Hansenula Polymorpha Research Articles

Characterizing methanol metabolism-related promoters for metabolic engineering of Ogataea polymorpha.

Promoters play an important role in regulating gene expression, and construction of microbial cell factories requires multiple promoters for balancing the metabolic pathways. However, there are only a limited number of characterized promoters for gene expression in the methylotrophic yeast Ogataea polymorpha, which hampers the extensive harnessing of this important yeast toward a cell factory. Here we characterized the promoters of methanol utilization pathway, precursor supply pathway, and reactive oxygen species (ROS) defense system, by using a green fluorescence protein variant (GFPUV) as a quantification signal. Finally, the characterized promoters were used for tuning a fatty alcohol biosynthetic pathway in O. polymorpha and realized fatty alcohol production from methanol. This promoter box should be helpful for gene expression and pathway optimization in the methylotrophic yeast O. polymorpha. KEY POINTS : • 22 promoters related to methanol metabolism were characterized in O. polymorpha. • Promoter truncation resulted shorter and compact promoters. • Promoters with various strengths were used for regulating a fatty alcohol biosynthesis from methanol.

Advantages of yeast-based recombinant protein technology as vaccine products against infectious diseases

The yeast expression system is widely used to produce functional recombinant proteins in the biopharmaceutical industry, such as vaccine products. The expression system choices using yeast as the host has many advantages. Various vaccines have been produced commercially using yeast expression systems. This review aims to explore the advantages of the yeast expression system in Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha, which emphasize vaccine products to prevent human infectious diseases. Selection of the appropriate expression system is carried out by identification at the genetic and fermentation levels, taking into account host features, vectors and expression strategies. We also demonstrate the development of a yeast expression system that can produce recombinant proteins, virus-like particles and yeast surface displays as a novel vaccine strategy against infectious diseases. The recombinant protein produced as a vaccine in the yeast system is cost-effective, immunogenic, and safe. In addition, this system has not introduced new microbe variants in nature that will be safe for the environment. Thus, it has the potential to become a commercial product used in vaccination programs to prevent human infectious diseases.

Effect of the compound bacterial agent on microbial community of the aerobic compost of food waste.

In our study, we used 16SrRNA and ITS to investigate the microbial community composition and the effect of compound bacterial agent on the microbial community composition in the aerobic composting process of food waste (FW). At the bacterial level, the main phyla of Group A (compost naturally) were Proteobacteria and Firmicutes, and the main species were Pseudomonas_sp._GR7, Bacillus licheniformis and Pediococcus acidilactici. The main phyla of Group B (compost with compound bacterial agent) were Proteobacteria, Firmicutes and Streptophyta, and the main species were Klebsiella pneumoniae, Cronobacter sakazakii, Macrococcus caseolyticus, Enterococcus faecalis, Citrobacter freundii and Bacillus velezensis. It is worth noting that M. caseolyticus may be able to improve the effect of odour which is an important sensory index during aerobic composting. At the fungal level, the main phylum of both Groups A and B was Ascomycota, and the main species of Group A were Paecilomyces variotii, Byssochlamys spectabilis and Aspergillus fumigatus. The main species of Group B were Ogataea polymorpha and Millerozyma farinosa. Finally, the degradation rate of Group B was 81% that was about 15% higher than that of Group A, indicating that the compound bacterial agent could effectively improve the degradation rate and the composting process, while the low abundance of the compound bacterial agent in the composting process might be due to the small initial addition or the inhibition of other bacteria or fungi in the composting process.

Perturbations in the Heme and Siroheme Biosynthesis Pathways Causing Accumulation of Fluorescent Free Base Porphyrins and Auxotrophy in Ogataea Yeasts.

