Screening of broad-host expression promoters for shuttle expression vectors in non-conventional yeasts and bacteria.

BACKGROUND: Non-conventional yeasts and bacteria gain significance in synthetic biology for their unique metabolic capabilities in converting low-cost renewable feedstocks into valuable products. Improving metabolic pathways and increasing bioproduct yields remain dependent on the strategically use of various promoters in these microbes. The development of broad-spectrum promoter libraries with varying strengths for different hosts is attractive for biosynthetic engineers. RESULTS: In this study, five Yarrowia lipolytica constitutive promoters (yl.hp4d, yl.FBA1in, yl.TEF1, yl.TDH1, yl.EXP1) and five Kluyveromyces marxianus constitutive promoters (km.PDC1, km.FBA1, km.TEF1, km.TDH3, km.ENO1) were selected to construct promoter-reporter vectors, utilizing α-amylase and red fluorescent protein (RFP) as reporter genes. The promoters' strengths were systematically characterized across Y. lipolytica, K. marxianus, Pichia pastoris, Escherichia coli, and Corynebacterium glutamicum. We discovered that five K. marxianus promoters can all express genes in Y. lipolytica and that five Y. lipolytica promoters can all express genes in K. marxianus with variable expression strengths. Significantly, the yl.TEF1 and km.TEF1 yeast promoters exhibited their adaptability in P. pastoris, E. coli, and C. glutamicum. In yeast P. pastoris, the yl.TEF1 promoter exhibited substantial expression of both amylase and RFP. In bacteria E. coli and C. glutamicum, the eukaryotic km.TEF1 promoter demonstrated robust expression of RFP. Significantly, in E. coli, The RFP expression strength of the km.TEF1 promoter reached ∼20% of the T7 promoter. CONCLUSION: Non-conventional yeast promoters with diverse and cross-domain applicability have great potential for developing innovative and dynamic regulated systems that can effectively manage carbon flux and enhance target bioproduct synthesis across diverse microbial hosts.

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.

Non-carrier immobilization of yeast cells by genipin crosslinking for the synthesis of prebiotic galactooligosaccharides from plant-derived galactose.

Galactooligosaccharides (GOS), as mimics of human milk oligosaccharides, are important prebiotics for modulating the ecological balance of intestinal microbiota. A novel carrier-free cell immobilization method was established using genipin to cross-link Kluyveromyces lactis CGMCC 2.1494, which produced ß-galactosidase, an enzyme essential for GOS synthesis. The resulting immobilized cells were characterized as stable by thermogravimetric analysis and confirmed to be crosslinked through scanning electron microscopy analysis (SEM) and Fourier transform infrared spectroscopy (FTIR). The Km and Vmax values of ß-galactosidase in immobilized cells towards o-nitrophenyl ß-D-galactoside were determined to be 3.446 mM and 2210 µmol min-1 g-1, respectively. The enzyme in the immobilized showed higher thermal and organic solvent tolerance compared to that in free cells. The immobilized cells were subsequently employed for GOS synthesis using plant-derived galactose as the substrate. The synthetic reaction conditions were optimized through both single-factor experiments and response surface methodology, resulting in a high yield of 49.1 %. Moreover, the immobilized cells showed good reusability and could be reused for at least 20 batches of GOS synthesis, with the enzyme activity remaining above 70 % at 35 °C.

Exploring volatile compounds and microbial dynamics: Kluyveromyces marxianus and Hanseniaspora opuntiae reduce Forastero cocoa fermentation time.

Traditional cocoa bean fermentation is a spontaneous process and can result in heterogeneous sensory quality. For this reason, yeast-integrated starter cultures may be an option for creating consistent organoleptic profiles. This study proposes the mixture of Hanseniaspora opuntiae and Kluyveromyces marxianus (from non-cocoa fermentation) as starter culture candidates. The microorganisms and volatile compounds were analyzed during the cocoa fermentation process, and the most abundant were correlated with predominant microorganisms. Results showed that Kluyveromyces marxianus, isolated from mezcal fermentation, was identified as the dominant yeast by high-throughput DNA sequencing. A total of 63 volatile compounds identified by HS-SPME-GC-MS were correlated with the more abundant bacteria and yeast using Principal Component Analysis and Agglomerative Hierarchical Clustering. This study demonstrates that yeasts from other fermentative processes can be used as starter cultures in cocoa fermentation and lead to the formation of more aromatic esters, decrease the acetic acid content.

