Recent progress on heterologous protein production in methylotrophic yeast systems.

Recombinant protein production technology is widely applied to the manufacture of biologics used as drug substances and industrial proteins such as recombinant enzymes and bioactive proteins. Various heterologous protein production systems have been developed using prokaryotic and eukaryotic hosts. Especially methylotrophic yeast in eukaryotic hosts is suggested to be particularly valuable because such systems have the following advantages: protein secretion into culture broth, eukaryotic quality control systems, a post-translational modification system, rapid growth, and established recombinant DNA tools and technologies such as strong promoters, effective selection markers, and gene knock-in and -out systems. Many methylotrophic yeasts such as the genera Candida, Ogataea, and Komagataella have been studied since methylotrophic yeast was first isolated in 1969. The methanol-consumption-related genes in methylotrophic yeast are strongly and strictly regulated under methanol-containing conditions. The well-regulated gene expression systems under the methanol-inducible gene promoter lead to the potential application of heterologous protein production in methylotrophic yeast. In this review, we describe the recent progress of heterologous protein production technology in methylotrophic yeast and introduce Ogataea minuta as an alternative production host as a substitute for K. phaffii and O. polymorpha.

Kodamaea ohmeri: A rare yeast causing invasive infections in immunocompromised patients.

INTRODUCTION: Kodamaea ohmeri is a rare, recognized pathogen that has previously been isolated from environmental sources. The patients commonly affected by this yeast include immunocompromised as well as immunocompetent patients having several associated risk factors. METHODOLOGY: We report three cases in which K. ohmeri was isolated from blood using Bact T/ALERT. Identification was carried out by MALDI-TOF MS (Vitek-MS, BioMérieux, Marcy-l'Etoile, France) in addition to color characteristics on chromogenic media. The patients had diminished immune response on account of a multitude of comorbidities. RESULTS: K. ohmeri can be misidentified as Candida tropicalis, Candida albicans, or Candida hemolounii by conventional methods; correct and timely identification can be achieved by MALDI-TOF MS. Antifungal susceptibility breakpoints for K. ohmeri are currently not defined. An Echinocandin was added to the treatment regimen of all three of the cases. CONCLUSIONS: Identification of K. ohmeri using conventional methods is difficult and unusual yeasts should be carefully observed, especially upon prolonged incubation.

Understanding exopolysaccharide byproduct formation in Komagataella phaffii fermentation processes for recombinant protein production.

BACKGROUND: Komagataella phaffii (Pichia pastoris) has emerged as a common and robust biotechnological platform organism, to produce recombinant proteins and other bioproducts of commercial interest. Key advantage of K. phaffii is the secretion of recombinant proteins, coupled with a low host protein secretion. This facilitates downstream processing, resulting in high purity of the target protein. However, a significant but often overlooked aspect is the presence of an unknown polysaccharide impurity in the supernatant. Surprisingly, this impurity has received limited attention in the literature, and its presence and quantification are rarely addressed. RESULTS: This study aims to quantify this exopolysaccharide in high cell density recombinant protein production processes and identify its origin. In stirred tank fed-batch fermentations with a maximal cell dry weight of 155 g/L, the polysaccharide concentration in the supernatant can reach up to 8.7 g/L. This level is similar to the achievable target protein concentration. Importantly, the results demonstrate that exopolysaccharide production is independent of the substrate and the protein production process itself. Instead, it is directly correlated with biomass formation and proportional to cell dry weight. Cell lysis can confidently be ruled out as the source of this exopolysaccharide in the culture medium. Furthermore, the polysaccharide secretion can be linked to a mutation in the HOC1 gene, featured by all derivatives of strain NRRL Y-11430, leading to a characteristic thinner cell wall. CONCLUSIONS: This research sheds light on a previously disregarded aspect of K. phaffii fermentations, emphasizing the importance of monitoring and addressing the exopolysaccharide impurity in biotechnological applications, independent of the recombinant protein produced.