The biosynthesis of cyclic tetrapyrrol chromophores such as heme, siroheme, and chlorophyll involves the formation of fluorescent porphyrin precursors or compounds, which become fluorescent after oxidation. To identify Ogataea polymorpha mutations affecting the final steps of heme or siroheme biosynthesis, we performed a search for clones with fluorescence characteristic of free base porphyrins. One of the obtained mutants was defective in the gene encoding a homologue of Saccharomyces cerevisiae Met8 responsible for the last two steps of siroheme synthesis. Same as the originally obtained mutation, the targeted inactivation of this gene in O. polymorpha and O. parapolymorpha led to increased porphyrin fluorescence and methionine auxotrophy. These features allow the easy isolation of Met8-defective mutants and can potentially be used to construct auxotrophic strains in various yeast species. Besides MET8, this approach also identified the HEM3 gene encoding porphobilinogen deaminase, whose increased dosage led to free base porphyrin accumulation.

Regulation of alcohol oxidase gene expression in methylotrophic yeast Ogataea minuta

Ogataea minuta is a methylotrophic yeast that is closely related to Ogataea (Hansenula) polymorpha. Like other methylotrophic yeasts, O. minuta possesses strongly methanol-inducible genes, such as AOX1. We have focused on O. minuta as a host for the production of heterologous glycoproteins. However, it remained unknown how the AOX1 promoter is regulated in O. minuta. To elucidate regulation mechanisms of the AOX1 promoter, we adopted an assay system to quantitate AOX1 promoter activity using the PHO5 gene, which encodes an acid phosphatase, of Saccharomyces cerevisiae. The promoter activity assay revealed that glycerol, as well as glucose, cause strong catabolite repression of AOX1 expression in O. minuta. To investigate what factors are involved in transcription of the AOX1 promoter in O. minuta, we cloned three putative transcription factor genes, TRM1, TRM2, and MPP1, as homologues of other methylotrophic yeast species. Deletion mutants of these genes all showed decreased induction of the AOX1 promoter when methanol was added as the sole carbon source, indicating that these genes are indeed involved in AOX1 promoter regulation in O. minuta. Double deletion and constitutive expression of these transcription factor genes indicated that TRM1 and MPP1 regulate the transcription of AOX1 in the same pathway, while TRM2 regulates it in another pathway. By reverse transcription-qPCR, we also found that these two pathways compensate for each other and have crosstalk mechanisms with each other. A possible model for regulation of the AOX1 promoter in O. minuta was shown.

Cellulosic biofuel production using emulsified simultaneous saccharification and fermentation (eSSF) with conventional and thermotolerant yeasts

BackgroundFuture expansion of corn-derived ethanol raises concerns of sustainability and competition with the food industry. Therefore, cellulosic biofuels derived from agricultural waste and dedicated energy crops are necessary. To date, slow and incomplete saccharification as well as high enzyme costs have hindered the economic viability of cellulosic biofuels, and while approaches like simultaneous saccharification and fermentation (SSF) and the use of thermotolerant microorganisms can enhance production, further improvements are needed. Cellulosic emulsions have been shown to enhance saccharification by increasing enzyme contact with cellulose fibers. In this study, we use these emulsions to develop an emulsified SSF (eSSF) process for rapid and efficient cellulosic biofuel production and make a direct three-way comparison of ethanol production between S. cerevisiae, O. polymorpha, and K. marxianus in glucose and cellulosic media at different temperatures.ResultsIn this work, we show that cellulosic emulsions hydrolyze rapidly at temperatures tolerable to yeast, reaching up to 40-fold higher conversion in the first hour compared to microcrystalline cellulose (MCC). To evaluate suitable conditions for the eSSF process, we explored the upper temperature limits for the thermotolerant yeasts Kluyveromyces marxianus and Ogataea polymorpha, as well as Saccharomyces cerevisiae, and observed robust fermentation at up to 46, 50, and 42 °C for each yeast, respectively. We show that the eSSF process reaches high ethanol titers in short processing times, and produces close to theoretical yields at temperatures as low as 30 °C. Finally, we demonstrate the transferability of the eSSF technology to other products by producing the advanced biofuel isobutanol in a light-controlled eSSF using optogenetic regulators, resulting in up to fourfold higher titers relative to MCC SSF.ConclusionsThe eSSF process addresses the main challenges of cellulosic biofuel production by increasing saccharification rate at temperatures tolerable to yeast. The rapid hydrolysis of these emulsions at low temperatures permits fermentation using non-thermotolerant yeasts, short processing times, low enzyme loads, and makes it possible to extend the process to chemicals other than ethanol, such as isobutanol. This transferability establishes the eSSF process as a platform for the sustainable production of biofuels and chemicals as a whole.

