresentatives, it turned out possible to use classic vectors and promoters for expression of recombinant protein in all cases. There exist expression systems based on other strains of the genus Komagataellaas well as the genus Candida. The potential of these microorganisms for genetic engineering is far from exhausted. Both improvement of existing expression systems and development of new ones on the basis of strains obtained from nature are advantageous. Historically, strains obtained on the southwest of the USA were used as expression systems up to 2009. Currently, expression systems based on strains obtained in Thailand are gaining popularity. Since this group of microorganisms is widely represented around the world both in nature and in urban environments, it may reasonably be expected that new expression systems for recombinant proteins based on strains obtained in other regions of the globe will appear.

Abstract The Hp Gcr1, a hexose transporter homologue from the methylotrophic yeast Hansenula (Ogataea) polymorpha , was previously identified as being involved in glucose repression. Intriguingly, potential Hp Gcr1 orthologues are found only in the genomes of a few yeasts phylogenetically closely related to H. polymorpha , but are absent in all other yeasts. The other closest Hp Gcr1 homologues are fungal high-affinity glucose symporters or putative transceptors suggesting a possible Hp Gcr1 origin due to a specific archaic gene retention or via horizontal gene transfer from Eurotiales fungi. Herein we report that, similarly to other yeast non-transporting glucose sensors, the substitution of the conserved arginine residue converts Hp Gcr1R165K into a constitutively signaling form. Synthesis of Hp Gcr1R165K in gcr1 Δ did not restore glucose transport or repression but instead profoundly impaired growth independent of carbon source used. Simultaneously, gcr1 Δ was impaired in transcriptional induction of repressible peroxisomal alcohol oxidase and in growth on methanol. Overexpression of the functional transporter Hp Hxt1 in gcr1 Δ partially restored growth on glucose and glucose repression but did not rescue impaired growth on methanol. Heterologous expression of Hp Gcr1 in a Saccharomyces cerevisiae hxt- null strain did not restore glucose uptake due to protein mislocalization. However, Hp Gcr1 overexpression in H. polymorpha led to increased sensitivity to extracellular 2-deoxyglucose, suggesting Hp Gcr1 is a functional glucose carrier. The combined data suggest that Hp Gcr1 represents a novel type of yeast glucose transceptor functioning also in the absence of glucose. [ABSTRACT FROM AUTHOR]

Analysis of the literature and GenBank data showed that the genus Ogataea is heterogeneous and includes at least five non-described genera. Comprehensive phylogenetic analysis of the present genus Ogataea may be conducted only based on multigene analysis of all the known species. Possibilities of molecular species identification in biogeographical and ecological studies of yeasts are discussed. [ABSTRACT FROM AUTHOR]

Azo dyes are extensively used in textile dyeing and other industries. Effluents of dying industries are specially colored and could cause severe damage to the environment. The anaerobic treatment of textile dying effluents is nowadays the preferred option, but it could generate carcinogenic aromatic amines. Recently, yeasts have become a promising alternative, combining unicellular growth with oxidative mechanisms. This work reports the characterization of the first methylotrophic yeast with dye decolorizing ability, Candida boidinii MM 4035 and some insights into its decoloration mechanism. The analysis of two selected media revealed a possible two stages mechanism of Reactive Black 5 decoloration. In glucose poor media, decoloration is incomplete and only the first stage proceeds, leading to the accumulation of a purple compound. In media with higher glucose concentrations, the yeast is able to decolorize totally an initial concentration of 200 mg/L. The entire process is co-metabolic, being largely dependent on glucose concentration but being able to proceed with several nitrogen sources. Manganese dependent peroxidase but not laccase activity could be detected during decoloration. Aromatic amines do not accumulate in culture media, supporting an oxidative decoloration mechanism of unknown ecophysiological relevance. [ABSTRACT FROM AUTHOR]

