Efficient and markerless gene integration with SlugCas9-HF in Kluyveromyces marxianus.

The nonconventional yeast Kluyveromyces marxianus has potential for industrial production, but the lack of advanced synthetic biology tools for precise engineering hinders its rapid development. Here, we introduce a CRISPR-Cas9-mediated multilocus integration method for assembling multiple exogenous genes. Using SlugCas9-HF, a high-fidelity Cas9 nuclease, we enhance gene editing precision. Specific genomic loci predisposed to efficient integration and expression of heterologous genes are identified and combined with a set of paired CRISPR-Cas9 expression plasmids and donor plasmids to establish a CRISPR-based biosynthesis toolkit. This toolkit enables genome integration of large gene modules over 12 kb and achieves simultaneous quadruple-locus integration in a single step with 20% efficiency. As a proof-of-concept, we apply the toolkit to screen for gene combinations that promote heme production, revealing the importance of HEM4Km and HEM12Sc. This CRISPR-based toolkit simplifies the reconstruction of complex pathways in K. marxianus, broadening its application in synthetic biology.

Coupling thermotolerance and high production of recombinant protein by CYR1N1546K mutation via cAMP signaling cascades.

In recombinant protein-producing yeast strains, cells experience high production-related stresses similar to high temperatures. It is possible to increase recombinant protein production by enhancing thermotolerance, but few studies have focused on this topic. Here we aim to identify cellular regulators that can simultaneously activate thermotolerance and high yield of recombinant protein. Through screening at 46 °C, a heat-resistant Kluyveromyces marxianus (K. marxianus) strain FDHY23 is isolated. It also exhibits enhanced recombinant protein productivity at both 30 °C and high temperatures. The CYR1N1546K mutation is identified as responsible for FDHY23's improved phenotype, characterized by weakened adenylate cyclase activity and reduced cAMP production. Introducing this mutation into the wild-type strain greatly enhances both thermotolerance and recombinant protein yields. RNA-seq analysis reveals that under high temperature and recombinant protein production conditions, CYR1 mutation-induced reduction in cAMP levels can stimulate cells to improve its energy supply system and optimize material synthesis, meanwhile enhance stress resistance, based on the altered cAMP signaling cascades. Our study provides CYR1 mutation as a novel target to overcome the bottleneck in achieving high production of recombinant proteins under high temperature conditions, and also offers a convenient approach for high-throughput screening of recombinant proteins with high yields.

Stress response and adaptation mechanisms in Kluyveromyces marxianus.

Kluyveromyces marxianus is a non-Saccharomyces yeast that has gained importance due to its great potential to be used in the food and biotechnology industries. In general, K. marxianus is a known yeast for its ability to assimilate hexoses and pentoses; even this yeast can grow in disaccharides such as sucrose and lactose and polysaccharides such as agave fructans. Otherwise, K. marxianus is an excellent microorganism to produce metabolites of biotechnological interest, such as enzymes, ethanol, aroma compounds, organic acids, and single-cell proteins. However, several studies highlighted the metabolic trait variations among the K. marxianus strains, suggesting genetic diversity within the species that determines its metabolic functions; this diversity can be attributed to its high adaptation capacity against stressful environments. The outstanding metabolic characteristics of K. marxianus have motivated this yeast to be a study model to evaluate its easy adaptability to several environments. This chapter will discuss overview characteristics and applications of K. marxianus and recent insights into the stress response and adaptation mechanisms used by this non-Saccharomyces yeast.

Transcriptome analysis of Kluyveromyces marxianus under succinic acid stress and development of robust strains.