Evolution of the interactions between GII.4 noroviruses and histo-blood group antigens: Insights from experimental and computational studies

Norovirus (NoV) is the major pathogen causing the outbreaks of the viral gastroenteritis across the world. Among the various genotypes of NoV, GII.4 is the most predominant over the past decades. GII.4 NoVs interact with the histo-blood group antigens (HBGAs) to invade the host cell, and it is believed that the receptor HBGAs may play important roles in selecting the predominate variants by the nature during the evolution of GII.4 NoVs. However, the evolution-induced changes in the HBGA-binding affinity for the GII.4 NoV variants and the mechanism behind the evolution of the NoV-HBGA interactions remain elusive. In the present work, the virus-like particles (VLPs) of the representative GII.4 NoV stains epidemic in the past decades were expressed by using the Hansenula polymorpha yeast expression platform constructed by our laboratory, and then the enzyme linked immunosorbent assay (ELISA)-based HBGA-binding assays as well as the molecular dynamics (MD) simulations combined with the molecular mechanics/generalized born surface area (MMGBSA) calculations were performed to investigate the interactions between various GII.4 strains and different types of HBGAs. The HBGA-binding assays show that for all the studied types of HBGAs, the evolution of GII.4 NoVs results in the increased NoV-HBGA binding affinities, where the early epidemic strains have the lower binding activity and the newly epidemic strains exhibit relative stronger binding intensity. Based on the MD simulation and MMGBSA calculation results, a physical mechanism that accounts for the increased HBGA-binding affinity was proposed. The evolution-involved residue mutations cause the conformational rearrangements of loop-2 (residues 390-396), which result in the narrowing of the receptor-binding pocket and thus tighten the binding of the receptor HBGAs. Our experimental and computational studies are helpful for better understanding the mechanism behind the evolution-induced increasing of HBGA-binding affinity, which may provide useful information for the drug and vaccine designs against GII.4 NoVs.

Recent trends in upgrading the performance of yeast as electrode biocatalyst in microbial fuel cells

Microbial fuel cell (MFC) is an optimistic fuel cell technology that applies microorganism's biochemical catalytic activities in consuming organic substrate and produce electricity. In the past, several researchers have reported power generation from Saccharomyces cerevisiae, but nowadays, most of the studies are centred around bacterial biofilms (prokaryotes) as anode biocatalyst. Yeast (a eukaryote) has also been applied as a biocatalyst in MFCs as they are non-pathogenic, easy to handle and tolerant to various environmental conditions. Yeast strains such as Arxula adeninvorans, Candida melibiosica, Hansenula polymorpha, Hansenula anomala, Kluyveromyces marxianus and Saccharomyces cerevisiae have been utilized in MFCs. This review summarizes the application of yeast as an anode biocatalyst together with a discussion on the mechanism of electron transfer from yeast cells to the anode and highlights the techniques applied in improving the efficiency of yeast-based MFCs. The recent challenges and benefits of utilizing yeast in MFCs have been also encapsulated in this review.

Genomic diversity, chromosomal rearrangements, and interspecies hybridization in the Ogataea polymorpha species complex.

The methylotrophic yeast Ogataea polymorpha has long been a useful system for recombinant protein production, as well as a model system for methanol metabolism, peroxisome biogenesis, thermotolerance, and nitrate assimilation. It has more recently become an important model for the evolution of mating-type switching. Here, we present a population genomics analysis of 47 isolates within the O. polymorpha species complex, including representatives of the species O. polymorpha, Ogataea parapolymorpha, Ogataea haglerorum, and Ogataea angusta. We found low levels of nucleotide sequence diversity within the O. polymorpha species complex and identified chromosomal rearrangements both within and between species. In addition, we found that one isolate is an interspecies hybrid between O. polymorpha and O. parapolymorpha and present evidence for loss of heterozygosity following hybridization.

