Single carbon metabolism - A new paradigm for microbial bioprocesses?

Increasing atmospheric carbon dioxide levels, a reduction of arable land area and the dependence of first and second generation biotechnology feedstocks on agricultural products, call for alternative, sustainable feedstock sources for industrial applications. The direct use of CO2 or conversion of CO2 into other single carbon (C1) sources have great potential as they might help to reduce carbon emissions and do not compete with agricultural land use. Here we discuss the microbial use of C1 carbon sources, their potential applications in biotechnology, and challenges towards sustainable C1-based industrial biotechnology processes. We focus on methanol, formic acid, methane, syngas, and CO2 as feedstocks for bioprocesses, their assimilation pathways, current and emerging applications, and limitations of their application. This mini-review is intended as a first introduction for researchers who are new to the field of C1 biotechnology.

Protein production dynamics and physiological adaptation of recombinant Komagataella phaffii at near-zero growth rates.

BACKGROUND: Specific productivity (qP) in yeast correlates with growth, typically peaking at intermediate or maximum specific growth rates (µ). Understanding the factors limiting productivity at extremely low µ might reveal decoupling strategies, but knowledge of production dynamics and physiology in such conditions is scarce. Retentostats, a type of continuous cultivation, enable the well-controlled transition to near-zero µ through the combined retention of biomass and limited substrate supply. Recombinant Komagataella phaffii (syn Pichia pastoris) secreting a bivalent single domain antibody (VHH) was cultivated in aerobic, glucose-limited retentostats to investigate recombinant protein production dynamics and broaden our understanding of relevant physiological adaptations at near-zero growth conditions. RESULTS: By the end of the retentostat cultivation, doubling times of approx. two months were reached, corresponding to µ = 0.00047 h-1. Despite these extremely slow growth rates, the proportion of viable cells remained high, and de novo synthesis and secretion of the VHH were observed. The average qP at the end of the retentostat was estimated at 0.019 mg g-1 h-1. Transcriptomics indicated that genes involved in protein biosynthesis were only moderately downregulated towards zero growth, while secretory pathway genes were mostly regulated in a manner seemingly detrimental to protein secretion. Adaptation to near-zero growth conditions of recombinant K. phaffii resulted in significant changes in the total protein, RNA, DNA and lipid content, and lipidomics revealed a complex adaptation pattern regarding the lipid class composition. The higher abundance of storage lipids as well as storage carbohydrates indicates that the cells are preparing for long-term survival. CONCLUSIONS: In conclusion, retentostat cultivation proved to be a valuable tool to identify potential engineering targets to decouple growth and protein production and gain important insights into the physiological adaptation of K. phaffii to near-zero growth conditions.

Fermenting the future - on the benefits of a bioart collaboration.

In this article we explore the intersection of science and art through a collaboration between us scientists and the bioartists Anna Dimitriu and Alex May, focusing on the interface of yeast biotechnology and art. The collaboration, originally initiated in 2018, resulted in three major artworks: CULTURE, depicting the evolution of yeast and human societies; FERMENTING FUTURES, illustrating a synthetic autotrophic yeast and its link to lactic acid production; and WOOD SPIRIT-AMBER ACID, inspired by the VIVALDI project targeting CO2 reduction to methanol. We emphasize the reciprocal nature of the collaboration, detailing the scientific insights gained and the impact of artistic perspectives on us as researchers. We also highlight the historical connection between art and science, particularly in the Renaissance periods, and underscore the educational value of integrating art into science not only to support public engagement and science dissemination, but also to widen our own perceptions in our research.

A native phosphoglycolate salvage pathway of the synthetic autotrophic yeast Komagataella phaffii.

Synthetic autotrophs can serve as chassis strains for bioproduction from CO2 as a feedstock to take measures against the climate crisis. Integration of the Calvin-Benson-Bassham (CBB) cycle into the methylotrophic yeast Komagataella phaffii (Pichia pastoris) enabled it to use CO2 as the sole carbon source. The key enzyme in this cycle is ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzing the carboxylation step. However, this enzyme is error prone to perform an oxygenation reaction leading to the production of toxic 2-phosphoglycolate. Native autotrophs have evolved different recycling pathways for 2-phosphoglycolate. However, for synthetic autotrophs, no information is available for the existence of such pathways. Deletion of CYB2 in the autotrophic K. phaffii strain led to the accumulation of glycolate, an intermediate in phosphoglycolate salvage pathways, suggesting that such a pathway is enabled by native K. phaffii enzymes. 13C tracer analysis with labeled glycolate indicated that the yeast pathway recycling phosphoglycolate is similar to the plant salvage pathway. This orthogonal yeast pathway may serve as a sensor for RuBisCO oxygenation, and as an engineering target to boost autotrophic growth rates in K. phaffii.

