Engineering peroxisomal surface display for enhanced biosynthesis in the emerging yeast Kluyveromyces marxianus.

The non-conventional yeast Kluyveromyces marxianus is a promising microbial host for industrial biomanufacturing. With the recent development of Cas9-based genome editing systems and other novel synthetic biology tools for K. marxianus, engineering of this yeast has become far more accessible. Enzyme colocalization is a proven approach to increase pathway flux and the synthesis of non-native products. Here, we engineer K. marxianus to enable peroxisomal surface display, an enzyme colocalization technique for displaying enzymes on the peroxisome membrane via an anchoring motif from the peroxin Pex15. The native KmPex15 anchoring motif was identified and fused to GFP, resulting in successful localization to the surface of the peroxisomes. To demonstrate the advantages for pathway localization, the Pseudomonas savastanoi IaaM and IaaH enzymes were co-displayed on the peroxisome surface; this increased production of indole-acetic acid 7.9-fold via substrate channeling effects. We then redirected pathway flux by displaying the violacein pathway enzymes VioE and VioD from Chromobacterium violaceum, increasing selectivity of proviolacein to prodeoxyviolacein by 2.5-fold. Finally, we improved direct access to peroxisomal acetyl-CoA and increased titers of the polyketide triacetic acid lactone (TAL) by 2-fold through concurrent display of the proteins Cat2, Acc1, and the type III PKS 2-pyrone synthase from Gerbera hybrida relative to the same three enzymes diffusing in the cytosol. We further improved TAL production by up to 2.1-fold through engineering peroxisome morphology and lifespan. Our findings demonstrate that peroxisomal surface display is an efficient enzyme colocalization strategy in K. marxianus and applicable for improving production of a wide range of non-native products.

Unlocking the potential of Kluyveromyces marxianus in the definition of aroma composition of cheeses.

Introduction: The cheese microbiota is very complex and is made up of technologically-relevant, spoilage, opportunistic and pathogenic microorganisms. Among them lactic acid bacteria and yeasts are the main ones. One of the most interesting dairy yeasts is Kluyveromyces marxianus because of its technological properties including the ability to produce aroma compounds. Methods: This study investigated the contribution of Kluyveromyces marxianus to the gross composition and aroma profile of cow cheeses. Experimental cheeses were prepared by inoculating a co-culture of K. marxianus FM09 and a commercial strain of Lacticaseibacillus casei and compared with cheeses obtained with only L. casei. The gross composition was determined by a FoodScan™ 2 Dairy Analyser, and free amino acids were evaluated at 507 nm after reaction with Cd-ninhydrin. The volatile organic compounds were extracted by head-space solid phase micro-extraction and analyzed by gas chromatography-mass spectrometry coupled with odor activity values. qRT-PCR was applied to determine the expression of genes involved in esters synthesis and degradation. Results: The inoculation of K. marxianus induced an increase of pH and a reduction of protein content of cheeses, in agreement with the stronger proteolysis detected in these cheeses. K. marxianus influenced the content of aroma compounds both quantitatively and qualitatively. In particular, an increase of higher alcohols, esters and organic acids was observed. Moreover, 12 compounds were detected only in cheeses obtained with the co-culture. These differences were in agreement with the odor activity values (OAV). In fact, only 11 compounds showed OAV > 1 in cheeses obtained with the commercial strain, and 24 in those obtained with the co-culture. The qPCR analysis revealed an over expression of ATF1, EAT1, and IAH1 genes. Conclusion: Kluyveromyces marxianus could act as an important auxiliary starter for cheese production through the development and diversification of compounds related to flavor in short-aged cow cheeses.

Biocontrol activity of Kluyveromyces marxianus YG-4 against Penicillium expansum LPH9 on apples.

Penicillium expansum (P. expansum), a widespread fungal pathogen, causes serious economic loss and public health concerns. The aim of this research is to investigate the antifungal effect of Kluyveromyces marxianus YG-4 (K. marxianus YG-4) against P. expansum and possible mechanism. The results showed that competition for nutrients and space, as well as the release of volatile organic compounds (VOCs), are the antifungal mechanisms. Citronellol may be the antifungal component of K. marxianus YG-4 VOCs based on GC-MS analysis. Further experiments had shown that citronellol inhibited the growth of P. expansum LPH9 by damaging the cell structure, disrupting the redox system, reducing antioxidant enzyme activity, and causing oxidative damage. K. marxianus YG-4, K. marxianus YG-4 VOCs and citronellol can effectively inhibit the spore germination of P. expansum on apples. The above results indicated that K. marxianus YG-4 had strong biocontrol activity and can be used as an excellent candidate strain for fruit preservation.

