Kluyveromyces as promising yeast cell factories for industrial bioproduction: From bio-functional design to applications.

As the two most widely used Kluyveromyces yeast, Kluyveromyces marxianus and K. lactis have gained increasing attention as microbial chassis in biocatalysts, biomanufacturing and the utilization of low-cost raw materials owing to their high suitability to these applications. However, due to slow progress in the development of molecular genetic manipulation tools and synthetic biology strategies, Kluyveromyces yeast cell factories as biological manufacturing platforms have not been fully developed. In this review, we provide a comprehensive overview of the attractive characteristics and applications of Kluyveromyces cell factories, with special emphasis on the development of molecular genetic manipulation tools and systems engineering strategies for synthetic biology. In addition, future avenues in the development of Kluyveromyces cell factories for the utilization of simple carbon compounds as substrates, the dynamic regulation of metabolic pathways, and for rapid directed evolution of robust strains are proposed. We expect that more synthetic systems, synthetic biology tools and metabolic engineering strategies will adapt to and optimize for Kluyveromyces cell factories to achieve green biofabrication of multiple products with higher efficiency.

Utilization of delactosed whey permeate for the synthesis of ethyl acetate with Kluyveromyces marxianus.

Ethyl acetate is an important organic solvent and currently produced from fossil carbon resources. Microbial synthesis of this ester from sugar-rich waste could be an interesting alternative. Therefore, synthesis of ethyl acetate by Kluyveromyces marxinanus DSM 5422 from delactosed whey permeate (DWP) was studied in an aerated stirred bioreactor at 40 °C. DWP is mainly composed of residual lactose and minerals. The minerals inhibited yeast growth, as witnessed by an increased lag period, a reduced growth rate, and an extended process duration. All experiments were therefore carried out with diluted DWP. In a series of batch experiments, the pH of iron-deficient DWP medium varied between 4.8 and 5.9. The pH of the cultivation medium significantly influenced cell growth and product syntheses, with the highest ethyl acetate yield of 0.347 g g-1 and lowest by-product formation achieved at pH 5.1. It is likely that this effect is due to pH-dependent iron chelation, which affects the iron bioavailability and the intracellular iron content, thus affecting growth and metabolite synthesis. The viability of yeast cells was always high despite the harsh conditions in DWP medium, which enabled extended usage of the biomass in repeated-batch and fed-batch cultivations. These two culture techniques increased the volume of DWP processed per time by 32 and 84% for the repeated-batch and the fed-batch cultivation, respectively, without a drop of the ester yield. KEY POINTS: • Delactosed whey permeate was converted to ethyl acetate with a high rate and yield. • The formation of ethyl acetate in DWP medium at iron limitation is pH-dependent. • Highly active yeasts from batch processes enabled extension as fed and repeated batch.

Identification of the main proteins secreted by Kluyveromyces marxianus and their possible roles in antagonistic activity against fungi.

Lytic enzymes secreted by Kluyveromyces marxianus can lyse Saccharomyces cerevisiae cells. Their ability to hydrolyze yeast cell walls can be used in biotechnological applications, such as the production of glucans and protoplasts, as well as a biological control agent against plant pathogenic fungi. Herein, 27 proteins secreted by K. marxianus were identified by mass spectrometry analyses. Importantly, 14 out of the 27 proteins were classified as hydrolases. Indeed, the enzyme extract secreted by K. marxianus caused damage to S. cerevisiae cells and reduced yeast cell viability. Moreover, K marxianus inhibited spore germination and mycelial growth of the phytopathogenic fungus Botrytis cinerea in simultaneous cocultivation assays. We suggest that this inhibition may be partially related to the yeast's ability to secrete lytic enzymes. Consistent with the in vitro antagonistic tests, K. marxianus was able to protect strawberry fruits inoculated with B. cinerea. Therefore, these findings suggest that K. marxianus possesses potential as a biocontrol agent against strawberry gray mold during the postharvest stage and may also have potential against other phytopathogenic fungi by means of its lytic enzymatic arsenal.

Physiological and transcriptome analyses of Kluyveromyces marxianus reveal adaptive traits in stress response.