The first and limiting step of metabolism of glucose, the most widespread carbon and energy source for majority of cells, is the transport of this sugar across cytoplasmic membrane. In the eukaryotic cells glucose controls and ensures its own effective metabolism, acting as extracellular effector, regulating on transcriptional and translational levels amount, type and activity of corresponding hexose transporters. In this study we investigated regulation of expression of genes encoding hexose transporter homologues – НрHxt1, НрHxt2, НрHxt3 and putative fructose transporter – НрFrt1 by two glucose sensors НрGcr1 and НрHxs1 in methylotrophic yeast Hansenula polymorpha. We have demonstrated that putative glucose sensor НрGcr1 is involved in repression of the gene of functional low affinity hexose transporter НрHxt1 under glucose deficient conditions and participates in repression of genes of putative high affinity glucose transporters НрHxt2 and НрHxt3 under excess of this carbon source. It was shown that glucose sensor НрHxs1 is involved in induction of НрHxt1 expression. As a result of deletion of HpGCR1, but not НрHxS1, the gene of putative high affinity fructose transporter НрFRT1 is constitutively expressed independently of glucose pre­sence and its concentration in growth medium. Therefore, regulation of hexose (glucose and fructose) transport in methylotrophic yeast H. polymorpha at the level of gene expression is a complicated system of interac­ting regulatory and transporting elements where glucose sensors НрGcr1 and НрHxs1 act as the key factors.

The review analyzes and summarizes the present state of art and trends of application of the unique metabolism of multifunctional methylotrophic bacteria and yeasts in various areas of modern biotechnology: biosynthesis, biocatalysis, bioanalytics and bioremediation.

In the yeasts, as in the majority of microorganisms, glucose is the main source of carbon and energy, as well as a key effector molecule involved in transcriptional regulation. Many genes are repressed in the presence of glucose, others are instead induced by glucose. In the mutants of bakers’ yeast Sacharomyces cerevisiae impaired in hexo­se transport, the proficiency of transport determins the strength of the repression signal. In the mutants of methylotrophic yeast Hansenula polymorpha with deleted glucose sensors НрGcr1 and НрHxs1, efficiency of glucose and fructose uptake and the effect of hexose transport on catabolite repression of peroxisomal alcohol oxidase was investigated. Contrary to S. cerevisiae, in the mutants ∆gcr1 and ∆hxs1 impairment glucose and fructose transport did not always corelated with the rate of catabolite repression of metabolic genes of alternative carbon sources. For instance, in the recessive gcr1-2 mutant with one amino acid substitution S85F glucose transport was less impaired relative to corresponding deletion mutant, whereas defect of glucose repression was more pronounced. Also, in the mutants ∆hxs1 and ∆gcr1, the defect of fructose transport was similar, when fructose repression defect was stronger in ∆hxs1. Therefore, specific impairment of hexose transport upon deletion of alternative hexose sensors in H. polymorpha is not an only cause that determins profiociency of hexose catabolite repression.

UDP-glucose:sterol glucosyltransferase (EC 2.4.1.173) catalyses the biosynthesis of ergosterol glucoside, a minor membrane lipid present in yeasts and plants. The sterol glucosyltransferases from different yeast species, including the methylotrophic yeast Pichia pastoris, display narrow substrate specificity with respect to both activated sugar and glycosyl acceptor. We isolated the mutant of the methanol-utilizing yeast Hansenula polymorpha defective in the ATG26 gene (encoding sterol glucosyltransferase) and analysed its phenotype. Similarly to P. pastoris product of the H. polymorpha ATG26 gene was required for pexophagy, the process of selective autophagic peroxisome degradation in vacuoles. Thus, sterol glucosyltransferases in different species of methylotrophic yeasts have a similar function in pexophagic process probably related to peculiarities of peroxisome morphogenesis and regulation of their homeostasis in these yeasts.