Cell growth and nutrient availability control the mitotic exit signaling network in budding yeast.

Cell growth is required for cell cycle progression. The amount of growth required for cell cycle progression is reduced in poor nutrients, which leads to a reduction in cell size. In budding yeast, nutrients can influence cell size by modulating the extent of bud growth, which occurs predominantly in mitosis. However, the mechanisms are unknown. Here, we used mass spectrometry to identify proteins that modulate bud growth in response to nutrient availability. This led to the discovery that nutrients regulate numerous components of the mitotic exit network (MEN), which controls exit from mitosis. A key component of the MEN undergoes gradual multisite phosphorylation during bud growth that is dependent upon bud growth and correlated with the extent of growth. Furthermore, activation of the MEN is sufficient to override a growth requirement for mitotic exit. The data suggest a model in which the MEN ensures that mitotic exit occurs only when an appropriate amount of bud growth has occurred.

Hemolymph metabolism of black soldier fly (Diptera: Stratiomyidae), response to different supplemental fungi.

The black soldier fly, Hermetia illucens L. (Diptera: Stratiomyidae), is commonly used for organic waste recycling and animal feed production. However, the often inadequate nutrients in organic waste necessitate nutritional enhancement of black soldier fly larvae, e.g., by fungal supplementation of its diet. We investigated the amino acid composition of two fungi, Candida tropicalis (Castell.) Berkhout (Saccharomycetales: Saccharomycetaceae) and Pichia kudriavzevii Boidin, Pignal & Besson (Saccharomycetales: Pichiaceae), from the black soldier fly gut, and commercial baker's yeast, Saccharomyces cerevisiae Meyen ex E.C. Hansen (Saccharomycetales: Saccharomycetaceae), and their effects on larval growth and hemolymph metabolites in fifth-instar black soldier fly larvae. Liquid chromatography-mass spectrometry was used to study the effect of fungal metabolites on black soldier fly larval metabolism. Amino acid analysis revealed significant variation among the fungi. Fungal supplementation led to increased larval body mass and differential metabolite accumulation. The three fungal species caused distinct metabolic changes, with each over-accumulating and down-accumulating various metabolites. We identified significant alteration of histidine metabolism, aminoacyl-tRNA biosynthesis, and glycerophospholipid metabolism in BSF larvae treated with C. tropicalis. Treatment with P. kudriavzevii affected histidine metabolism and citrate cycle metabolites, while both P. kudriavzevii and S. cerevisiae treatments impacted tyrosine metabolism. Treatment with S. cerevisiae resulted in down-accumulation of metabolites related to glycine, serine, and threonine metabolism. This study suggests that adding fungi to the larval diet significantly affects black soldier fly larval metabolomics. Further research is needed to understand how individual amino acids and their metabolites contributed by fungi affect black soldier fly larval physiology, growth, and development, to elucidate the interaction between fungal nutrients and black soldier fly physiology.

Enhancing the expression of chondroitin 4-O-sulfotransferase for one-pot enzymatic synthesis of chondroitin sulfate A.

Chondroitin sulfate (CS) stands as a pivotal compound in dietary supplements for osteoarthritis treatment, propelling significant interest in the biotechnological pursuit of environmentally friendly and safe CS production. Enzymatic synthesis of CS for instance CSA has been considered as one of the most promising methods. However, the bottleneck consistently encountered is the active expression of chondroitin 4-O-sulfotransferase (C4ST) during CSA biosynthesis. This study meticulously delved into optimizing C4ST expression through systematic enhancements in transcription, translation, and secretion mechanisms via modifications in the 5' untranslated region, the N-terminal encoding sequence, and the Komagataella phaffii chassis. Ultimately, the active C4ST expression escalated to 2713.1 U/L, representing a striking 43.7-fold increase. By applying the culture broth supernatant of C4ST and integrating the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) biosynthesis module, we constructed a one-pot enzymatic system for CSA biosynthesis, achieving a remarkable sulfonation degree of up to 97.0 %. The substantial enhancement in C4ST expression and the development of an engineered one-pot enzymatic synthesis system promises to expedite large-scale CSA biosynthesis with customizable sulfonation degrees.