Kluyveromyces marxianus is a non-conventional yeast with outstanding physiological characteristics and a high potential for lignocellulosic ethanol production. However, achieving high ethanol productivity requires overcoming several biotechnological challenges due to the cellular inhibition caused by the inhibitors present in the medium. In this work, K. marxianus SLP1 was adapted to increase its tolerance to a mix of inhibitory compounds using the adaptive laboratory evolution strategy to study the adaptation and stress response mechanisms used by this non-Saccharomyces yeast. The fermentative and physiological parameters demonstrated that the adapted K. marxianus P8 had a better response against the synergistic effects of multiple inhibitors because it reduced the lag phase from 12 to 4 h, increasing the biomass by 40% and improving the volumetric ethanol productivity 16-fold than the parental K. marxianus SLP1. To reveal the effect of adaptation process in P8, transcriptome analysis was carried out; the result showed that the basal gene expression in P8 changed, suggesting the biological capability of K. marxianus to activate the adaptative prediction mechanism. Similarly, we carried out physiologic and transcriptome analyses to reveal the mechanisms involved in the stress response triggered by furfural, the most potent inhibitor in K. marxianus. Stress response studies demonstrated that P8 had a better physiologic response than SLP1, since key genes related to furfural transformation (ALD4 and ALD6) and stress response (STL1) were upregulated. Our study demonstrates the rapid adaptability of K. marxianus to stressful environments, making this yeast a promising candidate to produce lignocellulosic ethanol. KEY POINTS: • K. marxianus was adapted to increase its tolerance to a mix of inhibitory compounds • The basal gene expression of K. marxianus changed after the adaptation process • Adapted K. marxianus showed a better physiological response to stress by inhibitors • Transcriptome analyses revealed key genes involved in the stress response.

Comparative impact of exogenous phenylalanine on oenological isolates of Kluyveromyces marxianus and Saccharomyces cerevisiae.

AIMS: Kluyveromyces marxianus' high production of 2-phenylethyl acetate (2-PEA) via L-phenylalanine (Phe) catabolism makes it relevant for industries relying on the production of aroma compounds through fermentation processes. This study assessed the physiological impact of exogenous supplementation of Phe on cell viability, fermentation performance, and, by extension, on lipid and amino acid metabolism in a wine isolate of this yeast. METHODS AND RESULTS: The data showed that Phe exerted cytotoxic effects on K. marxianus IWBT Y885, which were minimal on Saccharomyces cerevisiae and impacted amino acid metabolism and aroma production. We demonstrated that K. marxianus strains fermented sugars more effectively in the absence of Phe. While lipid supplementation did not mitigate any deleterious effects of Phe, it supported viability maintenance and fermentation performance in the absence of Phe. Phe supplementation succeeded in augmenting the production of 2-PE and 2-PEA. CONCLUSIONS: The enhanced production of 2-PEA in K. marxianus suggests that this transesterification may be, at least in part, a compensatory detoxification mechanism for this yeast.

Immobilization of Kluyveromyces lactis ß-Galactosidase on Meso-macroporous Silica: Use of Infrared Spectroscopy to Rationalize the Catalytic Efficiency.

Enzyme immobilization on adequate carriers is a challenging strategy. Understanding the enzyme-carrier interactions and their effects on enzyme conformation and bioactivity is critical. In this study, a meso-macropores silica (MMS) was used to immobilize ß-galactosidase from the yeast Kluyveromyces lactis (ß-gal-KL) by physical adsorption. The bioactivity of the immobilized ß-gal-KL was altered, evidenced by the increased Km , decreased Vmax and kcat , and increased activity at alkaline values. By performing infrared spectroscopy analysis and subsequent secondary structure assessment from the amide I band, the immobilized ß-gal-KL suffered a ß-sheet (∼31-35 %) to α-helix (∼15-19 %) transition with increased turns (∼21-22 %) with respect to the free ß-gal-KL having ∼12 % α-helix, ∼42 % ß-sheet, and ∼17 % turns. These findings led us to correlate the observed bioactivity performance to structural alterations to a non-native conformation. The presented line of thought can lead to a better understanding of the reasons causing bioactivity alterations upon enzyme immobilization.

