Hyaluronic Acid (HA) is a biopolymer composed by the monomers Glucuronic Acid (GlcUA) and N-Acetyl Glucosamine (GlcNAc). It has a broad range of applications in the field of medicine, being marketed between USD 1000-5000/kg. Its primary sources include extraction of animal tissue and fermentation using pathogenic bacteria. However, in both cases, extensive purification protocols are required to prevent toxin contamination. In this study, aiming at creating a safe HA producing microorganism, the generally regarded as safe (GRAS) yeast Kluyveroymyces lactis is utilized. Initially, the hasB (UDP-Glucose dehydrogenase) gene from Xenopus laevis (xlhasB) is inserted. After that, four strains are constructed harboring different hasA (HA Synthase) genes, three of humans (hshasA1, hshasA2, and hshasA3) and one with the bacteria Pasteurella multocida (pmhasA). Transcript values analysis confirms the presence of hasA genes only in three strains. HA production is verified by scanning electron microscopy in the strain containing the pmHAS isoform. The pmHAS strain is grown in a 1.3 l bioreactor operating in a batch mode, the maximum HA levels are 1.89 g/L with a molecular weight of 2.097 MDa. This is the first study that reports HA production in K. lactis and it has the highest HA titers reported among yeast.

Kluyveromyces marxianus is a non-conventional yeast whose physiology and metabolism lends itself to diverse biotechnological applications. While the wild-type yeast is already in use for producing fragrances and fermented products, the lack of standardised tools for its genetic and metabolic engineering prevent it from being used as a next-generation cell factory for bio-based chemicals. In this paper, we bring together and characterise a set of native K. marxianus parts for the expression of multiple genes for metabolic engineering and synthetic biology. All parts are cloned and stored according to the MoClo/Yeast Tool Kit standard for quick sharing and rapid construction. Using available genomic and transcriptomic data, we have selected promoters and terminators to fine-tune constitutive and inducible gene expression. The collection includes a number of known centromeres and autonomously replication sequences (ARS). We also provide a number of chromosomal integration sites selected for efficiency or visible phenotypes for rapid screening. Finally, we provide a single-plasmid CRISPR/Cas9 platform for genome engineering and facilitated gene targeting, and rationally create auxotrophic strains to expand the common range of selection markers available to K. marxianus. The curated and characterised tools we have provided in this kit will serve as a base to efficiently build next-generation cell factories from this alternative yeast. Plasmids containing all parts are available at Addgene for public distribution.

Spermidine is a polyamine molecule that performs various cellular functions, such as DNA and RNA stabilization, autophagy modulation, and eIF5A formation, and is generated from putrescine by aminopropyltransferase spermidine synthase (SpdS). During synthesis, the aminopropyl moiety is donated from decarboxylated S-adenosylmethionine to form putrescine, with 5'-deoxy-5'-methylthioadenosine being produced as a byproduct. Although the molecular mechanism of SpdS function has been well-established, its structure-based evolutionary relationships remain to be fully understood. Moreover, only a few structural studies have been conducted on SpdS from fungal species. Here, we determined the crystal structure of an apo-form of SpdS from Kluyveromyces lactis (KlSpdS) at 1.9 Å resolution. Structural comparison with its homologs revealed a conformational change in the α6 helix linked to the gate-keeping loop, with approximately 40° outward rotation. This change caused the catalytic residue Asp170 to move outward, possibly due to the absence of a ligand in the active site. These findings improve our understanding of the structural diversity of SpdS and provide a missing link that expands our knowledge of the structural features of SpdS in fungal species.