Wickerhamiella lachancei f.a. sp. nov., a novel ascomycetous yeast species isolated from flowers of Lantana camara in India.

Two isolates representing a novel species of the genus Wickerhamiella were obtained in India from nectar of flowers of Lantana camara, an ornamental exotic species native to Central and South America. Phylogenetic analyses of the D1/D2 domain of the 26S large subunit (LSU) rRNA gene, internal transcribed spacer (ITS) region, and physiological characteristics, supported the recognition of the novel species, that we designate Wickerhamiella lachancei sp. nov (MycoBank no. MB851709), with MCC 9929T as the holotype and PYCC 10003T as the isotype. Considering pairwise sequence similarity, the type strain of the novel species differs from the type strain of the most closely related species, Wickerhamiella drosophilae CBS 8459T, by 16 nucleotide substitutions and two gaps (3.9 % sequence variation) in the D1/D2 region (560 bp compared) and 28 nucleotide substitutions and five gaps (7.22 % sequence variation) in the ITS region (444 bp compared).

Enhancing the expression of the unspecific peroxygenase in Komagataella phaffii through a combination strategy.

The unspecific peroxygenase (UPO) from Cyclocybe aegerita (AaeUPO) can selectively oxidize C-H bonds using hydrogen peroxide as an oxygen donor without cofactors, which has drawn significant industrial attention. Many studies have made efforts to enhance the overall activity of AaeUPO expressed in Komagataella phaffii by employing strategies such as enzyme-directed evolution, utilizing appropriate promoters, and screening secretion peptides. Building upon these previous studies, the objective of this study was to further enhance the expression of a mutant of AaeUPO with improved activity (PaDa-I) by increasing the gene copy number, co-expressing chaperones, and optimizing culture conditions. Our results demonstrated that a strain carrying approximately three copies of expression cassettes and co-expressing the protein disulfide isomerase showed an approximately 10.7-fold increase in volumetric enzyme activity, using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. After optimizing the culture conditions, the volumetric enzyme activity of this strain further increased by approximately 48.7%, reaching 117.3 U/mL. Additionally, the purified catalytic domain of PaDa-I displayed regioselective hydroxylation of R-2-phenoxypropionic acid. The results of this study may facilitate the industrial application of UPOs. KEY POINTS: • The secretion of the catalytic domain of PaDa-I can be significantly enhanced through increasing gene copy numbers and co-expressing of protein disulfide isomerase. • After optimizing the culture conditions, the volumetric enzyme activity can reach 117.3 U/mL, using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. • The R-2-phenoxypropionic acid can undergo the specific hydroxylation reaction catalyzed by catalytic domain of PaDa-I, resulting in the formation of R-2-(4-hydroxyphenoxy)propionic acid.

Complete genome sequencing of Hortaea werneckii M-3 for identifying polyester polyurethane degrading enzymes.

Hortaea werneckii M-3, a black yeast isolated from the marine sediment of the West Pacific, can utilize polyester polyurethane (PU, Impranil DLN) as a sole carbon source. Here, we present the complete genome of Hortaea werneckii M-3 with the focus on PU degradation enzymes. The total genome size is 38,167,921 bp, consisting of 186 contigs with a N50 length of 651,266 bp and a GC content of 53.06%. Genome annotation analysis predicts a total of 13,462 coding genes, which include 99 tRNAs and 105 rRNAs. Some genes encoding PU degrading enzymes including cutinase and urease are identified in this genome. The genome analysis of Hortaea werneckii M-3 will be helpful for further understanding the degradation mechanism of polyester PU by marine yeasts.

Ceramide sorting into non-vesicular transport is independent of acyl chain length in budding yeast.