New Saccharomyces cerevisiae-Kluyveromyces marxianus fusant shows enhanced alcoholic fermentation performance.

Kluyveromyces marxianus has a promising enological potential because of strong extracellular pectinase activity and the copious production of specific higher alcohols and esters. However, K. marxianus is unable to complete alcoholic fermentation on its own. For this reason it is only used in co- or sequential fermentation in conjunction with Saccharomyces spp. Here we applied the protoplast fusion technique to two commercial Saccharomyces cerevisiae strains (Lalvin EC1118® and Lalvin QA23®) and K. marxianus isolate IWBT Y885, to obtain fusants that possess pre-selected, enologically relevant features of K. marxianus, but with improved fermentation performance. After an extensive selection process, performed using non-auxotrophic markers, we identified one fusant, BF2020, that shows enhanced fermentation performance compared to the parental strain K. marxianus Y885, but that still exhibits strong pectinase activity and may be suitable for industrial production. The genome of the selected hybrid was further investigated and characterized by RAPD-PCR analysis and whole genome sequencing.

Improving glycerol utilization during high-temperature xylitol production with Kluyveromyces marxianus using a transient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system.

Glycerol is an ideal co-substrate for xylitol production with Kluyveromyces marxianus. This study demonstrated that K. marxianus catabolizes glycerol through the Gut1-Gut2 pathway instead of the previously speculated NADPH-dependent Gcy1-Dak1 pathway using the transient clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9 (CRISPR/Cas9) system. Additionally, Utr1p was demonstrated to mediate NADPH generation through NADH phosphorylation. YZB392, which was constructed by integrating Utr1 into the Ypr1 site in the strain overexpressing NcXyl1 and CiGxf1 and harboring disrupted Xyl2, exhibited enhanced glycerol utilization for xylitol production (from 2.50- to 3.30- g/L after consuming 1 g/L glycerol). Fed-batch fermentation at 42 °C with YZB392 yielded 322.07 g/L xylitol, which is the highest known xylitol titer obtained via biological method. Feeding crude glycerol, xylose mother liquor, and corn steep liquor powder into a bioreactor resulted in the production of 235.69 g/L xylitol. This study developed a platform for xylitol production from industrial by-products.

Functional Interactions of Kluyveromyces lactis Telomerase Reverse Transcriptase with the Three-Way Junction and the Template Domains of Telomerase RNA.

The ribonucleoprotein telomerase contains two essential components: telomerase RNA (TER) and telomerase reverse transcriptase (TERT, Est2 in yeast). A small portion of TER, termed the template, is copied by TERT onto the chromosome ends, thus compensating for sequence loss due to incomplete DNA replication and nuclease action. Although telomerase RNA is highly divergent in sequence and length across fungi and mammals, structural motifs essential for telomerase function are conserved. Here, we show that Est2 from the budding yeast Kluyveromyces lactis (klEst2) binds specifically to an essential three-way junction (TWJ) structure in K. lactis TER, which shares a conserved structure and sequence features with the essential CR4-CR5 domain of vertebrate telomerase RNA. klEst2 also binds specifically to the template domain, independently and mutually exclusive of its interaction with TWJ. Furthermore, we present the high-resolution structure of the klEst2 telomerase RNA-binding domain (klTRBD). Mutations introduced in vivo in klTRBD based on the solved structure or in TWJ based on its predicted RNA structure caused severe telomere shortening. These results demonstrate the conservation and importance of these domains and the multiple protein-RNA interactions between Est2 and TER for telomerase function.

Effects of autochthonous Kluyveromyces lactis and commercial Enterococcus faecium adjunct cultures on the volatile profile and the sensory characteristics of short-ripened acid-curd Cebreiro cheese.