Genetic hybridization analysis revealed that industrially important species Komagataella kurtzmanii has reproductive postzygotic isolation from K. pastoris, K. phaffii, K. populi, K. pseudopastoris, and K. ulmi. Therefore, it represents a new biological species of the genus Komagataella. The genetic data are in perfect agreement with the molecular taxonomy of the genus Komagataella. [ABSTRACT FROM AUTHOR]

The group of methylotrophic yeast Hansenula (Ogataea) polymorpha attracts attention as suitable model organism for studying various cellular processes and serves as a producer of medically important heterologous proteins. This group comprises of three independently isolated strains: CBS4732T, NCYC495 and DL-1. The genomes of all strains are sequenced, but its taxonomic positions are matter of debates. The results of a systematic study aimed to understand the relationship between these strains are presented here. On the basis of sequence homology as well as according to our data from analysis of mating and sporulation abilities CBS4732T and NCYC495 strains are regarded as the same species. We also attempted to identify the species position of the strain DL-1. Our results suggests that although DL-1 differed in chromosomal organization and sequence divergence from CBS4732T and NCYC495 strains no clear cases of reproductive isolation have been found. This means that the boundary between these species is hard to define and the group of methylotrophic yeast O. (H.) polymorpha is promising model system for elucidating evolutionary processes acting on yeast genomes. [ABSTRACT FROM AUTHOR]

Apart from the toxic methanol, relatively safe formic acid and its salts (formates) were shown to act as efficient inducers of the AOX1 promoter in methylotrophic yeasts Komagataella kurtzmanii and K. phaffii. In order to investigate the mechanism of formate induction in the cells of these species, deletion of the formaldehyde dehydrogenase (FLD) gene was carried out, resulting in disruption of the metabolic pathway of methanol dissimilation and thus preventing the intracellular reduction of formate to methanol. The AOX1-lacZ reporter cassette was used to demonstrate that the fld mutation resulted in enhanced induction of the AOX1 by both methanol and formate. This result casts doubt on the hypothesis that methanol is the real inducer of the AOX1 promoter. Alternative mechanisms of induction are discussed. [ABSTRACT FROM AUTHOR]

Background Hansenula polymorpha DL1 is a methylotrophic yeast, widely used in fundamental studies of methanol metabolism, peroxisome biogenesis and function, and also as a microbial cell factory for production of recombinant proteins and metabolic engineering towards the goal of high temperature ethanol production. Results We have sequenced the 9 Mbp H. polymorpha DL1 genome and performed whole-genome analysis for the H. polymorpha transcriptome obtained from both methanol- and glucosegrown cells. RNA-seq analysis revealed the complex and dynamic character of the H. polymorpha transcriptome under the two studied conditions, identified abundant and highly unregulated expression of 40% of the genome in methanol grown cells, and revealed alternative splicing events. We have identified subtelomerically biased protein families in H. polymorpha, clusters of LTR elements at G + C-poor chromosomal loci in the middle of each of the seven H. polymorpha chromosomes, and established the evolutionary position of H. polymorpha DL1 within a separate yeast clade together with the methylotrophic yeast Pichia pastoris and the non-methylotrophic yeast Dekkera bruxellensis. Intergenome comparisons uncovered extensive gene order reshuffling between the three yeast genomes. Phylogenetic analyses enabled us to reveal patterns of evolution of methylotrophy in yeasts and filamentous fungi. Conclusions Our results open new opportunities for in-depth understanding of many aspects of H. polymorpha life cycle, physiology and metabolism as well as genome evolution in methylotrophic yeasts and may lead to novel improvements toward the application of H. polymorpha DL-1 as a microbial cell factory. [ABSTRACT FROM AUTHOR]

Highlights: [•] A recombinant capsid protein (rp26) of Equine Infectious Anemia Virus was expressed in Pichia pastoris. [•] The rp26 was evaluated in an enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) test. [•] EIAV rp26 protein was identified by OIE standard polyclonal antibody anti EIAV. [•] This is the first report of EIAV p26 protein production in yeast cells. [Copyright &y& Elsevier]