A convenient broad-host counterselectable system endowing rapid genetic manipulations of Kluyveromyces lactis and other yeast species.

Being generally regarded as safe, Kluyveromyces lactis has been widely taken for food, feed, and pharmaceutical applications, owing to its ability to achieve high levels of protein secretion and hence being suitable for industrial production of heterologous proteins. Production platform strains can be created through genetic engineering; while prototrophic cells without chromosomally accumulated antibiotics resistance genes have been generally preferred, arising the need for dominant counterselection. We report here the establishment of a convenient counterselection system based on a Frs2 variant, Frs2v, which is a mutant of the alpha-subunit of phenylalanyl-tRNA synthase capable of preferentially incorporating a toxic analog of phenylalanine, r-chloro-phenylalanine (4-CP), into proteins to bring about cell growth inhibition. We demonstrated that expression of Frs2v from an episomal plasmid in K. lactis could make the host cells sensitive to 2 mM 4-CP, and a Frs2v-expressing plasmid could be efficiently removed from the cells immediately after a single round of cell culturing in a 4-CP-contianing YPD medium. This Frs2v-based counterselection helped us attain scarless gene replacement in K. lactis without any prior engineering of the host cells. More importantly, counterselection with this system was proven to be functionally efficient also in Saccharomyces cerevisiae and Komagataella phaffii, suggestive of a broader application scope of the system in various yeast hosts. Collectively, this work has developed a strategy to enable rapid, convenient, and high-efficiency construction of prototrophic strains of K. lactis and possibly many other yeast species, and provided an important reference for establishing similar methods in other industrially important eukaryotic microbes.

CRISPR/Cas9-Based Genome Editing for Protein Expression and Secretion in Kluyveromyces lactis.

The budding yeast Kluyveromyces lactis has emerged as a promising microbial chassis in industrial biotechnology. However, a lack of efficient molecular genetic manipulation tools and strategies has hindered the development of K. lactis as a biomanufacturing platform. In this study, we developed and applied a CRISPR/Cas9-based genome editing method to K. lactis. Single-gene editing efficiency was increased to 80% by disrupting the nonhomologous end-joining-related gene KU80 and performing a series of process optimizations. Subsequently, the CRISPR/Cas9 system was explored based on different sgRNA delivery modes for simultaneous multigene editing. With the aid of the color indicator, the editing efficiencies of two and three genes reached 73.3 and 36%, respectively, in the KlΔKU80 strain. Furthermore, the CRISPR/Cas9 system was used for multisite integration to enhance lactase production and combinatorial knockout of TMED10 and HSP90 to characterize the extracellular secretion of lactase in K. lactis. Generally, genome editing is a powerful tool for constructing K. lactis cell factories for protein and chemical production.

Integrated biorefinery approach for utilization of wood waste into levulinic acid and 2-Phenylethanol production under mild treatment conditions.

In a bid to explore the on-site biorefinery approach for conversion of forestry residues, lignocellulosic biomass into value-added products was studied. The bark white pine wood was subjected to the microwave technique of fast and slow hydrolysis under varying acid and biomass concentrations to produce levulinic acid (LA). The HCl (2% v/v) and plant biomass (1% w/v) were identified as the optimum conditions for fast wood hydrolysis (270 ºC for 12 sec), which led to maximum LA yield of 446.68 g/kgPB. The proposed sustainable approach is mild, quick, and utilized a very low concentration of the HCl for the production of LA. The hydrolysate was used as a medium for Kluyveromyces marxianus growth to produce 2-phenylethanol (2-PE). K. marxianus used 74-95% of furfural from hydrolysate as a co-substrate to grow. The proposed model of the integrated biorefinery is an affordable on-site approach of using forest waste into localized solutions to produce LA and 2-PE.