In yeasts, proteins of the Major Superfamily Transporter selectively bind and allow the uptake of sugars to permit growth on varied substrates. The genome of brewer's yeast, Saccharomyces cerevisiae, encodes multiple hexose transporters (Hxt) to transport glucose and other MFS proteins for maltose, galactose, and other monomers. For sugar uptake, the dairy yeast, Kluyveromyces lactis, uses Rag1p for glucose, Hgt1 for glucose and galactose, and Lac12 for lactose. In the related industrial species Kluyveromyces marxianus, there are four genes encoding Lac12-like proteins but only one of them, Lac12, can transport lactose. In this study, which initiated with efforts to investigate possible functions encoded by the additional LAC12 genes in K. marxianus, a genome-wide survey of putative MFS sugar transporters was performed. Unexpectedly, it was found that the KHT and the HGT genes are present as tandem arrays of five to six copies, with the precise number varying between isolates. Heterologous expression of individual genes in S. cerevisiae and mutagenesis of single and multiple genes in K. marxianus was performed to establish possible substrates for these transporters. The focus was on the sugar galactose since it was already reported in K. lactis that this hexose was a substrate for both Lac12 and Hgt1. It emerged that three of the four copies of Lac12, four Hgt-like proteins and one Kht-like protein have some capacity to transport galactose when expressed in S. cerevisiae and inactivation of all eight genes was required to completely abolish galactose uptake in K. marxianus. Analysis of the amino acid sequence of all known yeast galactose transporters failed to identify common residues that explain the selectivity for galactose. Instead, the capacity to transport galactose has arisen three different times in K. marxianus via polymorphisms in proteins that are probably ancestral glucose transporters. Although, this is analogous to S. cerevisiae, in which Gal2 is related to glucose transporters, there are not conserved amino acid changes, either with Gal2, or among the K. marxianus galactose transporters. The data highlight how gene duplication and functional diversification has provided K. marxianus with versatile capacity to utilise sugars for growth.

Even before having its genome sequence published in 2004, Kluyveromyces lactis had long been considered a model organism for studies in genetics and physiology. Research on Kluyveromyces lactis is quite advanced and this yeast species is one of the few with which it is possible to perform formal genetic analysis. Nevertheless, until now, no complete metabolic functional annotation has been performed to the proteins encoded in the Kluyveromyces lactis genome.

Glucose phosphorylating enzymes are crucial in the regulation of basic cellular processes, including metabolism and gene expression. Glucokinases and hexokinases provide a pool of phosphorylated glucose in an adenosine diphosphate (ADP)- and ATP-dependent manner to shape the cell metabolism. The glucose processing enzymes from Kluyveromyces lactis are poorly characterized despite the emerging contribution of this yeast strain to industrial and laboratory scale biotechnology. The first reports on K. lactis glucokinase (KlGlk1) positioned the enzyme as an essential component required for glucose signaling. Nevertheless, no biochemical and structural information was available until now. Here, we present the first crystal structure of KlGlk1 together with biochemical characterization, including substrate specificity and enzyme kinetics. Additionally, comparative analysis of the presented structure and the prior structures of lactis hexokinase (KlHxk1) demonstrates the potential transitions between open and closed enzyme conformations upon ligand binding.

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.

Kluyveromyces marxianus is a thermotolerant, crabtree-negative yeast, which preferentially directs metabolism (e.g., from the tricarboxylic acid cycle) to aerobic alcoholic fermentation. Thus K. marxianus has great potential for engineering to produce various materials under aerobic cultivation conditions. In this study, we engineered K. marxianus to produce and secrete a single-chain antibody (scFv), a product that is highly valuable but has historically proven difficult to generate at large scale. scFv production was obtained with strains carrying either plasmid-borne or genomically integrated constructs using various combinations of promoters (P MDH1 or P ACO1 ) and secretion signal peptides (KmINUss or Scα-MFss). As the wild-type K. marxianus secretes endogenous inulinase predominantly, the corresponding INU1 gene was disrupted using a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein (CRISPR-Cas9) system to re-direct resources to scFv production. Genomic integration was used to replace INU1 with sequences encoding a fusion of the INU1 signal peptide to scFv; the resulting construct yielded the highest scFv production among the strains tested. Optimization of growth conditions revealed that scFv production by this strain was enhanced by incubation at 30 °C in xylose medium containing 200 mM MgSO4. These results together demonstrate that K. marxianus has the potential to serve as a host strain for antibody production.