A high-throughput approach was used to assess the effect of mono- and disaccharides on MOX, FMD, MPP1 and MAL1 promoters in Hansenula polymorpha. Site-specifically designed strains deficient for (1) hexokinase, (2) hexokinase and glucokinase, (3) maltose permease or (4) maltase were used as hosts for reporter plasmids in which β-glucuronidase ( Gus) expression was controlled by these promoters. The reporter strains were grown on agar plates containing varied carbon sources and Gus activity was measured in permeabilized cells on microtitre plates. We report that monosaccharides (glucose, fructose) repress studied promoters only if phosphorylated in the cell. Glucose-6-phosphate was proposed as a sugar repression signalling metabolite for H. polymorpha. Intriguingly, glucose and fructose strongly activated expression from these promoters in strains lacking both hexokinase and glucokinase, indicating that unphosphorylated monosaccharides have promoter-derepressing effect. We also show that maltose and sucrose must be internalized and split into monosaccharides to exert repression on MOX promoter. We demonstrate that at yeast growth on glucose-containing agar medium, glucose-limitation is rapidly created that promotes derepression of methanol-specific promoters and that derepression is specifically enhanced in hexokinase-negative strain. We recommend double kinase-negative and hexokinase-negative mutants as hosts for heterologous protein production from MOX and FMD promoters. [ABSTRACT FROM AUTHOR]

Ogataea ( Hansenula) polymorpha and related yeasts were studied to clarify their taxonomy and phylogenetic relationships. The molecular analyses based on ribosomal DNA sequences revealed that (1) ATCC 14755, type strain of Pichia angusta, is phylogenetically distinguished from the majority of O. polymorpha strains including ATCC 34438T (=NRRL Y-5445T=CBS 4732T), type of the species; (2) Ogataea thermophila is conspecific to O. polymorpha; and (3) two of the strains, ATCC 26012 and ATCC 58401, are a novel Candida species closely related to O. polymorpha. The conclusions were also supported by physiological characteristics and other taxonomic features of these strains. Therefore, we propose here two novel species, Ogataea angusta comb. nov. (ATCC 14755T=CBS 7073T=NRRL Y-2214T) and Candida parapolymorpha sp. nov. (ATCC 26012T=NRRL Y-7560T), and conclude that O. thermophila is a synonym of O. polymorpha. [ABSTRACT FROM AUTHOR]

Modified methylotrophic yeast Hansenula polymorpha ( HP A16 ) that was obtained by repressing leucine oxotrophic yeast; a wild type of Hansenula polymorpha CB4732 was used in this study. The yeast is grown with methanol, which is used as a sole carbon source, using various methanol concentrations and temperatures, and methanol oxidase (MOX) which is a key enzyme of methanol metabolism; production is maximized. Whole yeast cells were cultivated under optimized inoculation conditions; they were separated into two portions. One portion of these cells was directly used in bioconversion of methanol to formaldehyde. The second portion of the free cells was broken into pieces and a crude enzyme extract was obtained. The MOX enzyme in this extract was purified via salt precipitation, dialysis, and chromatographic methods. The purified MOX enzyme of yeast ( HP A16) oxidized the methanol to formaldehyde. Optimization of bioconversion conditions was studied to reach maximum activity of enzyme. The optimum temperature and pH were found to be 35°C and pH 8.0 in boric acid/NaOH buffer, and it was stable over the pH range of 6–9, at the 20°C 15 min. A suitable reaction period was found as 50 min. The enzyme indicated low carbon primary alcohols (C 2 to C 4 ), as well as methanol. Initially, MOX activity increased with the increase of methanol concentration, but enzyme activity decreased. The apparent K m and V max values for methanol substrate of HP A1 6 MOX were 0.25 mM and 30 U/mg, respectively. The purified MOX enzyme was applied onto sodium dodecyl sulphate‐polyacrylamide gel electrophoresis; molecular weight of the enzyme was calculated to be about 670 kDa. Each MOX enzyme is composed of eight identical subunits, each of whose molecular weight is around 82 kDa and which contain eight moles of FAD as the prosthetic group, and the pI of the natural enzyme is found to be 6.4. The purified MOX enzyme was used in the bioconversion of methanol to formaldehyde as a catalyst; this conversion was compared to the conversion percentages of whole cells in our previous article in terms of catalytic performances. [ABSTRACT FROM AUTHOR]