Kluyveromyces marxianus has become an attractive non-conventional yeast cell factory due to its advantageous properties such as high thermal tolerance and rapid growth. Succinic acid (SA) is an important platform molecule that has been applied in various industries such as food, material, cosmetics, and pharmaceuticals. SA bioproduction may be compromised by its toxicity. Besides, metabolite-responsive promoters are known to be important for dynamic control of gene transcription. Therefore, studies on global gene transcription under various SA concentrations are of great importance. Here, comparative transcriptome changes of K. marxianus exposed to various concentrations of SA were analyzed. Enrichment and analysis of gene clusters revealed repression of the tricarboxylic acid cycle and glyoxylate cycle, also activation of the glycolysis pathway and genes related to ergosterol synthesis. Based on the analyses, potential SA-responsive promoters were investigated, among which the promoter strength of IMTCP2 and KLMA_50231 increased 43.4% and 154.7% in response to 15 g/L SA. In addition, overexpression of the transcription factors Gcr1, Upc2, and Ndt80 significantly increased growth under SA stress. Our results benefit understanding SA toxicity mechanisms and the development of robust yeast for organic acid production. KEY POINTS: • Global gene transcription of K. marxianus is changed by succinic acid (SA) • Promoter activities of IMTCP2 and KLMA_50123 are regulated by SA • Overexpression of Gcr1, Upc2, and Ndt80 enhanced SA tolerance.

Enhancing simultaneous saccharification and co-fermentation of corncob by Kluyveromyces marxianus through overexpression of putative transcription regulator.

Overexpression of a gene with unknown function in Kluyveromyces marxianus markedly improved tolerance to lignocellulosic biomass-derived inhibitors. This overexpression also enhanced tolerance to elevated temperatures, ethanol, and high concentrations of NaCl and glucose. Inhibitor degradation and transcriptome analyses related this K. marxianusMultiple Stress Resistance (KmMSR) gene to the robustness of yeast cells. Nuclear localization and DNA-binding domain analyses indicate that KmMsr is a putative transcriptional regulator. Overexpression of a mutant protein with deletion in the flexible region between amino acids 100 and 150 further enhanced tolerance to multiple inhibitors during fermentation, with ethanol production and productivity increasing by 36.31 % and 80.22 %, respectively. In simultaneous saccharification co-fermentation of corncob without detoxification, expression of KmMSR with the deleted flexible region improved ethanol production by 5-fold at 42 °C and 2-fold at 37 °C. Overexpression of the KmMSR mutant provides a strategy for constructing robust lignocellulosic biomass using strains.

CRISPRi-induced transcriptional regulation of IAH1 gene and its influence on volatile compounds profile in Kluyveromyces marxianus DU3.

Mezcal is a traditional Mexican distilled beverage, known for its marked organoleptic profile, which is influenced by several factors, such as the fermentation process, where a wide variety of microorganisms are present. Kluyveromyces marxianus is one of the main yeasts isolated from mezcal fermentations and has been associated with ester synthesis, contributing to the flavors and aromas of the beverage. In this study, we employed CRISPR interference (CRISPRi) technology, using dCas9 fused to the Mxi1 repressor factor domain, to down-regulate the expression of the IAH1 gene, encoding for an isoamyl acetate-hydrolyzing esterase, in K. marxianus strain DU3. The constructed CRISPRi plasmid successfully targeted the IAH1 gene, allowing for specific gene expression modulation. Through gene expression analysis, we assessed the impact of IAH1 down-regulation on the metabolic profile of volatile compounds. We also measured the expression of other genes involved in volatile compound biosynthesis, including ATF1, EAT1, ADH1, and ZWF1 by RT-qPCR. Results demonstrated successful down-regulation of IAH1 expression in K. marxianus strain DU3 using the CRISPRi system. The modulation of IAH1 gene expression resulted in alterations in the production of volatile compounds, specifically ethyl acetate, which are important contributors to the beverage's aroma. Changes in the expression levels of other genes involved in ester biosynthesis, suggesting that the knockdown of IAH1 may generate intracellular alterations in the balance of these metabolites, triggering a regulatory response. The application of CRISPRi technology in K. marxianus opens the possibility of targeted modulation of gene expression, metabolic engineering strategies, and synthetic biology in this yeast strain.