The potential of CO2-based production cycles in biotechnology to fight the climate crisis.

Rising CO2 emissions have pushed scientists to develop new technologies for a more sustainable bio-based economy. Microbial conversion of CO2 and CO2-derived carbon substrates into valuable compounds can contribute to carbon neutrality and sustainability. Here, we discuss the potential of C1 carbon sources as raw materials to produce energy, materials, and food and feed using microbial cell factories. We provide an overview of potential microbes, natural and synthetic C1 utilization pathways, and compare their metabolic driving forces. Finally, we sketch a future in which C1 substrates replace traditional feedstocks and we evaluate the costs associated with such an endeavor.

The oxygen-tolerant reductive glycine pathway assimilates methanol, formate and CO2 in the yeast Komagataella phaffii.

The current climatic change is predominantly driven by excessive anthropogenic CO2 emissions. As industrial bioprocesses primarily depend on food-competing organic feedstocks or fossil raw materials, CO2 co-assimilation or the use of CO2-derived methanol or formate as carbon sources are considered pathbreaking contributions to solving this global problem. The number of industrially-relevant microorganisms that can use these two carbon sources is limited, and even fewer can concurrently co-assimilate CO2. Here, we search for alternative native methanol and formate assimilation pathways that co-assimilate CO2 in the industrially-relevant methylotrophic yeast Komagataella phaffii (Pichia pastoris). Using 13C-tracer-based metabolomic techniques and metabolic engineering approaches, we discover and confirm a growth supporting pathway based on native enzymes that can perform all three assimilations: namely, the oxygen-tolerant reductive glycine pathway. This finding paves the way towards metabolic engineering of formate and CO2 utilisation to produce proteins, biomass, or chemicals in yeast.

Carbon efficient production of chemicals with yeasts.

Microbial metabolism offers a wide variety of opportunities to produce chemicals from renewable resources. Employing such processes of industrial biotechnology provides valuable means to fight climate change by replacing fossil feedstocks by renewable substrate to reduce or even revert carbon emission. Several yeast species are well suited chassis organisms for this purpose, illustrated by the fact that the still largest microbial production of a chemical, namely bioethanol is based on yeast. Although production of ethanol and some other chemicals is highly efficient, this is not the case for many desired bulk chemicals. One reason for low efficiency is carbon loss, which decreases the product yield and increases the share of total production costs that is taken by substrate costs. Here we discuss the causes for carbon loss in metabolic processes, approaches to avoid carbon loss, as well as opportunities to incorporate carbon from CO2 , based on the electron balance of pathways. These aspects of carbon efficiency are illustrated for the production of succinic acid from a diversity of substrates using different pathways.

Systematic sequence engineering enhances the induction strength of the glucose-regulated GTH1 promoter of Komagataella phaffii.

The promoter of the high-affinity glucose transporter Gth1 (PGTH1) is tightly repressed on glucose and glycerol surplus, and strongly induced in glucose-limitation, thus enabling regulated methanol-free production processes in the yeast production host Komagataella phaffii. To further improve this promoter, an intertwined approach of nucleotide diversification through random and rational engineering was pursued. Random mutagenesis and fluorescence activated cell sorting of PGTH1 yielded five variants with enhanced induction strength. Reverse engineering of individual point mutations found in the improved variants identified two single point mutations with synergistic action. Sequential deletions revealed the key promoter segments for induction and repression properties, respectively. Combination of the single point mutations and the amplification of key promoter segments led to a library of novel promoter variants with up to 3-fold higher activity. Unexpectedly, the effect of gaining or losing a certain transcription factor binding site (TFBS) was highly dependent on its context within the promoter. Finally, the applicability of the novel promoter variants for biotechnological production was proven for the secretion of different recombinant model proteins in fed batch cultivation, where they clearly outperformed their ancestors. In addition to advancing the toolbox for recombinant protein production and metabolic engineering of K. phaffii, we discovered single nucleotide positions and correspondingly affected TFBS that distinguish between glycerol- and glucose-mediated repression of the native promoter.