Positive effects of yeast soluble cell wall polysaccharide on fruit postharvest control through resistance response.

Yeast-derived cell wall polysaccharides possess numerous biological activities, but their application in postharvest preservation is rarely reported. The aim of this research was to investigate the effects of Kluyveromyces marxianus soluble cell wall polysaccharide (SCWP) on preventing the infection of Penicillium expansum in pear fruit. The results showed that K. marxianus SCWP treatment could significantly improve the resistance of pear fruit to P. expansum, with respect to Saccharomyces cerevisiae-derived SCWP. Composition of both SCWPs was mannan with the main chains consisting of a â†’ 6)-α-D-Manp-(1 â†’ unit and the branch structure formed by → 2)-α-D-Manp-(1 except that K. marxianus SCWP took on a shorter side chain and a rougher surface than S. cerevisiae SCWP. In addition, mechanisms of K. marxianus SCWP on stimulating resistance response were associated with the apparent oxidative burst, increased gene expression and enzyme activity of antioxidant and defense systems in pear fruit. Our findings suggest that K. marxianus SCWP can be used as an innovative and promising candidate for preventing postharvest fungal decay and extending fruit shelf life.

Heterologous expressing melittin in a probiotic yeast to evaluate its function for promoting NSC-34 regeneration.

Melittin is a bioactive peptide and the predominant component in bee venom (BV), studied for its many medical properties, such as antibacterial, anti-inflammatory, anti-arthritis, nerve damage reduction, and muscle cell regeneration. Melittin is primarily obtained through natural extraction and chemical synthesis; however, both methods have limitations and cannot be used for mass production. This study established a heterologous melittin expression system in the probiotic yeast Kluyveromyces marxianus. This yeast was selected for its advantages in stress tolerance and high secreted protein yields, simplifying purification. A > 95% high-purity melittin (MET) and its precursor promelittin (ProMET) were successfully produced and purified at 1.68 µg/mL and 3.33 µg/mL concentrations and verified through HPLC and mass spectrum. The functional test of the NSC-34 cell regeneration revealed that MET achieved the best activity compared to ProMET and the natural-extracted BV groups. Growth-related gene expressions were evaluated, including microtubule-associated protein 2 (MAP2), microtubule-associated protein Tau (MAPT), growth-associated protein 43 (GAP-43), choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), and acetylcholine esterase (AChE). The results indicated that treating MET increased MAP2, GAP-43, and VAChT expressions, in which cholinergic signaling is related to neurological functions. A heterologously expressed melittin in a probiotic yeast and its potential for promoting NSC-34 regeneration described here facilitate commercial and therapeutic use. KEY POINTS: • MET and its precursor ProMET were successfully hetero-expressed in K. marxianus • > 95% high-purity MET and ProMET were purified at 1.68 µg/mL and 3.33 µg/mL • MET has no cytotoxicity toward NSC-34 and significantly promotes NSC-34 growth.

Impact of Kluyveromyces marxianus VM004 culture conditions on the cell wall structure and its influence on aflatoxin B1 binding.

The aim of this study was to determine the impact of Kluyveromyces marxianus VM004 culture conditions on the cell wall (CW) structure and its influence on aflatoxin B1 binding. The yeast was inoculated into two types of culture media: yeast extract-peptone-dextrose (YPD) broth and dried distiller's grains with solubles (DDG). The CW was extracted from the biomass produced in these media. AFB1 (150ng/ml) adsorption tests using the biomass (1×107cells/ml) and the CW (0.001g) were performed at pH 2 and pH 8. Transmission electron microscopy (TEM) evaluated the CW thickness, and infrared spectroscopy (IR) determined the CW composition. Biomass production in YPD was higher than that in DDG. Cell diameter (µm) and CW thickness (µm) increased in the DDG medium. The CW percentage obtained in DDG was higher than that in YPD. The absorbance of carbohydrates by IR was higher in YPD. pH influenced AFB1 adsorption, which was lower at pH 8. The proportion of ß-glucan and chitin present in CW was higher in the YPD medium. The IR method allowed to study the CW carbohydrate variation under the influence of these carbon sources. In conclusion, the culture media composition influenced the ß-glucan and chitin composition and consequently, AFB1 adsorption.

