Yeast Kluyveromyces Marxianus Research Articles

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.

Dietary Kluyveromyces marxianus hydrolysate alters humoral immunity, jejunal morphology, cecal microbiota and metabolic pathways in broiler chickens raised under a high stocking density

This study investigated the impact of dietary supplementation with hydrolyzed yeast (Kluyveromyces marxianus) on growth performance, humoral immunity, jejunal morphology, cecal microbiota and metabolic pathways in broilers raised at 45 kg/m2. A total of 1,176 mixed sex 1-day-old Ross 308 broilers were distributed into 42 pens and randomly assigned to either the control group, the control + 250 g hydrolyzed yeast (HY)/ton, 250HY group, or the control + 500 g HY/ton, 500HY group for 42 d. HY did not affect growth performance. However, HY reduced (P < 0.05) mortality at 25 to 35 d. Dietary HY lowered the heterophil/lymphocyte ratio and enhanced the villus height/crypt depth ratio and Newcastle disease titer (P < 0.05). Compared with HY250 and the control, HY500 upregulated (P < 0.05) IL-10. HY enhanced the α diversity, inferring the richness and evenness of the ceca microbiota. HY500 had greater β diversity than the control (P < 0.05). Six bacterial phyla, namely, Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Verrucomicrobia, and Cyanobacteria, were found. The relative abundance of Firmicutes was greater in the HY500 treatment group than in the HY250 and control groups. HY decreased the abundance of Actinobacteria. HY supplementation altered (P < 0.05) the abundance of 8 higher-level taxa consisting of 2 classes (Bacilli and Clostridia), 1 order (Lactobacillales), 1 family (Streptococcaceae), and five genera (Streptococcus, Lachnospiraceae_uc, Akkermansiaceae, PACO01270_g, and LLKB_g). HY500 improved (P < 0.05) the abundance of Bacilli, Clostridia, Lactobacillales, Streptococcaceae, Streptococcus, PACO01270_g, and Lachnospiraceae_uc, while HY250 enhanced (P < 0.05) the abundance of Akkermansiaceae and LLKB_g. HY improved the abundance of Lactobacillus and Akkermansia spp. Minimal set of pathway analyses revealed that compared with the control, both HY250 and HY500 regulated 20 metabolic pathways. These findings suggest that dietary K. marxianus hydrolysate, especially HY500, improved humoral immunity and jejunal morphology and beneficially altered the composition and metabolic pathways of the cecal microbiota in broilers raised at 45 kg/m2.

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.

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, PIT350 and PIN450, 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 PIN450 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 PIN450 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 PIN450 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.

Co‐encapsulated of potential probiotic yeast Kluyveromyces marxianus and peanut skin polyphenolic extract as a functional ingredient

SummaryThe aim of this study was to develop and evaluate the bioactive properties and storage stability of microcapsules (MCs) obtained by co‐encapsulation of the potential probiotic yeast Kluyveromyces marxianus VM004 and peanut skin polyphenolic extract (PSE) by spray drying, using whey protein concentrate (WPC) and water‐soluble chitosan (WSCh) as wall materials. The results showed that the selected wall materials provided protection to yeast during spray drying, storage, and simulated gastrointestinal conditions, obtaining better results for WPC and higher concentrations of PSE. Moreover, all formulations demonstrated cytoprotective effects against menadione‐induced oxidative stress in normal rat ileum epithelial cells (IEC‐18) at all concentrations and storage temperatures evaluated. These results suggest that the obtained MCs could be potential functional food ingredients, considering their antioxidant, cytoprotective, and potential probiotic properties.

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.

Microbiological, physicochemical and sensory changes throughout ripening of an experimental soft smear-ripened cheese in relation to salt concentrations