Repurposing plant hormone receptors as chemically-inducible genetic switches for dynamic regulation in yeast.

Precise control of gene expression is critical for optimizing cellular metabolism and improving the production of valuable biochemicals. However, hard-wired approaches to pathway engineering, such as optimizing promoters, can take time and effort. Moreover, limited tools exist for controlling gene regulation in non-conventional hosts. Here, we develop a two-channel chemically-regulated gene expression system for the multi-stress tolerant yeast Kluyveromyces marxianus and use it to tune ethyl acetate production, a native metabolite produced at high titers in this yeast. To achieve this, we repurposed the plant hormone sensing modules (PYR1ABA/HAB1 and PYR1*MANDI/HAB1*) for high dynamic-range gene activation and repression controlled by either abscisic acid (ABA) or mandipropamid (mandi). To redirect metabolic flux towards ethyl acetate biosynthesis, we simultaneously repress pyruvate dehydrogenase (PDA1) and activate pyruvate decarboxylase (PDC1) to enhance ethyl acetate titers. Thus, we have developed new tools for chemically tuning gene expression in K. marxianus and S. cerevisiae that should be deployable across many non-conventional eukaryotic hosts.

Characterization and optimization of 5´ untranslated region containing poly-adenine tracts in Kluyveromyces marxianus using machine-learning model.

BACKGROUND: The 5´ untranslated region (5´ UTR) plays a key role in regulating translation efficiency and mRNA stability, making it a favored target in genetic engineering and synthetic biology. A common feature found in the 5´ UTR is the poly-adenine (poly(A)) tract. However, the effect of 5´ UTR poly(A) on protein production remains controversial. Machine-learning models are powerful tools for explaining the complex contributions of features, but models incorporating features of 5´ UTR poly(A) are currently lacking. Thus, our goal is to construct such a model, using natural 5´ UTRs from Kluyveromyces marxianus, a promising cell factory for producing heterologous proteins. RESULTS: We constructed a mini-library consisting of 207 5´ UTRs harboring poly(A) and 34 5´ UTRs without poly(A) from K. marxianus. The effects of each 5´ UTR on the production of a GFP reporter were evaluated individually in vivo, and the resulting protein abundance spanned an approximately 450-fold range throughout. The data were used to train a multi-layer perceptron neural network (MLP-NN) model that incorporated the length and position of poly(A) as features. The model exhibited good performance in predicting protein abundance (average R2 = 0.7290). The model suggests that the length of poly(A) is negatively correlated with protein production, whereas poly(A) located between 10 and 30 nt upstream of the start codon (AUG) exhibits a weak positive effect on protein abundance. Using the model as guidance, the deletion or reduction of poly(A) upstream of 30 nt preceding AUG tended to improve the production of GFP and a feruloyl esterase. Deletions of poly(A) showed inconsistent effects on mRNA levels, suggesting that poly(A) represses protein production either with or without reducing mRNA levels. CONCLUSION: The effects of poly(A) on protein production depend on its length and position. Integrating poly(A) features into machine-learning models improves simulation accuracy. Deleting or reducing poly(A) upstream of 30 nt preceding AUG tends to enhance protein production. This optimization strategy can be applied to enhance the yield of K. marxianus and other microbial cell factories.

The interaction among Kluyveromyces marxianus G-Y4, Lacticaseibacillus paracasei GL1, and Lactobacillus helveticus SNA12 and signaling molecule AI-2 participate in the formation of biofilm.