The transport of ceramide from the endoplasmic reticulum (ER) to the Golgi is a key step in the synthesis of complex sphingolipids, the main building blocks of the plasma membrane. In yeast, ceramide is transported to the Golgi either through ATP-dependent COPII vesicles of the secretory pathway or by ATP-independent non-vesicular transport that involves tethering proteins at ER-Golgi membrane contact sites. Studies in both mammalian and yeast cells reported that vesicular transport mainly carries ceramide containing very long chain fatty acids, while the main mammalian non-vesicular ceramide transport protein CERT only transports ceramides containing short chain fatty acids. However, if non-vesicular ceramide transport in yeast similarly favors short chain ceramides remained unanswered. Here we employed a yeast GhLag1 strain in which the endogenous ceramide synthase is replaced by the cotton-derived GhLag1 gene, resulting in the production of short chain C18 rather than C26 ceramides. We show that block of vesicular transport through ATP-depletion or the use of temperature-sensitive sec mutants caused a reduction in inositolphosphorylceramide (IPC) synthesis to similar extent in WT and GhLag1 backgrounds. Since the remaining IPC synthesis is a readout for non-vesicular ceramide transport, our results indicate that non-vesicular ceramide transport is neither blocked nor facilitated when only short chain ceramides are present. Therefore, we propose that the sorting of ceramide into non-vesicular transport is independent of acyl chain length in budding yeast.

Updated component analysis method for naturally occurring sophorolipids from Starmerella bombicola.

Sophorolipids (SLs) are promising glycolipid biosurfactants as they are easily produced and functional. SLs from microorganisms are comprised of mixtures of multiple derivatives that have different structures and properties, including well-known acidic and lactonic SL (ASLs and LSLs, respectively). In this study, we established a method for analyzing all SL derivatives in the products of Starmerella bombicola, a typical SL-producing yeast. Detailed component analyses of S. bombicola products were carried out using reversed-phase high-performance liquid chromatography and mass spectrometry. Methanol was used as the eluent as it is a good solvent for all SL derivatives. With this approach, it was possible to not only quantify the ratio of the main components of ASL, LSL, and SL glycerides but also confirm trace components such as SL mono-glyceride and bola-form SL (sophorose at both ends); notably, this is the first time these components have been isolated and identified successfully in naturally occurring SLs. In addition, our results revealed a novel SL derivative in which a fatty acid is bonded in series to the ASL, which had not been reported previously. Using the present analysis method, it was possible to easily track compositional changes in the SL components during culture. Our results showed that LSL and ASL are produced initially and that SL glycerides accumulate from the middle stage during the fermentation process. KEY POINTS: • An easy and detailed component analysis method for sophorolipids (SLs) is introduced. • Multiple SL derivatives were identified different from known SLs. • A novel hydrophobic acidic SL was isolated and characterized.

Evaluation of heterologous expression in Pichia pastoris of Pine Weevil TRPA1 by GFP and flow cytometry.

BACKGROUND: The wasabi receptor, also known as the Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel, is a potential target for development of repellents for insects, like the pine weevil (Hylobius abietis) feeding on conifer seedlings and causing damage in forestry. Heterologous expression of TRPA1 from pine weevil in the yeast Pichia pastoris can potentially provide protein for structural and functional studies. Here we take advantage of the Green Fluorescent Protein (GFP) tag to examine the various steps of heterologous expression, to get more insight in clone selection, expression and isolation of the intact purified protein. RESULTS: The sequence of HaTRPA1 is reported and GFP-tagged constructs were made of the full-length protein and a truncated version (Δ1-708 HaTRPA1), lacking the N-terminal ankyrin repeat domain. Clones were screened on GFP expression plates, induced in small liquid cultures and in fed-batch cultures, and evaluated by flow cytometry and fluorescence microscopy. The screening on plates successfully identifies low-expression clones, but fails to predict the ranking of the best performing clones in small-scale liquid cultures. The two constructs differ in their cellular localization. Δ1-708 HaTRPA1 is found in a ring at the perimeter of cell, whereas HaTRPA1 is forming highly fluorescent speckles in interior regions of the cell. The pattern is consistent in different clones of the same construct and persists in fed-batch culture. The expression of Δ1-708 HaTRPA1 decreases the viability more than HaTRPA1, and in fed-batch culture it is clear that intact cells first express Δ1-708 HaTRPA1 and then become damaged. Purifications show that both constructs suffer from degradation of the expressed protein, but especially the HaTRPA1 construct. CONCLUSIONS: The GFP tag makes it possible to follow expression by flow cytometry and fluorescence microscopy. Analyses of localization, cell viability and expression show that the former two parameters are specific for each of the two evaluated constructs, whereas the relative expression of the constructs varies with the cultivation method. High expression is not all that matters, so taking damaged cells into account, something that may be linked to protein degradation, is important when picking the most suitable construct, clone, and expression scheme.