Customizing amino acid metabolism of Pichia pastoris for recombinant protein production.

Amino acids are the building blocks of proteins. In this respect, a reciprocal effect of recombinant protein production on amino acid biosynthesis as well as the impact of the availability of free amino acids on protein production can be anticipated. In this study, the impact of engineering the amino acid metabolism on the production of recombinant proteins was investigated in the yeast Pichia pastoris (syn Komagataella phaffii). Based on comprehensive systems-level analyses of the metabolomes and transcriptomes of different P. pastoris strains secreting antibody fragments, cell engineering targets were selected. Our working hypothesis that increasing intracellular amino acid levels could help unburden cellular metabolism and improve recombinant protein production was examined by constitutive overexpression of genes related to amino acid metabolism. In addition to 12 genes involved in specific amino acid biosynthetic pathways, the transcription factor GCN4 responsible for regulation of amino acid biosynthetic genes was overexpressed. The production of the used model protein, a secreted carboxylesterase (CES) from Sphingopyxis macrogoltabida, was increased by overexpression of pathway genes for alanine and for aromatic amino acids, and most pronounced, when overexpressing the regulator GCN4. The analysis of intracellular amino acid levels of selected clones indicated a direct linkage of improved recombinant protein production to the increased availability of intracellular amino acids. Finally, fed batch cultures showed that overexpression of GCN4 increased CES titers 2.6-fold, while the positive effect of other amino acid synthesis genes could not be transferred from screening to bioreactor cultures.

Efficient production of bacterial antibiotics aminoriboflavin and roseoflavin in eukaryotic microorganisms, yeasts.

BACKGROUND: Actinomycetes Streptomyces davaonensis and Streptomyces cinnabarinus synthesize a promising broad-spectrum antibiotic roseoflavin, with its synthesis starting from flavin mononucleotide and proceeding through an immediate precursor, aminoriboflavin, that also has antibiotic properties. Roseoflavin accumulation by the natural producers is rather low, whereas aminoriboflavin accumulation is negligible. Yeasts have many advantages as biotechnological producers relative to bacteria, however, no recombinant producers of bacterial antibiotics in yeasts are known. RESULTS: Roseoflavin biosynthesis genes have been expressed in riboflavin- or FMN-overproducing yeast strains of Candida famata and Komagataella phaffii. Both these strains accumulated aminoriboflavin, whereas only the latter produced roseoflavin. Aminoriboflavin isolated from the culture liquid of C. famata strain inhibited the growth of Staphylococcus aureus (including MRSA) and Listeria monocytogenes. Maximal accumulation of aminoriboflavin in shake-flasks reached 1.5 mg L- 1 (C. famata), and that of roseoflavin was 5 mg L- 1 (K. phaffii). Accumulation of aminoriboflavin and roseoflavin by K. phaffii recombinant strain in a bioreactor reached 22 and 130 mg L- 1, respectively. For comparison, recombinant strains of the native bacterial producer S. davaonensis accumulated near one-order less of roseoflavin while no recombinant producers of aminoriboflavin was reported at all. CONCLUSIONS: Yeast recombinant producers of bacterial antibiotics aminoriboflavin and roseoflavin were constructed and evaluated.

Tailored extraction and ion mobility-mass spectrometry enables isotopologue analysis of tetrahydrofolate vitamers.