In this study, two strains of lactic acid bacteria (Lacticaseibacillus paracasei GL1 and Lactobacillus helveticus SNA12) and one yeast strain of Kluyveromyces marxianus G-Y4 (G-Y4) isolated from Tibetan kefir grains were co-cultured. It was found that the addition of G-Y4 could not only promote the growth of lactic acid bacteria, but also increase the release of metabolites (lactic acid, ethanol, and amino nitrogen). Furthermore, the addition of live cells and cell-free fermentation supernatant (CFS) of G-Y4 could increase the ability of biofilm formation. Morever, the surface characteristics results showed that the addition of G-Y4 live cells could enhance the aggregation ability and hydrophobicity of LAB. Meanwhile, adding live cells and CFS of G-Y4 could promote the release of signaling molecule AI-2 and enhance the expression of the LuxS gene related to biofilm formation. In addition, Fourier-transform infrared spectroscopy and chemical composition analysis were used to investigate the composition of the biofilm, and the results indicated that the biofilm was mainly composed of a small amount of protein but it was rich in polysaccharides including glucose, galactose, and mannose with different ratios. Finally, the formation of biofilm could delay the decline of the number of viable bacteria in storage fermented milk.

Genome engineering of Kluyveromyces marxianus for high D-( -)-lactic acid production under low pH conditions.

Saccharomyces cerevisiae is the workhorse of fermentation industry. Upon engineering for D-lactate production by a series of gene deletions, this yeast had deficiencies in cell growth and D-lactate production at high substrate concentrations. Complex nutrients or high cell density were thus required to support growth and D-lactate production with a potential to increase medium and process cost of industrial-scale D-lactate production. As an alternative microbial biocatalyst, a Crabtree-negative and thermotolerant yeast Kluyveromyces marxianus was engineered in this study to produce high titer and yield of D-lactate at a lower pH without growth defects. Only pyruvate decarboxylase 1 (PDC1) gene was replaced by a codon-optimized bacterial D-lactate dehydrogenase (ldhA). Ethanol, glycerol, or acetic acid was not produced by the resulting strain, KMΔpdc1::ldhA. Aeration rate at 1.5 vvm and culture pH 5.0 at 30 °C provided the highest D-lactate titer of 42.97 ± 0.48 g/L from glucose. Yield and productivity of D-lactate, and glucose-consumption rate were 0.85 ± 0.01 g/g, 0.90 ± 0.01 g/(L·h), and 1.06 ± 0.00 g/(L·h), respectively. Surprisingly, D-lactate titer, productivity, and glucose-consumption rate of 52.29 ± 0.68 g/L, 1.38 ± 0.05 g/(L·h), and 1.22 ± 0.00 g/(L·h), respectively, were higher at 42 °C compared to 30 °C. Sugarcane molasses, a low-value carbon, led to the highest D-lactate titer and yield of 66.26 ± 0.81 g/L and 0.91 ± 0.01 g/g, respectively, in a medium without additional nutrients. This study is a pioneer work of engineering K. marxianus to produce D-lactate at the yield approaching theoretical maximum using simple batch process. Our results support the potential of an engineered K. marxianus for D-lactate production on an industrial scale. KEY POINTS: • K. marxianus was engineered by deleting PDC1 and expressing codon-optimized D-ldhA. • The strain allowed high D-lactate titer and yield under pH ranging from 3.5 to 5.0. • The strain produced 66 g/L D-lactate at 30 °C from molasses without any additional nutrients.

Targeted gene manipulation of Leloir pathway genes for the constitutive expression of ß-galactosidase and its transgalactosylation product galacto-oligosaccharides from Kluyveromyces lactis GG799 and knockout strains.