Scalable protein production by Komagataella phaffii enabled by ARS plasmids and carbon source-based selection.

BACKGROUND: Most recombinant Komagataella phaffii (Pichia pastoris) strains for protein production are generated by genomic integration of expression cassettes. The clonal variability in gene copy numbers, integration loci and consequently product titers limit the aptitude for high throughput applications in drug discovery, enzyme engineering or most comparative analyses of genetic elements such as promoters or secretion signals. Circular episomal plasmids with an autonomously replicating sequence (ARS), an alternative which would alleviate some of these limitations, are inherently unstable in K. phaffii. Permanent selection pressure, mostly enabled by antibiotic resistance or auxotrophy markers, is crucial for plasmid maintenance and hardly scalable for production. The establishment and use of extrachromosomal ARS plasmids with key genes of the glycerol metabolism (glycerol kinase 1, GUT1, and triosephosphate isomerase 1, TPI1) as selection markers was investigated to obtain a system with high transformation rates that can be directly used for scalable production processes in lab scale bioreactors. RESULTS: In micro-scale deep-well plate experiments, ARS plasmids employing the Ashbya gossypii TEF1 (transcription elongation factor 1) promoter to regulate transcription of the marker gene were found to deliver high transformation efficiencies and the best performances with the reporter protein (CalB, lipase B of Candida antarctica) for both, the GUT1- and TPI1-based, marker systems. The GUT1 marker-bearing strain surpassed the reference strain with integrated expression cassette by 46% upon re-evaluation in shake flask cultures regarding CalB production, while the TPI1 system was slightly less productive compared to the control. In 5 L bioreactor methanol-free fed-batch cultivations, the episomal production system employing the GUT1 marker led to 100% increased CalB activity in the culture supernatant compared to integration construct. CONCLUSIONS: For the first time, a scalable and methanol-independent expression system for recombinant protein production for K. phaffii using episomal expression vectors was demonstrated. Expression of the GUT1 selection marker gene of the new ARS plasmids was refined by employing the TEF1 promoter of A. gossypii. Additionally, the antibiotic-free marker toolbox for K. phaffii was expanded by the TPI1 marker system, which proved to be similarly suited for the use in episomal plasmids as well as integrative expression constructs for the purpose of recombinant protein production.

Genome and secretome insights: unravelling the lignocellulolytic potential of Myceliophthora verrucosa for enhanced hydrolysis of lignocellulosic biomass.

Lignocellulolytic enzymes from a novel Myceliophthora verrucosa (5DR) strain was found to potentiate the efficacy of benchmark cellulase during saccharification of acid/alkali treated bagasse by ~ 2.24 fold, indicating it to be an important source of auxiliary enzymes. The De-novo sequencing and analysis of M. verrucosa genome (31.7 Mb) revealed to encode for 7989 putative genes, representing a wide array of CAZymes (366) with a high proportions of auxiliary activity (AA) genes (76). The LC/MS QTOF based secretome analysis of M. verrucosa showed high abundance of glycosyl hydrolases and AA proteins with cellobiose dehydrogenase (CDH) (AA8), being the most prominent auxiliary protein. A gene coding for lytic polysaccharide monooxygenase (LPMO) was expressed in Pichia pastoris and CDH produced by M. verrucosa culture on rice straw based solidified medium were purified and characterized. The mass spectrometry of LPMO catalyzed hydrolytic products of avicel showed the release of both C1/C4 oxidized products, indicating it to be type-3. The lignocellulolytic cocktail comprising of in-house cellulase produced by Aspergillus allahabadii strain spiked with LPMO & CDH exhibited enhanced and better hydrolysis of mild alkali deacetylated (MAD) and unwashed acid pretreated rice straw slurry (UWAP), when compared to Cellic CTec3 at high substrate loading rate.