To evaluate the sodium chloride content effect on microbiological, biochemical, physicochemical and sensorial characteristics, Munster cheeses were prepared from pasteurized milk seeded with 3 yeasts (Kluyveromyces marxianus, Debaryomyces hansenii, Geotrichum candidum) and 5 ripening bacteria (Arthrobacter arilaitensis, Brevibacterium aurantiacum, Corynebacterium casei, Hafnia alvei, and Staphylococcus equorum). Experiments were performed under 1.0%, 1.7% and 2.4% NaCl levels in cheese in triplicate. Ripening (d2 - d27) was carried under 12°C and 96% RH. These kinetics were both reproducible and repeatable at 99% confidence level. For each microbial, biochemical and physicochemical parameter, 2 kinetic descriptors (maximal or minimal rate and its occurrence time) were defined. On d2 the physicochemical variables (water activity, dry matter, water content) were strongly dependent on the salting level. From d2 to d27 K. lactis was insensitive to salt while D. hansenii was stimulated. G. candidum growth appeared very sensitive to salt in cheese: at 1.0% NaCl G. candidum exhibited overgrowth, negatively impacting rind appearance, underrind consistency and thickness and off-flavor flaws. Salt concentration of 2.4% induced death of G. candidum. Four bacteria (A. arilaitensis, B. aurantiacum, C. casei, and H. alvei) were moderately sensitive to salt while S. equorum was insensitive to it. Salt level in cheese had a significant effect on carbon substrate consumption rates. Lactate consumption rate in 1.0% salted cheeses was approximately twice higher than under 2.4% NaCl. Data analysis of microorganism, biochemical, and physicochemical kinetics and sensory analysis showed that the best salt level in Munster-type cheeses to achieve an optimum balance between cheese characteristics, sensory qualities and marketability was 1.7% NaCl.

Metabolic coupling of acetate promotes xylose utilization in Kluyveromyces marxianus

Lignocellulosic biomass is a typical agriculture waste and a potential feedstock for the green biomanufacturing. However, insufficient utilization of carbon sources caused by inhibitors, especially acetic acid, hinders the economic bioconversion. Here, we demonstrated the acetate metabolic pathway via glyoxylate shunt in nonconventional yeast Kluyveromyces marxianus. Acetate metabolism increased to 122.7 % at 40 °C after overexpressing ICL1 and MLS1, key genes of glyoxylate shunt revealed by transcriptomic data. Strengthening GPD2 and FDH1 further enhanced the robustness of the K. marxianus, acetate metabolism was significantly increased. Finally, the modified K. marxianus 1727–5-ICL1-MLS1-GPD2-FDH1 boosted the efficiency for xylose uptake up to 1.3 folds in non-detoxified lignocellulosic hydrolysate, benefiting from a 3.9-fold increase in acetate consumption. Our results propose a novel strategy for xylose assimilation by coupling acetate metabolism, contributing the bioconversion of sustainable lignocellulosic biomass. It also provides a new scheme for the total carbon utilization of lignocellulosic biomass, including carbon dioxide (CO2) released from the biofuel production to achieve a circular economy.

A comparative evaluation of the kefir yeast Kluyveromyces marxianus A4 and sulfasalazine in ulcerative colitis: anti-inflammatory impact and gut microbiota modulation.

Although only Saccharomyces boulardii has been studied for ulcerative colitis (UC), probiotic yeasts have immense therapeutic potential. Herein, we evaluated the kefir yeast Kluyveromyces marxianus A4 (Km A4) and its anti-inflammatory effect with sulfasalazine in BALB/c mice with dextran sulfate sodium (DSS)-induced colitis. Oral administration continued for 7 days after the mice were randomly divided into seven groups: control (CON, normal mice administered with saline), DSS-induced colitis mice administered saline (DSS), and DSS-induced colitis mice administered sulfasalazine only (S), Km A4 only (A4), Km A4 plus sulfasalazine (A4 + S), S. boulardii ATCC MYA-796 (Sb MYA-796) only (Sb), and Sb MYA-796 plus sulfasalazine (Sb + S). The β-glucan content of Km A4 was significantly higher than that of Sb MYA-796 (P < 0.05). Body weight gain (BWG) significantly correlated with colon length, cyclooxygenase-2 (Cox-2) levels, and Bacteroides abundance (P < 0.05). In colitis-induced mice, the A4 + S group had the lowest histological score (6.00) compared to the DSS group (12.67), indicating the anti-inflammatory effects of this combination. The A4 + S group showed significantly downregulated expression of interleukin (Il)-6, tumor necrosis factor-α (Tnf-α), and Cox-2 and upregulated expression of Il-10 and occludin (Ocln) compared to the DSS group. Mice treated with A4 + S had enhanced Bacteroides abundance in their gut microbiota compared with the DSS group (P < 0.05). Bacteroides were significantly correlated with all colitis biomarkers (BWG, colon length, Il-6, Tnf-α, Il-10, Cox-2, and Ocln; P < 0.05). The anti-inflammatory effects of Km A4 could be attributed to high β-glucan content and gut microbiota modulation. Thus, treatment with Km A4 and sulfasalazine could alleviate UC.