Process scale-up simulation and techno-economic assessment of ethanol fermentation from cheese whey.

BACKGROUND: Production of cheese whey in the EU exceeded 55 million tons in 2022, resulting in lactose-rich effluents that pose significant environmental challenges. To address this issue, the present study investigated cheese-whey treatment via membrane filtration and the utilization of its components as fermentation feedstock. A simulation model was developed for an industrial-scale facility located in Italy's Apulia region, designed to process 539 m3/day of untreated cheese-whey. The model integrated experimental data from ethanolic fermentation using a selected strain of Kluyveromyces marxianus in lactose-supplemented media, along with relevant published data. RESULTS: The simulation was divided into three different sections. The first section focused on cheese-whey pretreatment through membrane filtration, enabling the recovery of 56%w/w whey protein concentrate, process water recirculation, and lactose concentration. In the second section, the recovered lactose was directed towards fermentation and downstream anhydrous ethanol production. The third section encompassed anaerobic digestion of organic residue, sludge handling, and combined heat and power production. Moreover, three different scenarios were produced based on ethanol yield on lactose (YE/L), biomass yield on lactose, and final lactose concentration in the medium. A techno-economic assessment based on the collected data was performed as well as a sensitivity analysis focused on economic parameters, encompassing considerations on cheese-whey by assessing its economical impact as a credit for the simulated facility, dictated by a gate fee, or as a cost by considering it a raw material. The techno-economic analysis revealed different minimum ethanol selling prices across the three scenarios. The best performance was obtained in the scenario presenting a YE/L = 0.45 g/g, with a minimum selling price of 1.43 €/kg. Finally, sensitivity analysis highlighted the model's dependence on the price or credit associated with cheese-whey handling. CONCLUSIONS: This work highlighted the importance of policy implementation in this kind of study, demonstrating how a gate fee approach applied to cheese-whey procurement positively impacted the final minimum selling price for ethanol across all scenarios. Additionally, considerations should be made about the implementation of the simulated process as a plug-in addition in to existing processes dealing with dairy products or handling multiple biomasses to produce ethanol.

High-Yield Expressed Human Ferritin Heavy-Chain Nanoparticles in K. marxianus for Functional Food Development.

The use of Generally Recognized as Safe (GRAS)-grade microbial cell factories to produce recombinant protein-based nutritional products is a promising trend in developing food and health supplements. In this study, GRAS-grade Kluyveromyces marxianus was employed to express recombinant human heavy-chain ferritin (rhFTH), achieving a yield of 11 g/L in a 5 L fermenter, marking the highest yield reported for ferritin nanoparticle proteins to our knowledge. The rhFTH formed 12 nm spherical nanocages capable of ferroxidase activity, which involves converting Fe2+ to Fe3+ for storage. The rhFTH-containing yeast cell lysates promoted cytokine secretion (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and -1ß (IL-1ß)) and enhanced locomotion, pharyngeal pumping frequency, egg-laying capacity, and lifespan under heat and oxidative stress in the RAW264.7 mouse cell line and the C. elegans model, respectively, whereas yeast cell lysate alone had no such effects. These findings suggest that rhFTH boosts immunity, holding promise for developing ferritin-based food and nutritional products and suggesting its adjuvant potential for clinical applications of ferritin-based nanomedicine. The high-yield production of ferritin nanoparticles in K. marxianus offers a valuable source of ferritin for the development of ferritin-based products.

Molecular Modification Enhances Xylose Uptake by the Sugar Transporter KM_SUT5 of Kluyveromyces marxianus.

This research cloned and expressed the sugar transporter gene KM_SUT5 from Kluyveromyces marxianus GX-UN120, which displayed remarkable sugar transportation capabilities, including pentose sugars. To investigate the impact of point mutations on xylose transport capacity, we selected four sites, predicted the suitable amino acid sites by molecular docking, and altered their codons to construct the corresponding mutants, Q74D, Y195K, S460H, and Q464F, respectively. Furthermore, we conducted site-directed truncation on six sites of KM_SUT5p. The molecular modification resulted in significant changes in mutant growth and the D-xylose transport rate. Specifically, the S460H mutant exhibited a higher growth rate and demonstrated excellent performance across 20 g L-1 xylose, achieving the highest xylose accumulation under xylose conditions (49.94 µmol h-1 gDCW-1, DCW mean dry cell weight). Notably, mutant delA554-, in which the transporter protein SUT5 is truncated at position delA554-, significantly increased growth rates in both D-xylose and D-glucose substrates. These findings offer valuable insights into potential modifications of other sugar transporters and contribute to a deeper understanding of the C-terminal function of sugar transporters.