Climate change directs the focus in biotechnology increasingly on one-carbon metabolism for fixation of CO2 and CO2-derived chemicals (e.g. methanol, formate) to reduce our reliance on both fossil and food-competing carbon sources. The tetrahydrofolate pathway is involved in several one-carbon fixation pathways. To study such pathways, stable isotope-labelled tracer analysis performed with mass spectrometry is state of the art. However, no such method is currently available for tetrahydrofolate vitamers. In the present work, we established a fit-for-purpose extraction method for the methylotrophic yeast Komagataella phaffii that allows access to intracellular methyl- and methenyl-tetrahydrofolate (THF) with demonstrated stability over several hours. To determine isotopologue distributions of methyl-THF, LC-QTOFMS provides a selective fragment ion with suitable intensity of at least two isotopologues in all samples, but not for methenyl-THF. However, the addition of ion mobility separation provided a critical selectivity improvement allowing accurate isotopologue distribution analysis of methenyl-THF with LC-IM-TOFMS. Application of these new methods for 13C-tracer experiments revealed a decrease from 83 ± 4 to 64 ± 5% in the M + 0 carbon isotopologue fraction in methyl-THF after 1 h of labelling with formate, and to 54 ± 5% with methanol. The M + 0 carbon isotopologue fraction of methenyl-THF was reduced from 83 ± 2 to 78 ± 1% over the same time when using 13C-methanol labelling. The labelling results of multiple strains evidenced the involvement of the THF pathway in the oxygen-tolerant reductive glycine pathway, the presence of the in vivo reduction of formate to formaldehyde, and the activity of the spontaneous condensation reaction of formaldehyde with THF in K. phaffii.

Synthetic two-species allodiploid and three-species allotetraploid Saccharomyces hybrids with euploid (complete) parental subgenomes.

Combination of the genomes of Saccharomyces species has great potential for the construction of new industrial strains as well as for the study of the process of speciation. However, these species are reproductively isolated by a double sterility barrier. The first barrier is mainly due to the failure of the chromosomes to pair in allodiploid meiosis. The second barrier ensures that the hybrid remains sterile even after genome duplication, an event that can restore fertility in plant interspecies hybrids. The latter is attributable to the autodiploidisation of the allotetraploid meiosis that results in sterile allodiploid spores (return to the first barrier). Occasionally, mating-competent alloaneuploid spores arise by malsegregation of MAT-carrying chromosomes. These can mate with cells of a third species resulting in aneuploid zygotes having at least one incomplete subgenome. Here we report on the construction of euploid three-species hybrids by making use of "rare mating" between a sterile S. kudriavzevii x S. uvarum allodiploid hybrid and a diploid S. cerevisiae strain. The hybrids have allotetraploid 2nScnSk nSu genomes consisting of complete sets of parental chromosomes. This is the first report on the production of euploid three-species Saccharomyces hybrids by natural mating, without genetic manipulation. The hybrids provide possibilities for studying the interactions of three allospecific genomes and their orthologous genes present in the same cell.

Synthetic activation of yeast stress response improves secretion of recombinant proteins.

Yeasts, such as Pichia pastoris (syn Komagataella spp.), are particularly suitable expression systems for emerging classes of recombinant proteins. Among them, recombinant antibody fragments, such as single-chain variable fragments (scFv) and single-domain antibodies (VHH), are credible alternatives to monoclonal antibodies. The availability of powerful genetic engineering and synthetic biology tools has facilitated improvement of this cell factory to overcome certain limitations. However, cell engineering to improve secretion often remains a trial-and-error approach and improvements are often specific to the protein produced. Where multiple genetic interventions are needed to remove bottlenecks in the process of recombinant protein secretion, this leads to a high number of combinatorial possibilities for creation of new production strains. Therefore, our aim was to exploit whole transcriptional programs (stress response pathways) in order to simplify the strain engineering of new production strains. Indeed, the artificial activation of the general stress response transcription factor Msn4, as well as synthetic versions thereof, could replace the secretion enhancing effect of several cytosolic chaperones. Greater than 4-fold improvements in recombinant protein secretion were achieved by overexpression of MSN4 or synMSN4, either alone or in combination with Hac1 or ER chaperones. With this concept we were able to successfully engineer strains reaching titers of more than 2.5 g/L scFv and 8 g/L VHH in bioreactor cultivations. This increased secretion capacity of different industrially relevant model proteins indicates that MSN4 overexpression most likely represents a general concept to improve recombinant protein production in yeast.

Conversion of CO2 into organic acids by engineered autotrophic yeast.