Antinociceptive and anti-inflammatory properties of α-D-mannan from the yeast Kluyveromyces marxianus: evidence for a role in interleukin-6 inhibition.

The management of inflammatory states typically involves non-steroidal anti-inflammatory drugs (NSAIDs) and opiates. Understanding the mechanisms underlying the processing of nociceptive information from potential alternatives such as some polysaccharides may enable new and meaningful therapeutic approaches. In this study, α-D-mannan isolated from the Kluyveromyces marxianus cell wall produced antinociceptive effects in models of inflammatory pain (formalin and complete Freund's adjuvant tests). Furthermore, α-D-mannan reduced paw edema and interleukin-6 (IL-6) production after carrageenan-induced inflammation. The polysaccharide α-D-mannan was characterized by gas chromatography-mass spectrometry, methylation analysis, and spectroscopic techniques. Moreover, the Doehlert experimental design was applied to find the optimal conditions for biomass production, with the best conditions being 10.8g/L and 117h for the glucose concentration and the fermentation time, respectively. These results indicate that α-D-mannan from K. marxianus exerts anti-inflammatory and antinociceptive effects in mice, possibly via a mechanism dependent on the inhibition of IL-6 production.

Monitoring β-Fructofuranosidase Activity through Kluyveromyces marxianus in Bioreactor Using a Lab-Made Sequential Analysis System

The yeast Kluyveromyces marxianus has shown the potential to produce β-fructofuranosidases, which are enzymes capable of hydrolyzing β-fructofuranosides links of fructans to obtain fructooligosaccharides. The thriving market for fructose syrup and the quality standards imposed by food and pharmaceutical industries have generated an increased search for improved, monitored, and controlled production processes. Monitoring β-fructofuranosidase activity in a bioprocess requires the use of adequate sensors and the processing of information using efficient software algorithms; nevertheless, currently, such a sensor does not exist for this purpose. In this contribution, a sequential injection analysis system (SIA) developed in our laboratory was adapted to monitor at-line β-fructofuranosidase activity produced by the yeast K. marxianus. Samples were taken out automatically from the bioreactor and analyzed using 3,5-dinitrosalicylic (DNS). An algorithm was designed to operate the overall components of the lab-made SIA system. The enzymatic activity error obtained with the automatic SIA compared to the off-line laboratory determinations varied from 0.07% at high enzyme concentrations to 20.39% at low β-fructofuranosidase activity. Further development is required to improve the performance of the lab-made SIA system; nevertheless, such a device must be considered as a potential method for monitoring β-fructofuranosidase activity in real time.

Antifungal activities of leaf extracts from the Capsicum genus

The emergence of microorganisms resistant to antimicrobials has driven the search for new substances aimed at the control and prevention of pathogens. Plant antimicrobial peptides have biotechnological applications in the development of drugs and agricultural inputs for this purpose. The present work aims to evaluate the antifungal activity of extracts containing peptides obtained from the leaves of Capsicum plants. The accessions UENF 1626, 1632 and 1381 and the cultivar Carioquinha were used. The leaves were macerated, and then extraction was performed using a 60% methanol and dichloromethane solution in a 1:1 ratio. The hydromethanolic phase was separated and used for testing after evaporation of methanol. All extracts have a similar electrophoretic profile on tricine gel stained with Coomassie Blue, showing 2 bands of approximately 6.5 and 3.5 kDa. Inhibition of fungal growth with 24 h of culture was performed in liquid culture medium in 96-well plates with reading at 620 nm using extracts with a protein concentration of 200 mg mL-1. The filamentous fungi Fusarium solani, Fusarium lateritium, and Colletotrichum lindemuthianum and the yeasts Candida albicans, Candida buinensis, Candida tropicalis, Saccharomyces cerevisiae, Pichia membranifaciens and Kluyveromyces marxianus were used. The extract with the greatest antifungal potential was the leaves of Capsicum annuum cv. Carioquinha, inhibiting the growth of F. solani by 29%, C. lindemuthianum by 15%, Candida buinensis by 22% and K. marxianus by 24%. Therefore, the extract has peptides with biotechnological potential.