Improving the Heterologous Production of Fungal Peroxygenases through an Episomal Pichia pastoris Promoter and Signal Peptide Shuffling System.

Fungal peroxygenases (UPOs) have emerged as oxyfunctionalization catalysts of tremendous interest in recent years. However, their widespread use in the field of biocatalysis is still hampered by their challenging heterologous production, substantially limiting the panel of accessible enzymes for investigation and enzyme engineering. Building upon previous work on UPO production in yeast, we have developed a combined promoter and signal peptide shuffling system for episomal high throughput UPO production in the industrially relevant, methylotrophic yeast Pichia pastoris. Eleven endogenous and orthologous promoters were shuffled with a diverse set of 17 signal peptides. Three previously described UPOs were selected as first test set, leading to the identification of beneficial promoter/signal peptide combinations for protein production. We applied the system then successfully to produce two novel UPOs: MfeUPO from Myceliophthora fergusii and MhiUPO from Myceliophthora hinnulea. To demonstrate the feasibility of the developed system to other enzyme classes, it was applied for the industrially relevant lipase CalB and the laccase Mrl2. In total, approximately 3200 transformants of eight diverse enzymes were screened and the best promoter/signal peptide combinations studied at various cofeeding, derepression, and induction conditions. High volumetric production titers were achieved by subsequent creation of stable integration lines and harnessing orthologous promoters from Hansenula polymorpha. In most cases promising yields were also achieved without the addition of methanol under derepressed conditions. To foster the use of the episomal high throughput promoter/signal peptide Pichia pastoris system, we made all plasmids available through Addgene.

The role of Mig1, Mig2, Tup1 and Hap4 transcription factors in regulation of xylose and glucose fermentation in the thermotolerant yeast Ogataea polymorpha.

Glucose is a preferred carbon source for most living organisms. The metabolism and regulation of glucose utilization are well studied mostly for Saccharomyces cerevisiae. Xylose is the main pentose sugar released from the lignocellulosic biomass, which has a high potential as a renewable feedstock for bioethanol production. The thermotolerant yeast Ogataea (Hansenula) polymorpha, in contrast to S. cerevisiae, is able to metabolize and ferment not only glucose but also xylose. However, in non-conventional yeasts, the regulation of glucose and xylose metabolism remains poorly understood. In this study, we characterize the role of transcriptional factors Mig1, Mig2, Tup1 and Hap4 in the natural xylose-fermenting yeast O. polymorpha. The deletion of MIG1 had no significant influence on ethanol production either from xylose or glucose, however the deletion of both MIG1 and MIG2 reduced the amount of ethanol produced from these sugars. The deletion of HAP4-A and TUP1 genes resulted in increased ethanol production from xylose. Inversely, the overexpression of HAP4-A and TUP1 genes reduced ethanol production during xylose alcoholic fermentation. Thus, HAP4-A and TUP1 are involved in repression of xylose metabolism and fermentation in yeast O. polymorpha and their deletion could be a viable strategy to improve ethanol production from this pentose.

A kinetic approach to the formation of two-mediator systems for developing microbial biosensors as exemplified by a rapid biochemical oxygen demand assay.

This work proposes a method of forming a microorganism-mediator(s) receptor system, in which the rates of separate stages of mediator bioelectrocatalysis are used as the basis for the development of biosensors for the biochemical oxygen demand (BOD) rapid assay. In the presence of a ferrocene mediator, the yeast Blastobotrys adeninivorans was shown to enable oxidation of a larger range of substrates as compared with other investigated microorganisms-bacteria Escherichia coli and yeast Ogataea polymorpha. The rate constants of the interaction of the yeast B. adeninivorans with nine compounds, electron transfer mediators, were determined; the best mediator for these microorganisms was found to be neutral red (k int = 0.681 ± 0.009dm3/gs). Neutral red possesses a high rate of interaction with the ferrocene mediator (14,200 ± 100dm3/mols) shown earlier to be the most promising acceptor of electrons at a carbon paste electrode (0.4 ± 0.1cm/s). These features enabled the formation of a two-mediator ferrocene-neutral red system to be used in a biosensor. A two-mediator-based biosensor had a higher sensitivity (the lower limit of detected BOD concentrations, 0.16mg/dm3) than that of a one-mediator system based on neutral red and ferrocene. Analysis of ten samples from surface water reservoirs showed the combination of ferrocene, neutral red and the yeast B. adeninivorans to enable the data that highly correlated (R = 0.9693) with those of the standard method.