Four batches of Cebreiro-type cheese were made in duplicate from pasteurized milk. A control batch was manufactured with only a commercial O-starter. The other three batches were made with the same starter plus: (i) a commercial culture of Enterococcus faecium; (ii) a selected Kluyveromyces lactis adjunct culture used in a cheese-milk pre-ripening step; and (iii) the combination of both adjunct cultures. The cheeses made with the yeast adjunct were characterized by higher values of overall proteolysis, pH and aw, and showed total and lactic acid bacteria (LAB) counts at least 2 log units than the batches made with only LAB. The volatile profiles of the cheeses made with added K. lactis were distinguished by high contents of esters, branched-chain alcohols, fatty acids, acetoin and 2-pnenylethanol. These batches had a more friable and sticky texture, and exhibited differential piquant, yeasty, alcoholic, acetic and fruity flavors. Furthermore, the addition of enterococci seemed to help achieve more desirable sensory characteristics. The batches manufactured with both adjunct cultures were awarded the highest scores for texture preference, flavor intensity, flavor preference, and overall sensory preference. The sensory profiles of the cheeses made with added yeast closely resembled those of traditional 'good quality' raw-milk Cebreiro cheese.

Ergosterol production at elevated temperatures by Upc2-overexpressing Kluyveromyces marxianus using Jerusalem artichoke tubers as feedstock.

Ergosterol is an important precursor in the pharmaceutical industry for the production of numerous drugs. In this study, Kluyveromyces marxianus that showed more potential for ergosterol production than some other yeasts was reported. The effects of transcription factors UPC2, MOT3, and ROX1 of K. marxianus on ergosterol synthesis were explored, and a Upc2-overexpressing strain produced 1.78 times more ergosterol (167.33 mg/L) than the wild-type strain (60.04 mg/L). A total of 239.98 mg/L ergosterol was produced when glucose was replaced with fructose to limit ethanol production. Enhanced aeration increased ergosterol titer from 63.09 mg/L to 128.46 mg/L at 42 °C. The ergosterol titer reached 304.37 mg/L in a shake flask at 37 °C, or 1124.38 and 948.32 mg/L at 37 °C and 42 °C, respectively, in a 5 L bioreactor, using Jerusalem artichoke tubers as the sole carbon source. This study establishes a platform for ergosterol biosynthesis using inexpensive materials.

Analysis of Prototheca and yeast species isolated from bulk tank milk collected in Tokachi District, Japan.

Bovine mastitis, a major infectious disease affecting milking cows, leads to reduced milk yield and quality, reduced animal welfare, and an increased need for culling. Although its major causative agents are bacteria, yeast species and achlorophyllous algae of the Prototheca genus are well known as causative agents of bovine refractory mastitis. Nevertheless, few studies have analyzed specific yeasts and Prototheca in this context. Herein, we present survey data of yeast species and Prototheca species isolated from bulk tank milk in the Tokachi district of Japan from April 2020 through March 2021. The species of 276 isolates were determined. Yeast species accounted for 184 isolates, of which Pichia kudriavzevii was the most prevalent species. Regarding Prototheca species, only Prototheca bovis was isolated (92 isolates). Prototheca bovis and Pichia kudriavzevii were detected throughout the year and were detected repeatedly on the same farm. Kluyveromyces marxianus was the second most frequently isolated yeast species after Pichia kudriavzevii. Candida parapsilosis, the fourth most frequently isolated yeast species, was found discontinuously. Analysis of monthly data indicated that Kluyveromyces marxianus and Candida parapsilosis were mainly found during the winter and summer months, respectively. Candida akabanensis and Pichia cactophila were the third and fifth most frequently isolated yeast species, respectively. They were detected repeatedly in bulk tank milk samples from the same farms. Results obtained from bulk tank milk underscore the prevalence of these species. These study results are expected to contribute to the elucidation of problematic yeast and Prototheca species.

Kluyveromyces marxianus as a microbial cell factory for lignocellulosic biomass valorisation.