Engineering Living Material for Controlled Fragrance Release Utilizing Kluyveromyces marxianus CBS6556 and Adaptive Hydrogel.

The demand for controllable fragrance materials is substantial owing to their potential to impart enduring scents in a variety of applications. However, the practical application of such materials has been limited by challenges in tunable morphogenesis, structural variability, and adaptability to diverse conditions. In our study, we introduce a hybrid living material that integrates a genetically engineered strain of Kluyveromyces marxianus CBS6556 with an adaptive hydrogel. The engineered K. marxianus achieved temperature stability in 2-phenylethanol (2-PE) and 2-phenylethyl acetate (2-PEAc) production by expressing relevant genes in the 2-PE metabolic pathway using the high-temperature preferential promoter SSE1. The enhanced water retention capacity supports the metabolic activities of the encapsulated yeast cells, ensuring their survival and functionality over an extended period. Fragrance-releasing living material (FLM) is designed to controllably emit fragrance 2-PE by adjusting the microbial concentration within the hydrogel matrix. The FLM exhibits versatile adhesion capabilities, effectively binding to a spectrum of surfaces such as wood, textiles, and glass as well as to natural substrates like leaves. This adaptability enhances the material's applicability across various settings. Furthermore, FLM can be crafted into various forms, including microbeads, fibers, and films. This research opens up new horizons for controlled fragrance release of living materials.

Lipase activity of recombinant KmYJR107Wp and KmLIP3p enzymes expressed in Saccharomyces cerevisiae BY4742 from Kluyveromyces marxianus L2029.

Lipases are used in many food, energy, and pharmaceutical processes. Thus, new systems have been sought to synthesize alternative lipases with potential biotechnological applications. Kluyveromyces marxianus is a yeast with recognized lipase activity; at least ten putative lipases/esterases in its genome have been detected, and two of them possess a signal peptide for extracellular secretion. The study of extracellular lipases becomes more relevant since they usually have higher activity rates than intracellular lipases and simpler purification mechanisms. For these reasons, this study aimed to characterize the production and lipase activity of the putative extracellular lipases of the K. marxianus L-2029 strain, encoded in the genes LIP3 and YJR107W. Both genes were heterologously expressed in Saccharomyces cerevisiae BY4742 (yeast strain without extracellular lipase activity) using a pYES2.1/V5-His-TOPO® plasmid. Herein, we show evidence that the strain transformed with the LIP3 gene did not show lipase activity during flask galactose induction. On the other hand, the strain transformed with the YJR107W gene showed a specific activity of 0.397 U/mg, with an optimum temperature of 37 °C and pH 6. For maximum cell production, glucose and yeast extract concentrations were evaluated by a 22 factorial design, followed by the validation of the best concentrations predicted by a statistical model; a 22 factorial design was also carried out to evaluate the concentration of the inducer galactose on the transformed strains, and the intracellular and extracellular lipase specific activities were quantified. Finally, the biomass and lipase production were determined for each strain, which was grown in a stirred tank bioreactor with a working volume of 1.5 L. The specific activities of the transformed strains obtained in the bioreactor were 1.36 U/mg for the LIP3 transformant and 1.25 U/mg for the YJR107W transformant, respectively.

Engineering a carbon source-responsive promoter for improved biosynthesis in the non-conventional yeast Kluyveromyces marxianus.