Kinetic modeling and optimization of ethanol fermentation by the marine yeast Wickerhamomyces subpelliculosus ZE75.

The study aims to enhance ethanol production by Wickerhamomyces subpelliculosus ZE75 isolated from marine sediment. In addition, analyzing the kinetic parameters of ethanol production and optimization of the fermentation conditions was performed. The marine yeast isolate ZE75 was selected as the front runner ethanol-producer, with an ethanol yield of 89.77 gL-1. ZE75 was identified relying on the phenotypic and genotypic characteristics of W. subpelliculosus. The genotypic characterization based on the Internal Transcribed Spacer (ITS) sequence was deposited in the GenBank database with the accession number OP715873. The maximum specific ethanol production rate (vmax) was 0.482 gg-1 h-1 at 175 gL-1 glucose concentration, with a high accuracy of R2 0.95. The maximum growth specific rates (µmax) were 0.141 h-1 obtained at 150 gL-1 glucose concentration with R2 0.91. Optimization of the fermentation parameters such as pH and salinity has been achieved. The highest ethanol yield 0.5637 gg-1 was achieved in a 100% natural seawater-based medium. The maximum ethanol production of 104.04 gL-1 was achieved at pH 4.5 with a specific ethanol rate of 0.1669 gg-1 h-1. The findings of the present study recommend the possibility of ethanol production from a seawater-based medium on a large scale using W. subpelliculosus ZE75.

Unlocking the genome of the non-sourdough Kazachstania humilis MAW1: insights into inhibitory factors and phenotypic properties.

BACKGROUND: Ascomycetous budding yeasts are ubiquitous environmental microorganisms important in food production and medicine. Due to recent intensive genomic research, the taxonomy of yeast is becoming more organized based on the identification of monophyletic taxa. This includes genera important to humans, such as Kazachstania. Until now, Kazachstania humilis (previously Candida humilis) was regarded as a sourdough-specific yeast. In addition, any antibacterial activity has not been associated with this species. RESULTS: Previously, we isolated a yeast strain that impaired bio-hydrogen production in a dark fermentation bioreactor and inhibited the growth of Gram-positive and Gram-negative bacteria. Here, using next generation sequencing technologies, we sequenced the genome of this strain named K. humilis MAW1. This is the first genome of a K. humilis isolate not originating from a fermented food. We used novel phylogenetic approach employing the 18 S-ITS-D1-D2 region to show the placement of the K. humilis MAW1 among other members of the Kazachstania genus. This strain was examined by global phenotypic profiling, including carbon sources utilized and the influence of stress conditions on growth. Using the well-recognized bacterial model Escherichia coli AB1157, we show that K. humilis MAW1 cultivated in an acidic medium inhibits bacterial growth by the disturbance of cell division, manifested by filament formation. To gain a greater understanding of the inhibitory effect of K. humilis MAW1, we selected 23 yeast proteins with recognized toxic activity against bacteria and used them for Blast searches of the K. humilis MAW1 genome assembly. The resulting panel of genes present in the K. humilis MAW1 genome included those encoding the 1,3-ß-glucan glycosidase and the 1,3-ß-glucan synthesis inhibitor that might disturb the bacterial cell envelope structures. CONCLUSIONS: We characterized a non-sourdough-derived strain of K. humilis, including its genome sequence and physiological aspects. The MAW1, together with other K. humilis strains, shows the new organization of the mating-type locus. The revealed here pH-dependent ability to inhibit bacterial growth has not been previously recognized in this species. Our study contributes to the building of genome sequence-based classification systems; better understanding of K.humilis as a cell factory in fermentation processes and exploring bacteria-yeast interactions in microbial communities.