The non-conventional yeast Kluyveromyces marxianus is widely used for several biotechnological applications, mainly due to its thermotolerance, high growth rate, and ability to metabolise a wide range of sugars. These cell traits are strategic for lignocellulosic biomass valorisation and strain diversity prompts the development of robust chassis, either with improved tolerance to lignocellulosic inhibitors or ethanol. This review summarises bioethanol and value-added chemicals production by K. marxianus from different lignocellulosic biomasses. Moreover, metabolic engineering and process optimization strategies developed to expand K. marxianus potential are also compiled, as well as studies reporting cell mechanisms to cope with lignocellulosic-derived inhibitors. The main lignocellulosic-based products are bioethanol, representing 71% of the reports, and xylitol, representing 17% of the reports. K. marxianus also proved to be a good chassis for lactic acid and volatile compounds production from lignocellulosic biomass, although the literature on this matter is still scarce. The increasing advances in genome editing tools and process optimization strategies will widen the K. marxianus-based portfolio products.

Transcriptomic analysis reveals process of autolysis of Kluyveromyces marxianus in vacuum negative pressure and the higher temperature

Kluyveromyces marxianus is expected to be used in the production of yeast extracts due to its good fermentation ability and nutritional properties. Yeast autolysis is a key process to produce yeast extract and vacuum negative pressure stress can be used as an effective way to assist autolysis. However, the molecular mechanism of initiating Kluyveromyces marxianus autolysis induced by vacuum negative pressure and the higher temperature is still unclear. In this study, RNA-seq technology was performed mainly to analyze autolytic processes in Kluyveromyces marxianus strains. Considerable differentially expressed genes (DEGs) of downregulation were significantly enriched in 7 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to synthesis and transport of RNA and ribosome, which indicated that abnormal protein translations had already occurred in autolytic process. Interestingly, due to obvious change of related DEGs, endoplasmic reticulum-associated degradation (ERAD) and autophagy were activated and cell wall integrity pathway was hindered. Under the continuous influence of the external stress environment, the long-term changes of the above pathways triggered a vicious circle of gradual damage to yeast cells, which is the main cause of yeast autolysis. These results may provide important clues for the in-depth interpretation of the yeast autolytic mechanism.(AU)

Effect of co-fermentation system with isolated new yeasts on soymilk: microbiological, physicochemical, rheological, aromatic, and sensory characterizations.

The beany flavor adversely influences consumer acceptance of soymilk (SM) products. Thus, in this work, the co-fermentation of isolated new yeasts (Kluyveromyces marxianus SP-1, Candida ethanolica ATW-1, and Pichia amenthionina Y) and Kluyveromyces marxianus K (a commercial yeast) along with an XPL-1 starter (including five strains of lactic acid bacteria (LAB)) was utilized to mend the beany flavor of fermented SM (FSM) beverages. Probiotic count, pH, titratable acidity, syneresis, water holding capacity, rheological characteristics, and sensory attributes were investigated. Furthermore, the free amino acids, nucleotides, and volatile compounds (VCs) were analyzed, also presenting the collected VC data by exploiting a principal component analysis (PCA) and a heatmap with a hierarchical cluster analysis. The co-fermentation with Kluyveromyces marxianus SP-1 and K remarkably enhanced the LAB strain growth and acid production, improving the rheological attributes, whereas that of yeast along with XPL-1 as a mullite starter could reduce the beany odor. PCA chart displayed that higher amounts of alcohols, ketones, acids, and esters that significantly improved the flavor quality of FSM beverages were generated throughout the co-fermentation process. The co-fermentation with Pichia amenthionina Y generated the highest acetoin (36.19%) and diacetyl (2.02%), thus improving the overall acceptance of FSM, as well as the sensory characteristics of FSM beverages with the highest umami, sweet, odorless amino acids, and umami nucleotides, and the lowest content of alcohol and inosine. Taken together, the co-fermentation of Pichia amenthionina Y along with XPL-1 within SM provides novel insights regarding the development of FSM and fermented beverages.

Downregulation of ammonium uptake improves the growth and tolerance of Kluyveromyces marxianus at high temperature.