The increase of CO2 emissions due to human activity is one of the preeminent reasons for the present climate crisis. In addition, considering the increasing demand for renewable resources, the upcycling of CO2 as a feedstock gains an extensive importance to establish CO2-neutral or CO2-negative industrial processes independent of agricultural resources. Here we assess whether synthetic autotrophic Komagataella phaffii (Pichia pastoris) can be used as a platform for value-added chemicals using CO2 as a feedstock by integrating the heterologous genes for lactic and itaconic acid synthesis. 13C labeling experiments proved that the resulting strains are able to produce organic acids via the assimilation of CO2 as a sole carbon source. Further engineering attempts to prevent the lactic acid consumption increased the titers to 600 mg L-1, while balancing the expression of key genes and modifying screening conditions led to 2 g L-1 itaconic acid. Bioreactor cultivations suggest that a fine-tuning on CO2 uptake and oxygen demand of the cells is essential to reach a higher productivity. We believe that through further metabolic and process engineering, the resulting engineered strain can become a promising host for the production of value-added bulk chemicals by microbial assimilation of CO2, to support sustainability of industrial bioprocesses.

Pushing and pulling proteins into the yeast secretory pathway enhances recombinant protein secretion.

Yeasts and especially Pichia pastoris (syn Komagataella spp.) are popular microbial expression systems for the production of recombinant proteins. One of the key advantages of yeast host systems is their ability to secrete the recombinant protein into the culture media. However, secretion of some recombinant proteins is less efficient. These proteins include antibody fragments such as Fabs or scFvs. We have recently identified translocation of nascent Fab fragments from the cytosol into the endoplasmic reticulum (ER) as one major bottleneck. Conceptually, this bottleneck requires engineering to increase the flux of recombinant proteins at the translocation step by pushing on the cytosolic side and pulling on the ER side. This engineering strategy is well-known in the field of metabolic engineering. To apply the push-and-pull strategy to recombinant protein secretion, we chose to modulate the cytosolic and ER Hsp70 cycles, which have a key impact on the translocation process. After identifying the relevant candidate factors of the Hsp70 cycles, we combined the push-and-pull factors in a single strain and achieved synergistic effects for antibody fragment secretion. With this concept we were able to successfully engineer strains and improve protein secretion up to 5-fold for different model protein classes. Overall, titers of more than 1.3 g/L Fab and scFv were reached in bioreactor cultivations.

From strain engineering to process development: monoclonal antibody production with an unnatural amino acid in Pichia pastoris.

BACKGROUND: Expansion of the genetic code is a frequently employed approach for the modification of recombinant protein properties. It involves reassignment of a codon to another, e.g., unnatural, amino acid and requires the action of a pair of orthogonal tRNA and aminoacyl tRNA synthetase modified to recognize only the desired amino acid. This approach was applied for the production of trastuzumab IgG carrying p-azido-L-phenylalanine (pAzF) in the industrial yeast Pichia pastoris. Combining the knowledge of protein folding and secretion with bioreactor cultivations, the aim of the work was to make the production of monoclonal antibodies with an expanded genetic code cost-effective on a laboratory scale. RESULTS: Co-translational transport of proteins into the endoplasmic reticulum through secretion signal prepeptide change and overexpression of lumenal chaperones Kar2p and Lhs1p improved the production of trastuzumab IgG and its Fab fragment with incorporated pAzF. In the case of Fab, a knockout of vacuolar targeting for protein degradation further increased protein yield. Fed-batch bioreactor cultivations of engineered P. pastoris strains increased IgG and IgGpAzF productivity by around 50- and 20-fold compared to screenings, yielding up to 238 mg L-1 and 15 mg L-1 of fully assembled tetrameric protein, respectively. Successful site-specific incorporation of pAzF was confirmed by mass spectrometry. CONCLUSIONS: Pichia pastoris was successfully employed for cost-effective laboratory-scale production of a monoclonal antibody with an unnatural amino acid. Applying the results of this work in glycoengineered strains, and taking further steps in process development opens great possibilities for utilizing P. pastoris in the development of antibodies for subsequent conjugations with, e.g., bioactive payloads.

Genotypic and phenotypic diversity among Komagataella species reveals a hidden pathway for xylose utilization.