Transcriptomic analysis reveals hub genes and pathways in response to acetic acid stress in Kluyveromyces marxianus during high-temperature ethanol fermentation

The thermotolerant yeast Kluyveromyces marxianus is known for its potential in high-temperature ethanol fermentation, yet it suffers from excess acetic acid production at elevated temperatures, which hinders ethanol production. To better understand how the yeast responds to acetic acid stress during high-temperature ethanol fermentation, this study investigated its transcriptomic changes under this condition. RNA sequencing (RNA-seq) was used to identify differentially expressed genes (DEGs) and enriched gene ontology (GO) terms and pathways under acetic acid stress. The results showed that 611 genes were differentially expressed, and GO and pathway enrichment analysis revealed that acetic acid stress promoted protein catabolism but repressed protein synthesis during high-temperature fermentation. Protein–protein interaction (PPI) networks were also constructed based on the interactions between proteins coded by the DEGs. Hub genes and key modules in the PPI networks were identified, providing insight into the mechanisms of this yeast's response to acetic acid stress. The findings suggest that the decrease in ethanol production is caused by the imbalance between protein catabolism and protein synthesis. Overall, this study provides valuable insights into the mechanisms of K. marxianus's response to acetic acid stress and highlights the importance of maintaining a proper balance between protein catabolism and protein synthesis for high-temperature ethanol fermentation.

Co-Fermentation of Agri-Food Residues Using a Co-Culture of Yeasts as a New Bioprocess to Produce 2-Phenylethanol.

Whey is a dairy residue generated during the production of cheese and yogurt. Whey contains mainly lactose and proteins, contributing to its high chemical oxygen demand (COD). Current environmental regulations request proper whey disposal to avoid environmental pollution. Whey components can be transformed by yeast into ethanol and biomolecules with aroma and flavor properties, for example, 2-phenyethanol (2PE), highly appreciated in the industry due to its organoleptic and biocidal properties. The present study aimed to valorize agri-food residues in 2PE by developing suitable bioprocess. Cheese whey was used as substrate source, whereas crab headshells, residual soy cake, and brewer's spent yeast (BSY) were used as renewable nitrogen sources for the yeasts Kluyveromyces marxianus and Debaryomyces hansenii. The BSYs promoted the growth of both yeasts and the production of 2PE in flask fermentation. The bioprocess scale-up to 2 L bioreactor allowed for obtaining a 2PE productivity of 0.04 g2PE/L·h, twofold better productivity results compared to the literature. The bioprocess can save a treatment unit because the whey COD decreased under the detection limit of the analytical method, which is lower than environmental requirements. In this way, the bioprocess prevents environmental contamination and contributes to the circular economy of the dairy industry.

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 ± 0.48g/L from glucose. Yield and productivity of D-lactate, and glucose-consumption rate were 0.85 ± 0.01g/g, 0.90 ± 0.01g/(L·h), and 1.06 ± 0.00g/(L·h), respectively. Surprisingly, D-lactate titer, productivity, and glucose-consumption rate of 52.29 ± 0.68g/L, 1.38 ± 0.05g/(L·h), and 1.22 ± 0.00g/(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 ± 0.81g/L and 0.91 ± 0.01g/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 66g/L D-lactate at 30°C from molasses without any additional nutrients.

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 60.39mg/L by ITD0049 and ITD 0136 yeasts respectively.

Creation of microbial bio‐aroma wheel design according to smell attributes and multivariate exploratory methods

AbstractMicrobial bio‐aroma synthesized from biowaste, and its marketing is an emerging aspect of future research due to their benefits. However, the sensory lexicon of bio‐aroma has not yet been developed so far. This study aims to identify the smell characteristics of bio‐aroma produced by fermentative yeast Kluyveromyces marxianus by solid‐state fermentation, where the sample was extracted from fermented sugarcane bagasse (FSB), sweet lemon peel (FSLP), and pomegranate peel (FPP). A group of 49 respondents participated in this sensory analysis; the generated data reveal that a total of 29, 35, and 26 aroma essences gets from FSB, FSLP, and FPP, respectively. Multiple factor analysis was performed using XLSTAT 2022.1 version, and hierarchical clustering was used to classify the different cluster levels of aroma attributes. An aroma wheel is designed according to the attributes of classified aroma (smell). This bio‐aroma has prospects for application in food, pharmaceuticals, and cosmetic products.Practical ApplicationsThis fermented bio‐waste aroma lexicon will be helpful to academicians, researchers, flavorist and consumers to understand the precise description and basic terms. It also make them aware about a natural alternative source of flavor and aroma ingredients. It can be used as a sustainable additive for food, pharmaceutical, and cosmetic industries in the future.