Eukaryotes, including the unicellular eukaryotes such as yeasts, employ multiple levels of gene regulation. Regulation of chromatin structure through chromatin compaction cascades, and influenced by transcriptional insulators, might play a role in the coordinated regulation of genes situated at adjacent loci and expressed as a co-regulated cluster. Subtelomeric gene silencing, which has previously been described in the yeast Saccharomyces cerevisiae, is an example of this phenomenon. Transcription from a common regulatory element located around a shared intergenic region is another factor that could coordinate the transcription of genes at adjacent loci. Additionally, the presence of DNA binding sites for the same transcription factor may coordinate expression of multiple genes. Yeasts such as the industrially important Kluyveromyces marxianus may also display these modes of regulation, but this has not been explored to date. An exploration was done using a complete genome and RNA-seq data from a previous study of the transcriptional response to glucose or xylose as the carbon source in a defined culture medium, and investigating whether the species displays clusters of co-localised differentially expressed genes. Regions of possible subtelomeric silencing were evident, but were non-responsive to the carbon sources tested here. Additionally, glucose or xylose responsive clusters were discovered far from telomeres which contained some of the most significantly differentially expressed genes, encoding enzymes involved in the utilisation of alternative carbon sources such as the industrially important inulinase gene INU1. These clusters contained putative binding sites for the carbon source responsive transcription factors Mig1 and Adr1. Additionally, we investigated the potential contribution of common intergenic regions in co-regulation. Some observations were also made in terms of the evolutionary conservation of these clusters among yeast species and the presence of potential transcriptional insulators at the periphery of these clusters.

Several micro-scale chromatography-based procedures for purification of the beta-galactosidase from the yeast Kluyveromyces lactis were assayed. Purified enzyme was suitable to be used as antigen to induce polyclonal antibodies production. Specific staining of non-denaturing PAGE gels with chromogenic substrates allowed the determination of the number of subunits forming the native enzyme.

The yeast Kluyveromyces marxianus SLP1 has the potential for application in biotechnological processes because it can metabolize several sugars and produce high-value metabolites. K. marxianus SLP1 is a thermotolerant yeast isolated from the mezcal process, and it is tolerant to several cell growth inhibitors such as saponins, furan aldehydes, weak acids, and phenolics compounds. The genomic differences between dairy and nondairy strains related to K. marxianus variability are a focus of research attention, particularly the pathways leading this species toward polyploidy. We report the diploid genome assembly of K. marxianus SLP1 nonlactide strain into 32 contigs to reach a size of ∼12 Mb (N50 = 1.3 Mb) and a ∼39% GC content. Genome size is consistent with the k-mer frequency results. Genome annotation by Funannotate estimated 5000 genes in haplotype A and 4910 in haplotype B. The enriched annotated genes by ontology show differences between alleles in biological processes and cellular component. The analysis of variants related to DMKU3 and between haplotypes shows changes in LAC12 and INU1, which we hypothesize can impact carbon source performance. This report presents the first polyploid K. marxianus strain recovered from nonlactic fermenting medium.

Kluyveromyces marxianus combines the ease of genetic manipulation and fermentation with the ability to efficiently secrete high molecular weight proteins, performing eukaryotic post-translational modifications. It is able to grow efficiently in a wide range of temperatures. The secretion performances were analyzed in the host K. marxianus L3 in the range between 5°C and 40°C by means of 3 different reporter proteins, since temperature appears a key parameter for production and secretion of recombinant proteins.

Illegitimate recombination (IR) is the process by which two DNA molecules not sharing homology to each other are joined. In Kluyveromyces lactis, integration of heterologous DNA occurred very frequently therefore constituting an excellent model organism to study IR. IR was completely dependent on the nonhomologous end-joining (NHEJ) pathway for DNA double strand break (DSB) repair and we detected no other pathways capable of mediating IR. NHEJ was very versatile, capable of repairing both blunt and non-complementary ends efficiently. Mapping the locations of genomic IR-events revealed target site preferences, in which intergenic regions (IGRs) and ribosomal DNA were overrepresented six-fold compared to open reading frames (ORFs). The IGR-events occurred predominantly within transcriptional regulatory regions. In a rad52 mutant strain IR still preferentially occurred at IGRs, indicating that DSBs in ORFs were not primarily repaired by homologous recombination (HR). Introduction of ectopic DSBs resulted in the efficient targeting of IR to these sites, strongly suggesting that IR occurred at spontaneous mitotic DSBs. The targeting efficiency was equal when ectopic breaks were introduced in an ORF or an IGR. We propose that spontaneous DSBs arise more frequently in transcriptional regulatory regions and in rDNA and such DSBs can be mapped by analyzing IR target sites.