The growth and tolerance of Kluyveromyces marxianus at high temperatures decreased significantly in the synthetic medium (SM), which is commonly used in industrial fermentations. After 100 days of adaptive laboratory evolution, a strain named KM234 exhibited excellent tolerance at a high temperature, without loss of its growth ability at a moderate temperature. Transcriptomic analysis revealed that the KM234 strain decreased the expression of the ammonium (NH4+ ) transporter gene MEP3 and increased the synthesis of the amino acid carbon backbone, which may contribute greatly to the high-temperature growth phenotype. High NH4+ content in SM significantly increased the reactive oxygen species (ROS) production at high temperatures and thus caused toxicity to yeast cells. Replacing NH4+ with organic nitrogen sources or increasing the concentration of potassium ions (K+ ) in the medium restored the growth of the wild-type K. marxianus at a high temperature in SM. We also showed that the NH4+ toxicity mitigated by K+ might closely depend on the KIN1 gene. Our results provide a practical solution to industrial fermentation under high-temperature conditions.

Reconstruction of a catalogue of genome-scale metabolic models with enzymatic constraints using GECKO 2.0.

Genome-scale metabolic models (GEMs) have been widely used for quantitative exploration of the relation between genotype and phenotype. Streamlined integration of enzyme constraints and proteomics data into such models was first enabled by the GECKO toolbox, allowing the study of phenotypes constrained by protein limitations. Here, we upgrade the toolbox in order to enhance models with enzyme and proteomics constraints for any organism with a compatible GEM reconstruction. With this, enzyme-constrained models for the budding yeasts Saccharomyces cerevisiae, Yarrowia lipolytica and Kluyveromyces marxianus are generated to study their long-term adaptation to several stress factors by incorporation of proteomics data. Predictions reveal that upregulation and high saturation of enzymes in amino acid metabolism are common across organisms and conditions, suggesting the relevance of metabolic robustness in contrast to optimal protein utilization as a cellular objective for microbial growth under stress and nutrient-limited conditions. The functionality of GECKO is expanded with an automated framework for continuous and version-controlled update of enzyme-constrained GEMs, also producing such models for Escherichia coli and Homo sapiens. In this work, we facilitate the utilization of enzyme-constrained GEMs in basic science, metabolic engineering and synthetic biology purposes.

Development of a ribosome profiling protocol to study translation in Kluyveromyces marxianus.

Kluyveromyces marxianus is an interesting and important yeast because of particular traits such as thermotolerance and rapid growth, and for applications in food and industrial biotechnology. For both understanding its biology and developing bioprocesses, it is important to understand how K. marxianus responds and adapts to changing environments. For this, a full suite of omics tools to measure and compare global patterns of gene expression and protein synthesis is needed. We report here the development of a ribosome profiling method for K. marxianus, which allows codon resolution of translation on a genome-wide scale by deep sequencing of ribosome locations on mRNAs. To aid in the analysis and sharing of ribosome profiling data, we added the K. marxianus genome as well as transcriptome and ribosome profiling data to the publicly accessible GWIPS-viz and Trips-Viz browsers. Users are able to upload custom ribosome profiling and RNA-Seq data to both browsers, therefore allowing easy analysis and sharing of data. We also provide a set of step-by-step protocols for the experimental and bioinformatic methods that we developed.

Extraction, isolation, structural characterization and prebiotic activity of cell wall polysaccharide from Kluyveromyces marxianus.

In this study, three yeast α-mannans (LZ-MPS, MC-MPS, and G-MPS) were extracted from different sources of Kluyveromyces marxianus. The total sugar content of the three α-mannans ranged from 91.13-97.10%, whereas no proteins were detected. A structural arrangement was proposed using ultraviolet spectroscopy, Fourier-transform infrared spectroscopy, and one-dimensional and two-dimensional nuclear magnetic resonance. The main chain of the three yeast α-mannans was formed by a →6)-α-D-Manp-(1→ unit, which was slightly different from the repeating unit of the branch structure. The prebiotic potential of LZ-MPS, MC-MPS, and G-MPS was assessed using in vitro fermentation with pure and faecal cultures. The three yeast α-mannans could be utilised as substrates for the growth of Lactobacillus and Lactococcus strains. In addition, the three yeast α-mannans markedly regulated the intestinal microbiota composition by increasing the relative abundances of Bacteroides, Parabacteroides, and Phascolarctobacterium and decreasing the abundance of pathogenic bacteria.