Random Transfer of Ogataea polymorpha Genes into Saccharomyces cerevisiae Reveals a Complex Background of Heat Tolerance.

Horizontal gene transfer, a process through which an organism acquires genes from other organisms, is a rare evolutionary event in yeasts. Artificial random gene transfer can emerge as a valuable tool in yeast bioengineering to investigate the background of complex phenotypes, such as heat tolerance. In this study, a cDNA library was constructed from the mRNA of a methylotrophic yeast, Ogataea polymorpha, and then introduced into Saccharomyces cerevisiae. Ogataea polymorpha was selected because it is one of the most heat-tolerant species among yeasts. Screening of S. cerevisiae populations expressing O. polymorpha genes at high temperatures identified 59 O. polymorpha genes that contribute to heat tolerance. Gene enrichment analysis indicated that certain S. cerevisiae functions, including protein synthesis, were highly temperature-sensitive. Additionally, the results confirmed that heat tolerance in yeast is a complex phenotype dependent on multiple quantitative loci. Random gene transfer would be a useful tool for future bioengineering studies on yeasts.

Screening neutral sites for metabolic engineering of methylotrophic yeast Ogataea polymorpha

Methylotrophic yeast Ogataea polymorpha is capable to utilize multiple carbon feedstocks especially methanol as sole carbon source and energy, making it an ideal host for bio-manufacturing. However, the lack of gene integration sites limits its systems metabolic engineering, in particular construction of genome-integrated pathway. We here screened the genomic neutral sites for gene integration without affecting cellular fitness, by genomic integration of an enhanced green fluorescent protein (eGFP) gene via CRISPR-Cas9 technique. After profiling the growth and fluorescent intensity in various media, 17 genome positions were finally identified as potential neutral sites. Finally, integration of fatty alcohol synthetic pathway genes into neutral sites NS2 and NS3, enabled the production of 4.5 mg/L fatty alcohols, indicating that these neutral sites can be used for streamline metabolic engineering in O. polymorpha. We can anticipate that the neutral sites screening method described here can be easily adopted to other eukaryotes.

Whole-Genome Transformation Promotes tRNA Anticodon Suppressor Mutations under Stress.

tRNAs are encoded by a large gene family, usually with several isogenic tRNAs interacting with the same codon. Mutations in the anticodon region of other tRNAs can overcome specific tRNA deficiencies. Phylogenetic analysis suggests that such mutations have occurred in evolution, but the driving force is unclear. We show that in yeast suppressor mutations in other tRNAs are able to overcome deficiency of the essential TRT2-encoded tRNAThrCGU at high temperature (40°C). Surprisingly, these tRNA suppressor mutations were obtained after whole-genome transformation with DNA from thermotolerant Kluyveromyces marxianus or Ogataea polymorpha strains but from which the mutations did apparently not originate. We suggest that transient presence of donor DNA in the host facilitates proliferation at high temperature and thus increases the chances for occurrence of spontaneous mutations suppressing defective growth at high temperature. Whole-genome sequence analysis of three transformants revealed only four to five nonsynonymous mutations of which one causing TRT2 anticodon stem stabilization and two anticodon mutations in non-threonyl-tRNAs, tRNALysCUU and tRNAeMetCAU, were causative. Both anticodon mutations suppressed lethality of TRT2 deletion and apparently caused the respective tRNAs to become novel substrates for threonyl-tRNA synthetase. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) data could not detect any significant mistranslation, and reverse transcription-quantitative PCR results contradicted induction of the unfolded protein response. We suggest that stress conditions have been a driving force in evolution for the selection of anticodon-switching mutations in tRNAs as revealed by phylogenetic analysis.IMPORTANCE In this work, we have identified for the first time the causative elements in a eukaryotic organism introduced by applying whole-genome transformation and responsible for the selectable trait of interest, i.e., high temperature tolerance. Surprisingly, the whole-genome transformants contained just a few single nucleotide polymorphisms (SNPs), which were unrelated to the sequence of the donor DNA. In each of three independent transformants, we have identified a SNP in a tRNA, either stabilizing the essential tRNAThrCGU at high temperature or switching the anticodon of tRNALysCUU or tRNAeMetCAU into CGU, which is apparently enough for in vivo recognition by threonyl-tRNA synthetase. LC-MS/MS analysis indeed indicated absence of significant mistranslation. Phylogenetic analysis showed that similar mutations have occurred throughout evolution and we suggest that stress conditions may have been a driving force for their selection. The low number of SNPs introduced by whole-genome transformation may favor its application for improvement of industrial yeast strains.

