The HpGcr1, a hexose transporter homologue from the methylotrophic yeast Hansenula (Ogataea) polymorpha, was previously identified as being involved in glucose repression. Intriguingly, potential ...HpGcr1 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 HpGcr1 homologues are fungal high-affinity glucose symporters or putative transceptors suggesting a possible HpGcr1 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 HpGcr1R165K into a constitutively signaling form. Synthesis of HpGcr1R165K in gcr1Δ did not restore glucose transport or repression but instead profoundly impaired growth independent of carbon source used. Simultaneously, gcr1Δ was impaired in transcriptional induction of repressible peroxisomal alcohol oxidase and in growth on methanol. Overexpression of the functional transporter HpHxt1 in gcr1Δ partially restored growth on glucose and glucose repression but did not rescue impaired growth on methanol. Heterologous expression of HpGcr1 in a Saccharomyces cerevisiae hxt-null strain did not restore glucose uptake due to protein mislocalization. However, HpGcr1 overexpression in H. polymorpha led to increased sensitivity to extracellular 2-deoxyglucose, suggesting HpGcr1 is a functional glucose carrier. The combined data suggest that HpGcr1 represents a novel type of yeast glucose transceptor functioning also in the absence of glucose.
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.
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
and the methylotrophic yeast
.
is further considered a convenient model system for functional studies of heterologously expressed proteins, while
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.
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.
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 glucose-grown 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.
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.
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.
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 altemative, 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.
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.
—
Molecular genetic study of 22 methanol-assimilating
Komagataella
strains maintained at the UCDFST collection (United States) was carried out. Multigene phylogenetic analysis revealed that strains ...previously assigned to “
Pichia pastoris
” based on the standard taxonomic tests actually belong to four different species:
K. pastoris
,
K. phaffii
,
K. mondaviorum
, and
K. ulmi
. Seven
Komagataella
species are practically indistinguishable by the standard morphological and physiological tests, and have similar D1/D2 sequences. Multigene phylogenetic analysis should be applied for reliable delineation of all seven
Komagataella
species. The biogeography of
Komagataella
yeasts is noteworthy.
K. mondaviorum
,
K. kurtzmanii
,
K. phaffii
,
K. populi
, and
K. ulmi
have been found only in North America, while
K. pastoris
and
K. pseudopastoris
are characteristic of Europe.
Functional biological materials are a growing research area with potential applicability in medicine and biotechnology. Using genetic engineering, the possibility to introduce additional functions ...into spider silk‐based materials has been realized. Recently, a recombinant spider silk fusion protein, Z‐4RepCT, was produced intracellularly in Escherichia coli and could after purification self‐assemble into silk‐like fibers with ability to bind antibodies via the IgG‐binding Z domain. In this study, the use of the methylotrophic yeast Pichia pastoris for production of Z‐4RepCT has been investigated. Temperature, pH and production time were influencing the amount of soluble Z‐4RepCT retrieved from the extracellular fraction. Purification of secreted Z‐4RepCT resulted in a mixture of full‐length and degraded silk proteins that failed to self‐assemble into fibers. A position in the C‐terminal domain of 4RepCT was identified as being subjected to proteolytic cleavage by proteases in the Pichia culture supernatant. Moreover, the C‐terminal domain was subjected to glycosylation during production in P. pastoris. These observed alterations of the CT domain are suggested to contribute to the failure in fiber assembly. As alternative approach, Z‐4RepCT retrieved from the intracellular fraction, which was less degraded, was used and shown to retain ability to assemble into silk‐like fibers after enzymatic deglycosylation.
The methylotrophic yeast Pichia pastoris was used to express the silk fusion protein Z‐4RepCT, which was then retrieved either from the intracellular or secreted fraction. Secreted silk fusion protein was observed to be both partially degraded and subjected to glycosylation on the CT silk domain, affecting its ability to assemble into fibers. In contrast, intracellular silk fusion protein was less subjected to cleavage and could assemble into Z‐4RepCT fibers after enzymatic deglycosylation. This is the first report on silk proteins expressed in yeast with maintained ability to spontaneously assemble into silk‐like fibers.
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.
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