Genetic conflicts in budding yeast: The 2µ plasmid as a model selfish element.

Antagonistic coevolution, arising from genetic conflict, can drive rapid evolution and biological innovation. Conflict can arise both between organisms and within genomes. This review focuses on budding yeasts as a model system for exploring intra- and inter-genomic genetic conflict, highlighting in particular the 2-micron (2µ) plasmid as a model selfish element. The 2µ is found widely in laboratory strains and industrial isolates of Saccharomyces cerevisiae and has long been known to cause host fitness defects. Nevertheless, the plasmid is frequently ignored in the context of genetic, fitness, and evolution studies. Here, I make a case for further exploring the evolutionary impact of the 2µ plasmid as well as other selfish elements of budding yeasts, discuss recent advances, and, finally, future directions for the field.

Making progress.

Mapping proteins in and associated with the Golgi apparatus reveals how this cellular compartment emerges in budding yeast and progresses over time.

Harnessing alkaline-pH regulatable promoters for efficient methanol-free expression of enzymes of industrial interest in Komagataella Phaffii.

BACKGROUND: The yeast Komagataella phaffii has become a very popular host for heterologous protein expression, very often based on the use of the AOX1 promoter, which becomes activated when cells are grown with methanol as a carbon source. However, the use of methanol in industrial settings is not devoid of problems, and therefore, the search for alternative expression methods has become a priority in the last few years. RESULTS: We recently reported that moderate alkalinization of the medium triggers a fast and wide transcriptional response in K. phaffii. Here, we present the utilization of three alkaline pH-responsive promoters (pTSA1, pHSP12 and pPHO89) to drive the expression of a secreted phytase enzyme by simply shifting the pH of the medium to 8.0. These promoters offer a wide range of strengths, and the production of phytase could be modulated by adjusting the pH to specific values. The TSA1 and PHO89 promoters offered exquisite regulation, with virtually no enzyme production at acidic pH, while limitation of Pi in the medium further potentiated alkaline pH-driven phytase expression from the PHO89 promoter. An evolved strain based on this promoter was able to produce twice as much phytase as the reference pAOX1-based strain. Functional mapping of the TSA1 and HSP12 promoters suggests that both contain at least two alkaline pH-sensitive regulatory regions. CONCLUSIONS: Our work shows that the use of alkaline pH-regulatable promoters could be a useful alternative to methanol-based expression systems, offering advantages in terms of simplicity, safety and economy.

Phosphorylation of the F-BAR protein Hof1 drives septin ring splitting in budding yeast.

A double septin ring accompanies cytokinesis in yeasts and mammalian cells. In budding yeast, reorganisation of the septin collar at the bud neck into a dynamic double ring is essential for actomyosin ring constriction and cytokinesis. Septin reorganisation requires the Mitotic Exit Network (MEN), a kinase cascade essential for cytokinesis. However, the effectors of MEN in this process are unknown. Here we identify the F-BAR protein Hof1 as a critical target of MEN in septin remodelling. Phospho-mimicking HOF1 mutant alleles overcome the inability of MEN mutants to undergo septin reorganisation by decreasing Hof1 binding to septins and facilitating its translocation to the actomyosin ring. Hof1-mediated septin rearrangement requires its F-BAR domain, suggesting that it may involve a local membrane remodelling that leads to septin reorganisation. In vitro Hof1 can induce the formation of intertwined septin bundles, while a phosphomimetic Hof1 protein has impaired septin-bundling activity. Altogether, our data indicate that Hof1 modulates septin architecture in distinct ways depending on its phosphorylation status.