An Aldolase-Based New Pathway for Bioconversion of Formaldehyde and Ethanol into 1,3-Propanediol in Escherichia coli

Formaldehyde (HCHO) is a reactive one-carbon compound that is interesting for biosynthesis. The assimilation of HCHO depends on the catalysis of aldolase. Here, we present a novel synthetic pathway in E.coli to convert HCHO and ethanol into 1,3-propanediol (PDO) using a deoxyribose-5-phosphate aldolase (DERA). DERA condenses HCHO and acetaldehyde to form 3-hydroxypropionaldehyde, the direct precursor of PDO formation. This new pathway opens up the possibility to synthesize an appealing C3 compound from a C1 compound and a C2 compound without carbon loss in contrast to all the other known PDO synthetic pathways where typically 30-50% of the carbons are lost as CO2 and other byproducts. The pathway is successfully demonstrated by elaborating three metabolic modules. First, DERA from Thermotoga maritima was found to be efficient for the aldol condensation and PDO production module. For the module of acetaldehyde supply from ethanol, an alcohol dehydrogenase from Hansenula polymorpha was selected. For the HCHO supply module, the control of HCHO concentration and its utilization were shown to be important for achieving the assimilation of HCHO in recombinant E.coli cells. By deleting the gene frmA for endogenous conversion of HCHO to formate and controlling HCHO at a level of about 0.6 mM, the concentration and yield of PDO were increased from initially 5.67 mM (0.43 g/L) and 0.057 mol/mol to 17.35 mM (1.32 g/L) and 0.096 mol/mol in bioconversion of ethanol and HCHO with resting E.coli cells. Further engineering of DERA and the HCHO supply module is necessary to realize the potential of this promising metabolic pathway.

Genome-scale model reconstruction of the methylotrophic yeast Ogataea polymorpha

BackgroundOgataea polymorpha is a thermotolerant, methylotrophic yeast with significant industrial applications. While previously mainly used for protein synthesis, it also holds promise for producing platform chemicals. O. polymorpha has the distinct advantage of using methanol as a substrate, which could be potentially derived from carbon capture and utilization streams. Full development of the organism into a production strain and estimation of the metabolic capabilities require additional strain design, guided by metabolic modeling with a genome-scale metabolic model. However, to date, no genome-scale metabolic model is available for O. polymorpha.ResultsTo overcome this limitation, we used a published reconstruction of the closely related yeast Komagataella phaffii as a reference and corrected reactions based on KEGG and MGOB annotation. Additionally, we conducted phenotype microarray experiments to test the suitability of 190 substrates as carbon sources. Over three-quarter of the substrate use was correctly reproduced by the model and 27 new substrates were added, that were not present in the K. phaffii reference model.ConclusionThe developed genome-scale metabolic model of O. polymorpha will support the engineering of synthetic metabolic capabilities and enable the optimization of production processes, thereby supporting a sustainable future methanol economy.