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.

Optimizing bioreactor conditions for Spirulina fermentation by Lactobacillus helveticus and Kluyveromyces marxianus: Impact on chemical & bioactive properties.

This study focused on optimizing the production of fermented Spirulina (FS) products using a bioactivity-guided strategy with Lactobacillus helveticus B-4526 and Kluyveromyces marxianus Y-329 in a 3-L bioreactor. Various operating conditions, including aeration rates and pH modes, were tested. While both microorganisms thrived under all conditions, the "cascade" mode, controlling dissolved oxygen, enhanced protein hydrolysis and antioxidant activity, as confirmed by SDS-PAGE and DPPH/TEAC assays, respectively. Screening revealed that "cascade" FS significantly decreased viability of colon cancer cells (HT-29) in a dose-dependent manner, with up to a 72 % reduction. Doses ≤ 500 µg mL-1 of "cascade" FS proved safe and effective in suppressing NO release without compromising cellular viability. Additionally, "cascade" FS exhibited diverse volatile organic compounds and reducing the characteristic "seaweed" aroma. These findings highlight "cascade" FS as a promising alternative food source with improved bioactive properties, urging further exploration of its bioactive compounds, particularly bioactive peptides.

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.

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.

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.

Fermentation of whey-derived matrices by Kluyveromyces marxianus: alcoholic beverage development from whey and fruit juice mixes.

This research paper addresses the hypotheses that Kluyveromyces marxianus can be cultured with good alcohol production on different whey-derived matrices, and that the fermented product can be used in order to develop alcoholic beverages with acceptable sensory characteristics by mixtures with yeast-fermented fruit-based matrices. Growth and fermentative characteristics of Kluyveromyces marxianus LFIQK1 in different whey-derived matrices were explored by culturing (24 h, 30°C) on reconstituted whey, demineralized whey, heat-treated whey and milk permeate media. High lactose consumption, ethanol production and yield were observed. Reconstituted whey matrix was selected for mixing with orange or strawberry juices fermented using Saccharomyces cerevisiae to obtain alcoholic beverages (W-OR and W-ST, respectively). Consumer evaluation of beverages was performed using acceptability and Check-All-That-Apply (CATA) questions. Good acceptance was observed, significantly higher for W-ST than for W-OR. CATA questions gave information about organoleptic characteristics of beverages. Penalty analysis showed W-R and W-ST were positively associated with smooth/refreshing and fruity/natural, respectively. Liking was represented, accordingly with penalty analysis, by natural/refreshing. A novel alternative for utilization of whey and whey-related matrices by alcoholic beverages production with natural ingredients is presented.

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.

Optimal Extraction and Deproteinization Method for Mannoprotein Purification from Kluyveromyces marxianus.

Background: Mannoproteins, mannose-glycosylated proteins, play an important role in biological processes and have various applications in industries. Several methods have been already used for the extraction of mannoproteins from yeast cell-wall. The aim of this study was to evaluate the extraction and deproteinization of mannan oligosaccharide from the Kluyveromyces (K.) marxianus mannoprotein. Methods: To acquire crude mannan oligosaccharides, K. marxianus mannoproteins were deproteinized by the Sevage, trichloroacetic acid, and hydrochloric acid (HCL) methods. Total nitrogen, crude protein content, fat, carbohydrate and ash content were measured according to the monograph prepared by the meeting of the Joint FAO/WHO Expert Committee and standard. Mannan oligosaccharide loss, percentage of deproteinization, and chemical composition of the product were assessed to check the proficiency of different methods. Results: Highly purified (95.4%) mannan oligosaccharide with the highest deproteinization (97.33 ± 0.4%) and mannan oligosaccharide loss (25.1 ± 0.6%) were obtained following HCl method. Conclusion: HCl, was the most appropriate deproteinization method for the removal of impurities. This preliminary data will support future studies to design scale-up procedures.

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 ± 0.48 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.