Many desired biobased chemicals exhibit a range of toxicity to microbial cell factories, making industry-level biomanufacturing more challenging. Separating microbial growth and production phases is known to be beneficial for improving production of toxic products. Here, we developed a novel synthetic carbon-responsive promoter for use in the rapidly growing, stress-tolerant yeast Kluyveromyces marxianus, by fusing carbon-source responsive elements of the native ICL1 promoter to the strong S. cerevisiae TDH3 or native NC1 promoter cores. Two hybrids, P IT350 and P IN450 , were validated via EGFP fluorescence and demonstrated exceptional strength, partial repression during growth, and late phase activation in glucose- and lactose-based medium, respectively. Expressing the Gerbera hybrida 2-pyrone synthase (2-PS) for synthesis of the polyketide triacetic acid lactone (TAL) under the control of P IN450 increased TAL more than 50% relative to the native NC1 promoter, and additional promoter engineering further increased TAL titer to 1.39 g/L in tube culture. Expression of the Penicillium griseofulvum 6-methylsalicylic acid synthase (6-MSAS) under the control of P IN450 resulted in a 6.6-fold increase in 6-MSA titer to 1.09 g/L and a simultaneous 1.5-fold increase in cell growth. Finally, we used P IN450 to express the Pseudomonas savastanoi IaaM and IaaH proteins and the Salvia pomifera sabinene synthase protein to improve production of the auxin hormone indole-3-acetic acid and the monoterpene sabinene, respectively, both extremely toxic to yeast. The development of carbon-responsive promoters adds to the synthetic biology toolbox and available metabolic engineering strategies for K. marxianus, allowing greater control over heterologous protein expression and improved production of toxic metabolites.

Transfer of disulfide bond formation modules via yeast artificial chromosomes promotes the expression of heterologous proteins in Kluyveromyces marxianus.

Kluyveromyces marxianus is a food-safe yeast with great potential for producing heterologous proteins. Improving the yield in K. marxianus remains a challenge and incorporating large-scale functional modules poses a technical obstacle in engineering. To address these issues, linear and circular yeast artificial chromosomes of K. marxianus (KmYACs) were constructed and loaded with disulfide bond formation modules from Pichia pastoris or K. marxianus. These modules contained up to seven genes with a maximum size of 15 kb. KmYACs carried telomeres either from K. marxianus or Tetrahymena. KmYACs were transferred successfully into K. marxianus and stably propagated without affecting the normal growth of the host, regardless of the type of telomeres and configurations of KmYACs. KmYACs increased the overall expression levels of disulfide bond formation genes and significantly enhanced the yield of various heterologous proteins. In high-density fermentation, the use of KmYACs resulted in a glucoamylase yield of 16.8 g/l, the highest reported level to date in K. marxianus. Transcriptomic and metabolomic analysis of cells containing KmYACs suggested increased flavin adenine dinucleotide biosynthesis, enhanced flux entering the tricarboxylic acid cycle, and a preferred demand for lysine and arginine as features of cells overexpressing heterologous proteins. Consistently, supplementing lysine or arginine further improved the yield. Therefore, KmYAC provides a powerful platform for manipulating large modules with enormous potential for industrial applications and fundamental research. Transferring the disulfide bond formation module via YACs proves to be an efficient strategy for improving the yield of heterologous proteins, and this strategy may be applied to optimize other microbial cell factories.

CRISPR-Cas9 mediated genome editing of Kluyveromyces marxianus for iterative, multiplexed gene disruption and pathway integration.

Kluyveromyces marxianus, a thermotolerant, fast-growing, Crabtree-negative yeast, is a promising chassis for the manufacture of various bioproducts. Although several genome editing tools are available for this yeast, these tools still require refinement to enable more convenient and efficient genetic modification. In this study, we engineered the K. marxianus NBRC 104275 strain by impairing the nonhomologous end joining and enhancing the homologous recombination machinery, which resulted in improved homology-directed repair effective on homology arms of up to 40 bp in length. Additionally, we simplified the CRISPR-Cas9 editing system by constructing a strain for integrative expression of Cas9 nuclease and plasmids bearing different selection markers for gRNA expression, thereby facilitating iterative genome editing without the need for plasmid curing. We demonstrated that tRNA was more effective than the hammerhead ribozyme for processing gRNA primary transcripts, and readily assembled tRNA-gRNA arrays were used for multiplexed editing of at least four targets. This editing tool was further employed for simultaneous scarless in vivo assembly of a 12-kb cassette from three fragments and marker-free integration for expressing a fusion variant of fatty acid synthase, as well as the integration of genes for starch hydrolysis. Together, the genome editing tool developed in this study makes K. marxianus more amenable to genetic modification and will facilitate more extensive engineering of this nonconventional yeast for chemical production.

Detoxification of Mycotoxin Patulin by the Yeast Kluyveromyces marxianus YG-4 in Apple Juice.