BACKGROUND: The yeast genus Komagataella currently consists of seven methylotrophic species isolated from tree environments. Well-characterized strains of K. phaffii and K. pastoris are important hosts for biotechnological applications, but the potential of other species from the genus remains largely unexplored. In this study, we characterized 25 natural isolates from all seven described Komagataella species to identify interesting traits and provide a comprehensive overview of the genotypic and phenotypic diversity available within this genus. RESULTS: Growth tests on different carbon sources and in the presence of stressors at two different temperatures allowed us to identify strains with differences in tolerance to high pH, high temperature, and growth on xylose. As Komagataella species are generally not considered xylose-utilizing yeasts, xylose assimilation was characterized in detail. Growth assays, enzyme activity measurements and 13C labeling confirmed the ability of K. phaffii to utilize D-xylose via the oxidoreductase pathway. In addition, we performed long-read whole-genome sequencing to generate genome assemblies of all Komagataella species type strains and additional K. phaffii and K. pastoris isolates for comparative analysis. All sequenced genomes have a similar size and share 83-99% average sequence identity. Genome structure analysis showed that K. pastoris and K. ulmi share the same rearrangements in difference to K. phaffii, while the genome structure of K. kurtzmanii is similar to K. phaffii. The genomes of the other, more distant species showed a larger number of structural differences. Moreover, we used the newly assembled genomes to identify putative orthologs of important xylose-related genes in the different Komagataella species. CONCLUSIONS: By characterizing the phenotypes of 25 natural Komagataella isolates, we could identify strains with improved growth on different relevant carbon sources and stress conditions. Our data on the phenotypic and genotypic diversity will provide the basis for the use of so-far neglected Komagataella strains with interesting characteristics and the elucidation of the genetic determinants of improved growth and stress tolerance for targeted strain improvement.

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

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

Adaptive laboratory evolution and reverse engineering enhances autotrophic growth in Pichia pastoris.

Synthetic biology offers several routes for CO2 conversion into biomass or bio-chemicals, helping to avoid unsustainable use of organic feedstocks, which negatively contribute to climate change. The use of well-known industrial organisms, such as the methylotrophic yeast Pichia pastoris (Komagataella phaffii), for the establishment of novel C1-based bioproduction platforms could wean biotechnology from feedstocks with alternative use in food production. Recently, the central carbon metabolism of P. pastoris was re-wired following a rational engineering approach, allowing the resulting strains to grow autotrophically with a µmax of 0.008 h-1, which was further improved to 0.018 h-1 by adaptive laboratory evolution. Using reverse genetic engineering of single-nucleotide (SNPs) polymorphisms occurring in the genes encoding for phosphoribulokinase and nicotinic acid mononucleotide adenylyltransferase after evolution, we verified their influence on the improved autotrophic phenotypes. The reverse engineered SNPs lead to lower enzyme activities in putative branching point reactions and in reactions involved in energy balancing. Beyond this, we show how further evolution facilitates peroxisomal import and increases growth under autotrophic conditions. The engineered P. pastoris strains are a basis for the development of a platform technology, which uses CO2 for production of value-added products, such as cellular biomass, technical enzymes and chemicals and which further avoids consumption of organic feedstocks with alternative use in food production. Further, the identification and verification of three pivotal steps may facilitate the integration of heterologous CBB cycles or similar pathways into heterotrophic organisms.

What makes Komagataella phaffii non-conventional?

The important industrial protein production host Komagataella phaffii (syn Pichia pastoris) is classified as a non-conventional yeast. But what exactly makes K. phaffii non-conventional? In this review, we set out to address the main differences to the 'conventional' yeast Saccharomyces cerevisiae, but also pinpoint differences to other non-conventional yeasts used in biotechnology. Apart from its methylotrophic lifestyle, K. phaffii is a Crabtree-negative yeast species. But even within the methylotrophs, K. phaffii possesses distinct regulatory features such as glycerol-repression of the methanol-utilization pathway or the lack of nitrate assimilation. Rewiring of the transcriptional networks regulating carbon (and nitrogen) source utilization clearly contributes to our understanding of genetic events occurring during evolution of yeast species. The mechanisms of mating-type switching and the triggers of morphogenic phenotypes represent further examples for how K. phaffii is distinguished from the model yeast S. cerevisiae. With respect to heterologous protein production, K. phaffii features high secretory capacity but secretes only low amounts of endogenous proteins. Different to S. cerevisiae, the Golgi apparatus of K. phaffii is stacked like in mammals. While it is tempting to speculate that Golgi architecture is correlated to the high secretion levels or the different N-glycan structures observed in K. phaffii, there is recent evidence against this. We conclude that K. phaffii is a yeast with unique features that has a lot of potential to explore both fundamental research questions and industrial applications.