Examines the regulation of pexophagic pathways by analyzing peroxisome degradation in cells induced under a variety of conditions. Distinct pexophagic pathways through which methylotrophic yeast Pichia pastoris can degrade peroxisomes; Distinction between micropexophagy and macropexophagy in living cells; Role of the intracellular adenosine triphosphate in regulating the pathways.

The cloning of 7.2- and 9.6-kbp fragments of the methylotrophic yeast Hansenula polymorpha DNA restored the wild-type phenotype Gsh+ in the glutathione-dependent gsh1 and gsh2 mutants of this yeast defective in glutathione (GSH) synthesis because of a failure of the γ-glutamylcysteine synthetase reaction. The 9.6-kbp DNA fragment was found to contain a 4.3-kbp subfragment, which complemented the Gsh– phenotype of the gsh2 mutant. The Gsh+ transformants of the gsh1 and gsh2 mutants, which bear plasmids pG1 and pG24, having the 7.2- and 4.3-kbp DNA fragments, respectively, had a completely restored wild-type phenotype with the ability to synthesize GSH and to grow in GSH-deficient synthetic media on various carbon sources, including methanol, and with acquired tolerance to cadmium ions. In addition, the 4.3-kbp DNA fragment borne by plasmid pG24 eliminated pleiotropic changes in the gsh2 mutants associated with methylotrophic growth in a semisynthetic (GSH-supplemented) medium (poor growth and alterations in the activity of the GSH-catabolizing enzyme γ-glutamyltransferase and the methanol-oxidizing enzyme alcohol oxidase). [ABSTRACT FROM AUTHOR]

Methylotrophic yeasts, named after their ability to grow on methanol as the sole carbon source, have raised large interest as recombinant protein factories. In this review, we explain the basic mechanisms underlying this interest and describe the minimal requirements to transform the two genera recognized as methylotrophic, Pichia and Candida, into a powerful protein production tool. We present a comparison between this group of yeasts and the conventional yeasts used as expression system in view of productivity, level of secretion and quality of post-translational modifications. Selected examples of recombinant protein produced by methylotrophic yeast are also included. [Copyright &y& Elsevier]

Nutritional changes can effect either the assembly or disassembly of yeast peroxisomes. In the past decade, insights regarding the molecular mechanisms of peroxisome assembly have been gained chiefly through the cloning of the PEX genes obtained by complementation of corresponding pex mutants in several yeast strains and Chinese hamster ovary cell lines. Depletion of these peroxins (proteins encoded by PEX genes) by deletion of the corresponding genes affects peroxisomal protein import biogenesis or proliferation. To complement these studies in the field, the authors undertook an investigation of the functions of a subset of Candida boidinii peroxisomal membrane proteins (PMPs), Pex11, Pmp47, and Pmp20, by analyzing strains of C. boidnii in which the genes encoding these proteins were deleted. The authors' studies show that Pex11p is involved in peroxisome proliferation; Pmp47 plays a role in the translocation, folding, or assembly of dihydroxyacetone synthase; and Pmp20 is probably involved in methanol metabolism. In contrast to the studies on peroxisome assembly, the molecular mechanisms of peroxisome degradation remain poorly understood. To shed light on this problem, the authors isolated Pichia pastoris mutants defective in peroxisome autopathy ( pag mutants). A novel, double-fluorescence method used for the characterization of wild-type cells and of pag mutants enabled us to dissect the microautophagic degradation of peroxisomes into several distinct stages. These studies show that specific PAG gene products are involved in multiple steps of the process. Future cloning and characterization of the functions of PAG genes will reveal the molecular basis of peroxisome degradation. [ABSTRACT FROM AUTHOR]