Patulin (PAT) is a mycotoxin produced by Penicillium species, which often contaminates fruit and fruit-derived products, posing a threat to human health and food safety. This work aims to investigate the detoxification of PAT by Kluyveromyces marxianus YG-4 (K. marxianus YG-4) and its application in apple juice. The results revealed that the detoxification effect of K. marxianus YG-4 on PAT includes adsorption and degradation. The adsorption binding sites were polysaccharides, proteins, and some lipids on the cell wall of K. marxianus YG-4, and the adsorption groups were hydroxyl groups, amino acid side chains, carboxyl groups, and ester groups, which were combined through strong forces (ion interactions, electrostatic interactions, and hydrogen bonding) and not easily eluted. The degradation active substance was an intracellular enzyme, and the degradation product was desoxypatulinic acid (DPA) without cytotoxicity. K. marxianus YG-4 can also effectively adsorb and degrade PAT in apple juice. The contents of organic acids and polyphenols significantly increased after detoxification, significantly improving the quality of apple juice. The detoxification ability of K. marxianus YG-4 toward PAT would be a novel approach for the elimination of PAT contamination.

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.

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.

Inhibitory action of antibiotics on Kluyveromyces marxianus.

A bioassay containing Kluyveromyces marxianus in microtiter plates was used to determine the inhibitory action of 28 antibiotics (aminoglycosides, beta-lactams, macrolides, quinolones, tetracyclines and sulfonamides) against this yeast in whey. For this purpose, the dose-response curve for each antibiotic was constructed using 16 replicates of 12 different concentrations of the antibiotic. The plates were incubated at 40°C until the negative samples exhibited their indicator (5-7h). Subsequently, the absorbances of the yeast cells in each plate were measured by the turbidimetric method (λ=600nm) and the logistic regression model was applied. The concentrations causing 10% (IC10) and 50% (IC50) of growth inhibition of the yeast were calculated. The results allowed to conclude that whey contaminated with cephalosporins, quinolones and tetracyclines at levels close to the Maximum Residue Limits inhibits the growth of K. marxianus. Therefore, previous inactivation treatments should be implemented in order to re-use this contaminated whey by fermentation with K. marxianus.

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.

Effects of Saccharomyces cerevisiae and Kluyveromyces marxianus on the Physicochemical, Microbial, and Flavor Changes of Sauce Meat during Storage.

Saccharomyces cerevisiae (S. cerevisiae) and Kluyveromyces marxianus (K. marxianus) are often used as fermenters in yogurt and alcohol, and have been less studied within meat products. The yeasts were added to sauce meat, and the uninoculated group served as a control in this study to examine and compare the changing patterns of physicochemical and flavor characteristics of S. cerevisiae and K. marxianus on sauce meat during storage. The changes in moisture content, aw, pH, thiobarbituric acid reactive substances (TBARS), and other flavor characteristics were measured in sauce meat during the first, second, fourth, and sixth months after production. The following factors were examined: moisture content, aw, pH, TBARS, peroxide value (POV), acid value (AV), soluble protein (SP), free amino acid (FAA), and volatile flavoring compounds. With VIP > 1 and p < 0.05 as the screening conditions, the partial least squares model (PLS-DA) was used to assess the distinctive flavor components in the sausages. The findings demonstrated that the three groups' changes in sauce meat were comparable during the first two months of storage but differed significantly between the 4th and 6th months. The moisture content, water activity, and pH of the sauce meat decreased gradually with the storage time; TBARS, AV, and FAA increased significantly; SP decreased significantly from 2.61 to 1.72, while POV increased to 0.03 and then decreased to 0.02. The POV and TBARS values of the yeast-infected meat were substantially lower than those of the control group, and the POV and TBARS values of the meat inoculated with S. cerevisiae were particularly decreased (p < 0.05). The POV and TBARS values of SC (S. cerevisiae group) decreased by 49.09% and 40.15%, respectively, compared to CK (the control group) at the time of storage until June. The experimental group (KM: K. marxianus group) significantly increased the SP and FAA values of the sauce meat (p < 0.05) by 32.4% and 29.84% compared to the CK group, respectively. Esters and olefins as well as alcohols and esters were much greater in meat that had been supplemented with S. cerevisiae and K. marxianus than in meat from the control group. In conclusion, inoculating sauce meat with S. cerevisiae can significantly enhance the quality and flavor of sauce meat while it is being stored.