Alternative secretory signal sequences for recombinant protein production in Pichia pastoris

Extracellular protein production is primarily preferred to facilitate the downstream processes in recombinant protein production. Secretion of recombinant proteins is mediated by the processing of signal peptides in their N-terminal portion by the secretory mechanism of host expression systems. These molecular elements involved in secretion are functionally interchangeable between different species and secretion sequence screening is one of the widely used approaches to improve extracellular protein production. In this study, α-mating and protein internal repeats (PIR) secretion sequences isolated from different yeasts (Kluyveromyces lactis, Kluyveromyces marxianus and Hansenula polymorpha) were tested in Pichia pastoris for the production of two different proteins (α-amylase and xylanase) and compared with the well-known secretory signals, S. cerevisiae α-mating factor (Sc-MF) and P. pastoris protein internal repeats PIR (PpPIR). The results obtained showed the potential of signal sequences tested. Among the tested peptides, the highest yields were achieved with H. polymorpha protein internal repeats (HpPIR) and K. lactis α-mating factor (Kl-MF) for xylanase and K. marxianus protein internal repeats (KmPIR) and K. lactis α-mating factor (Kl-MF) for amylase. In further studies, these sequences can be evaluated as alternatives in the production of different proteins in P. pastoris and in the production of recombinant proteins in different expression systems.

Ethanol production from Agave salmiana leaves by semi and simultaneous saccharification and fermentation at high temperature using Kluyveromycesmarxianus

Second-generation ethanol production by separate hydrolysis and fermentation (SHF) currently remains economically unviable, and thus more profitable alternatives are being sought. In the present study, the lignocellulosic biomass (LB) of Agave salmiana leaves was evaluated in the production of bioethanol through the processes of SHF, semi-simultaneous saccharification and fermentation (SSSF), and simultaneous saccharification and fermentation (SSF). The leaves of A. salmiana were subjected to acid pretreatment (1% H2SO4) and then to saccharification at temperatures of 40 and 50 °C. The processes of SHF, SSSF, and SSF were evaluated at 40 °C using the yeast Kluyveromyces marxianus OFF1. The results show that saccharification at 50 °C released up to 103.12 ± 1.07 g/L of reducing sugars, while at a temperature of 40 °C, the value was 78.41 ± 0.80 g/L, and thus the enzyme retained 76% of its activity at the latter temperature. In the fermentation processes, sugar consumption was up to 89% and ethanol yield up to 94%. The maximum ethanol production was recorded with the process of SSF, with concentrations of 51.5 g/L, which were significant and at least 13% higher compared to the other processes. The results show that the thermotolerance of K. marxianus OFF1 allows fermentation at 40 °C and a more efficient SSF process for transforming the sugars from this agro-industrial waste into ethanol.

High-Purity Bioactive Ingredient-3S,3'S-Astaxanthin: A New Preparation from Genetically Modified Kluyveromyces marxianus without Column Chromatography and Gel Filtration.

A highly efficient methodology for bioactive ingredient 3S,3'S-astaxanthin (3S,3'S-AST) preparation from genetically modified yeast (Kluyveromyces marxianus) with a combination of enzyme-assisted extraction and salt-assisted liquid-liquid extraction (SALLE) was achieved. The highest yield of 3S,3'S-AST indicated that FoodPro® CBL for yeast cell walls hydrolysis could significantly enhance extraction and obtain, with the help of SALLE procedure, quantified 3S,3'S-AST over 99% in purity through cation chelation. In the oxygen radical antioxidant capacity (ORAC) assay, the antioxidant capacity of high-purity 3S,3'S-AST products were 18.3 times higher than that of the original raw material extract. This new combination preparation may replace previous methods and has the potential to be scaled up in the manufacture of high-purity 3S,3'S-AST from low-value bioresources of raw materials to high-value products in the food and/or drug industries with lower cost and simple equipment.