A highly-efficient method for transformation of the methylotrophic yeast Hansenula polymorpha has been developed. Routinely, transformation frequencies of up to 1.7×10/μg plasmid DNA were obtained by applying an electric pulse of the exponential decay type of 7.5 kV/cm to a highly-concentrated cell mixture during 5 ms. Efficient transformation was dependent on: (1) pretreatment of the cells with the reducing agent dithiotreitol, (2) the use of sucrose as an osmotic stabilizer in an ionic electroporation buffer, and (3) the use of cells grown to the mid-logarithmic phase. Important parameters for optimizing the transformation frequencies were field strength, pulse duration, and cell concentration during the electric pulse. In contrast to electrotransformation protocols described for Saccharomyces cerevisiae and Candida maltosa, transformation frequencies (transformants per μg DNA) for H. polymorpha remained high when large amounts (up to 10μg) of plasmid DNA were added. This feature renders this procedure pre-eminently advantageous for gene cloning experiments when high numbers of transformants are needed. [ABSTRACT FROM AUTHOR]

We have isolated a mutant of the metholotrophic yeast Hansenula polymorpha defective in peroxisomal biosynthesis. The mutant strain has been derived by a selection procedure from cells of a high-copy number transformant that overproduces the major peroxisomal enzyme methanol oxidase (MOX) and forms enlarged peroxisomes. In contrast to the parental strain the mutant lacks intact peroxisomes in thin sections, but exhibits electron-dense particles that are devoid of intact membranes and crystalloid cores. Consequently, peroxisomal enzymes show severe proteolytic degradation in crude cell lysates. Complementation of this, and analogous mutations, will offer the possibility to identify genes that are required for peroxisome assembly. [ABSTRACT FROM AUTHOR]

Methylotrophic yeasts have been used as the platform for expression of heterologous proteins since the 1980's. They are highly productive and allow producing eukaryotic proteins with an acceptable glycosylation level. The first Pichia pastoris-based system for expression of recombinant protein was developed on the basis of the treeexudate- derived strain obtained in the US southwest. Being distributed free of charge for scientific purposes, this system has become popular around the world. As methylotrophic yeasts were classified in accordance with biomolecular markers, strains used for production of recombinant protein were reclassified as Komagataella phaffii. Although patent legislation suggests free access to these yeasts, they have been distributed on a contract basis. Whereas their status for commercial use is undetermined, the search for alternative stains for expression of recombinant protein continues. Strains of other species of methylotrophic yeasts have been adapted, among which the genus Ogataea representatives prevail. Despite the phylogenetic gap between the genus Ogataea and the genus Komagataella representatives, it turned out possible to use classic vectors and promoters for expression of recombinant protein in all cases. There exist expression systems based on other strains of the genus Komagataella as well as the genus Candida. The potential of these microorganisms for genetic engineering is far from exhausted. Both improvement of existing expression systems and development of new ones on the basis of strains obtained from nature are advantageous. Historically, strains obtained on the southwest of the USA were used as expression systems up to 2009. Currently, expression systems based on strains obtained in Thailand are gaining popularity. Since this group of microorganisms is widely represented around the world both in nature and in urban environments, it may reasonably be expected that new expression systems for recombinant proteins based on strains obtained in other regions of the globe will appear., (Copyright © AUTHORS, 2020.)

During the production of pectinases by a strain of Aspergillus niger isolated from rotten lemons, methanol was liberated into the medium due to the cleavage of the pectin molecule used as the carbon source. The methanol was subsequently consumed by the microorganism but neither the synthesis nor the activity of pectinesterase and polygalacturonase was affected. Although not studied in detail, the mechanism involved in the utilization of methanol is similar to that described for methylotrophic yeasts. [ABSTRACT FROM AUTHOR]