Galacto-oligosaccharides (GOS) are used as prebiotic ingredients in various food and pharmaceutical industry. At present, production of GOS involves the enzymatic transformation of lactose by transgalactosylation using ß-galactosidase. The yeast Kluyveromyces lactis can utilize lactose as its carbon and energy source. In this species lactose is hydrolyzed by an intracellular ß-galactosidase (EC 3.2.1.23) which is induced by its substrate and related compounds like galactose. The molecular details of gene regulation in kluyveromyces lactis, we have used multiple knockout approaches to study the constitutive expression by which galactose induces ß-galactosidase. The present study involved carrying out to a method of enhancing the constitutive expression of ß-galactosidase through galactose induction and its trans-galactosylation reaction for the production of galacto-oligosaccharides (GOS) in Kluyveromyces lactis (K. Lactis) by applying a knockout based approach on Leloir pathway genes based on fusion-overlap extension polymerase chain reaction and transformation into its genome. The k.lactis strain subjected to Leloir pathway genes knockout, resulted in the accumulation of galactose intracellularly and this internal galactose acts as an inducer of galactose regulon for constitutive expression of ß-galactosidase at early stationary phase was due to the positive regulatory function of mutant gal1p, gal7p and both. These resulted strains used for trans-galactosylation of lactose by ß - galactosidase is characterized for the production of galacto-oligosaccharides. Galactose-induced constitutive expression of ß-galactosidase during the early stationary phase of knockout strains was analysed qualitatively & quantitatively. The activity of ß-galactosidase of wild type, gal1z, gal7k and gal1z & gal7k strains were 7, 8, 9 and 11 U/ml respectively using high cell density cultivation medium. Based on these expression differences in ß-galactosidase, the trans-galactosylation reaction for GOS production and percentage yield of GOS were compared at 25% w/v of lactose. The percentage yield of GOS production of wild type, Δgal1z Lac4+, Δgal7k Lac4++ and Δgal1z Δgal7k Lac4+++mutants strains were 6.3, 13, 17 and 22 U/ml, respectively. Therefore, we propose that the availability of galactose can be used for constitutive over expression of ß - galactosidase in Leloir pathway engineering applications and also for GOS production. Further, increased expression of ß - galactosidases can be used in dairy industry by-products like whey to produce added value products such as galacto-oligosaccharides.

Reprogramming the Metabolic Network in Kluyveromyces lactis with a Transcriptional Switch for De Novo Lacto-N-biose Synthesis.

Lacto-N-biose (LNB) is a member of the human milk oligosaccharide (HMO) family and is synthesized via an enzymatic reaction in vitro with N-acetylglucosamine (GlcNAc) and cofactors. In this study, LNB was synthesized using a cell factory for the first time. First, three modules were constructed in Kluyveromyces lactis for producing LNB from lactose and GlcNAc without the addition of cofactors. Second, a de novo pathway was constructed in K. lactis for producing LNB from lactose without adding GlcNAc. Finally, a transcriptional switch was introduced into K. lactis to reprogram its metabolic network for improving the flux from GlcNAc-6-P to GlcNAc in the de novo pathway. Subsequently, a final LNB yield of 10.41 g/L, similar to the salvage pathway yield, was achieved through the de novo pathway. The engineered K. lactis provides a promising technology platform for the industrial scale production of LNB.

Characterization of mating type on aroma production and metabolic properties wild Kluyveromyces marxianus yeasts.

Kluyveromyces marxianus yeasts represent a valuable industry alternative due to their biotechnological potential to produce aromatic compounds. 2-phenylethanol and 2-phenylethylacetate are significant aromatic compounds widely used in food and cosmetics due to their pleasant odor. Natural obtention of these compounds increases their value, and because of this, bioprocesses such as de novo synthesis has become of great significance. However, the relationship between aromatic compound production and yeast's genetic diversity has yet to be studied. In the present study, the analysis of the genetic diversity in K. marxianus isolated from the natural fermentation of Agave duranguensis for Mezcal elaboration is presented. The results of strains in a haploid and diploid state added to the direct relationship between the mating type locus MAT with metabolic characteristics are studied. Growth rate, assimilate carbohydrates (glucose, lactose, and chicory inulin), and the production of aromatic compounds such as ethyl acetate, isoamyl acetate, isoamyl alcohol, 2-phenylethyl butyrate and phenylethyl propionate and the diversity in terms of the output of 2-phenylethanol and 2-phenylethylacetate by de novo synthesis were determinate, obtaining maximum concentrations of 51.30 and 60.39 mg/L by ITD0049 and ITD 0136 yeasts respectively.