Effects of Liquid Smoking on the Microbiological and Physicochemical Characteristics of Suan Yu, a Traditional Chinese Fermented Fish Product

ABSTRACT This work studied the changes in microbiological and physiochemical properties during the fermentation of Suan yu by liquid smoking and traditional smoking (batches B, C, and A). Lactobacillus plantarum, Pediococcus pentosaceus, Saccharomyces cerevisiae, and Hansenula polymorpha were the dominant microorganisms in the three samples. Only the acid-resistant Staphylococcus xylosus and Staphylococcus carnosus survived in the acid environment of the final fermented products. The amounts of Enterobacteriaceae, Enterococci, and Pseudomonades in the final fermented products were under the detection limit. Batch B and C samples had higher rate of reduction in pH (4.24 ± 0.17–4.39 ± 0.18 from 6.21 ± 0.24, respectively) and lower acetic acid generation rate (0.06%±0. 00% and 0.05%±0. 00%, respectively) than batch A samples. Large amounts of free amino acids were generated during the fermentation of the samples. Batches B and C had thiobarbituric acid reactive substances (TBARS) value between 0.88 ± 0.03 and 0.89 ± 0.03, total volatile basic nitrogen (TVB-N) value between 18.9 ± 0.34 and 18.8 ± 0.33 mg/kg, and benzo(a)pyrene (BaP) content of 0.1 ± 0.00 μg/kg, which was lower than those of fermented Suan yu prepared by traditional smoking. Sensory analysis indicates that these sample groups have unique smoking and fermentation flavor. Consequently, the fermented Suan yu produced through liquid smoking is a kind of fermented aquatic product with full-bodied smoking flavor, palatable acid flavor, abundant nutrition, and high safety.

Recombination machinery engineering for precise genome editing in methylotrophic yeast Ogataea polymorpha

SummaryMethanol biotransformation can expand biorefinery substrate spectrum other than biomass by using methylotrophic microbes. Ogataea (Hansenula) polymorpha, a representative methylotrophic yeast, attracts much attention due to its thermotolerance, but the low homologous recombination (HR) efficiency hinders its precise genetic manipulation during cell factory construction. Here, recombination machinery engineering (rME) is explored for enhancing HR activity together with establishing an efficient CRISPR-Cas9 system in O. polymorpha. Overexpression of HR-related proteins and down-regulation of non-homologous end joining (NHEJ) increased HR rates from 20%–30% to 60%–70%. With these recombination perturbation mutants, a competition between HR and NHEJ is observed. This HR up-regulated system has been applied for homologous integration of large fragments and in vivo assembly of multiple fragments, which enables the production of fatty alcohols in O. polymorpha. These findings will simplify genetic engineering in non-conventional yeasts and facilitate the adoption of O. polymorpha as an attractive cell factory for industrial application.

Evaluation of Ogataea (Hansenula) polymorpha for Hyaluronic Acid Production.

Hyaluronic acid (HA) is a biopolymer formed by UDP-glucuronic acid and UDP-N-acetyl-glucosamine disaccharide units linked by β-1,4 and β-1,3 glycosidic bonds. It is widely employed in medical and cosmetic procedures. HA is synthesized by hyaluronan synthase (HAS), which catalyzes the precursors’ ligation in the cytosol, elongates the polymer chain, and exports it to the extracellular space. Here, we engineer Ogataea (Hansenula) polymorpha for HA production by inserting the genes encoding UDP-glucose 6-dehydrogenase, for UDP-glucuronic acid production, and HAS. Two microbial HAS, from Streptococcus zooepidemicus (hasAs) and Pasteurella multocida (hasAp), were evaluated separately. Additionally, we assessed a genetic switch using integrases in O. polymorpha to uncouple HA production from growth. Four strains were constructed containing both has genes under the control of different promoters. In the strain containing the genetic switch, HA production was verified by a capsule-like layer around the cells by scanning electron microscopy in the first 24 h of cultivation. For the other strains, the HA was quantified only after 48 h and in an optimized medium, indicating that HA production in O. polymorpha is limited by cultivation conditions. Nevertheless, these results provide a proof-of-principle that O. polymorpha is a suitable host for HA production.