Relevance of aromatic and polar amino acids in the specificity of Inulinase ISO3 from Kluyveromyces marxianus: A molecular dynamics approach with an experimental verification.

The Inulinase from Kluyveromyces marxianus ISO3 (Inu-ISO3) is an enzyme able to hydrolyze linear fructans such as chicory inulin as well as branched fructans like agavin. This enzyme was cloned and expressed in Komagataella pastoris to study the role of selected aromatic and polar residues in the catalytic pocket by Alanine scanning. Molecular dynamics (MD) simulations and enzyme kinetics analysis were performed to study the functional consequences of these amino acid substitutions. Site-directed mutagenesis was used to construct the mutants of the enzyme after carrying out the MD simulations between Inu-ISO3 and its substrates. Mutation Trp79:Ala resulted in the total loss of activity when fructans were used as substrates, while with sucrose, the activity decreased by 98 %. In contrast, the mutations Phe113:Ala and Gln236:Ala increased the invertase activity when sucrose was used as a substrate. Although these amino acids are not part of the conserved motifs where the catalytic triad is located, they are essential for the enzyme's activity. In silico and experimental approaches corroborate the relevance of these residues for substrate binding and their influence on enzymatic activity.

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.

Purification and characterization of a fungal laccase expressed in Kluyveromyces lactis suitable for baking.

Laccase enzyme can replace chemical additives to improve texture properties and the volume of bread. Laccase encoding gene from Phlebia brevispora, a native fungus from Misiones, Argentina, was expressed in the generally recognized as safe yeast Kluyveromyces lactis. To improve laccase activity, medium conditions were optimized. The use of iron sulfate at a concentration of 1 mM led to optimum laccase activity (1289 U·L-1 ) on the fourth day of incubation. SDS-PAGE analysis revealed that the molecular mass of purified laccase was about 180 kDa. Optimum pH for the enzyme was 4 and optimum temperature was 40°C. Laccase exhibited high stability at low pH and high temperature. The application of recombinant laccase to bread decreased hardness, gumminess, and chewiness and increased bread volume. Based on these results, recombinant laccase from P. brevispora with improved yield is a good option for application as an improver of the physicochemical quality of bread at the industrial level. Besides, it will allow us to advance toward our goal of developing healthy alternatives for the bakery industry. No previous work has been reported concerning the heterologous expression of the laccase gene native to the province of Misiones, Argentina, with an aim for application in baking. PRACTICAL APPLICATION: Healthy bakeries became a trend in recent years. The use of the laccase enzyme increases the specific volume and decreases the hardness of bread, being thus an alternative for the replacement of chemical additives in the bakery industry.

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.

Enhancement of biocontrol efficacy of Kluyveromyces marxianus induced by N-acetylglucosamine against Penicillium expansum.

This research aimed to investigate the effect of N-acetylglucosamine (GlcNAc) on the biocontrol activity of Kluyveromyces marxianus and involved possible mechanisms. The results indicated that 0.5% GlcNAc significantly improved the antagonistic efficacy of K. marxianus against Penicillium expansum. GlcNAc supplementation led to increases of biocontrol-related enzymes and stress-resistant substances in K. marxianus including chitinase, ß-1,3-glucanase, trehalose and proline, being beneficial for yeasts vigor maintenance under adverse circumstances. The antioxidative-related enzyme activities in K. marxianus, such as superoxide dismutase (SOD), catalase (CAT) and others, were increased by GlcNAc along with more quickly excess reactive oxygen species (ROS) scavenging. GlcNAc resulted in an enhancement of proliferative capacity in K. marxianus according to the transcriptomic analysis. Consequently, GlcNAc contributed to improvements of K. marxianus in the environmental adaptability and stress resistance. Our study demonstrated that both GlcNAc and K. marxianus could be regarded as potential candidates for postharvest technology application.

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.

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.