L-ascorbic acid (L-AA) is naturally synthesized in plants from D-glucose by 10 steps pathway. The pathway branch to synthesize L-galactose, the key intermediate for L-ascorbic acid biosynthesis, has been recently elucidated. Budding yeast produces an 5-carbon ascorbic acid analogue Dehydro-D-arabinono 1,4-lactone (D-DAL), which is synthesized from D-arabinose. Yeast is able to synthesize L-ascorbic acid only if it is cultivated in the presence of one of its precursors: L-galactose, L-galactono 1,4-lactone, or L-gulono 1,4-lactone extracted from plants or animals. To avoid feeding the yeast culture with this "L" enantiomer, we engineered Kluyveromyces lactis with L-galactose biosynthesis pathway genes: GDP-mannose 3,5-epimerase (GME), GDP-L-galactose phosphorylase (VTC2) and L-galactose-1-phosphate phosphatase (VTC4) isolated from Arabidopsis thaliana.

Kluyveromyces marxianus is a yeast that could be identified from kefir and can use a broad range of substrates, such as glucose and lactate, as carbon sources. The lactate produced in kefir culture can be a substrate for K. marxianus. However, the complexity of the kefir microbiota makes the traits of K. marxianus difficult to study. In this research, we focused on K. marxianus cultured with lactate as the sole carbon source. The optimal growth and released protein in lactate culture were determined under different pH conditions, and the LC-MS/MS-identified proteins were associated with the tricarboxylic acid cycle, glycolysis pathway, and cellular stress responses in cells, indicating that autolysis of K. marxianus had occurred under the culture conditions. The abundant glyceraldehyde-3-phosphate dehydrogenase 1 (GAP1) was cocrystallized with other proteins in the cell-free fraction, and the low transcription level of the GAP1 gene indicated that the protein abundance under autolysis conditions was dependent on protein stability. These results suggest that lactate induces the growth and autolysis of K. marxianus, releasing proteins and peptides. These findings can be fundamental for K. marxianus probiotic and kefir studies in the future.

Kluyveromyces lactis killer strains secrete a zymocin complex that inhibits proliferation of sensitive yeast genera including Saccharomyces cerevisiae. In search of the putative toxin target (TOT), we used mTn3:: tagging to isolate zymocin-resistant tot mutants from budding yeast. Of these we identified the TOT1, TOT2 and TOT3 genes (isoallelic with ELP1, ELP2 and ELP3, respectively) coding for the histone acetyltransferase (HAT)-associated Elongator complex of RNA polymerase II holoenzyme. Other than the typical elp ts-phenotype, tot phenocopies hypersensitivity towards caffeine and Calcofluor White as well as slow growth and a G(1) cell cycle delay. In addition, TOT4 and TOT5 (isoallelic with KTI12 and IKI1, respectively) code for components that associate with ELONGATOR: Intriguingly, strains lacking non-Elongator HATs (gcn5, hat1, hpa3 and sas3) or non-Elongator transcription elongation factors TFIIS (dst1) and Spt4p (spt4) cannot confer resistance towards the K.lactis zymocin, thus providing evidence that Elongator equals TOT and that Elongator plays an important role in signalling toxicity of the K.lactis zymocin.

Kluyveromyces marxianus is traditionally associated with fermented dairy products, but can also be isolated from diverse non-dairy environments. Because of thermotolerance, rapid growth and other traits, many different strains are being developed for food and industrial applications but there is, as yet, little understanding of the genetic diversity or population genetics of this species. K. marxianus shows a high level of phenotypic variation but the only phenotype that has been clearly linked to a genetic polymorphism is lactose utilisation, which is controlled by variation in the LAC12 gene. The genomes of several strains have been sequenced in recent years and, in this study, we sequenced a further nine strains from different origins. Analysis of the Single Nucleotide Polymorphisms (SNPs) in 14 strains was carried out to examine genome structure and genetic diversity. SNP diversity in K. marxianus is relatively high, with up to 3% DNA sequence divergence between alleles. It was found that the isolates include haploid, diploid, and triploid strains, as shown by both SNP analysis and flow cytometry. Diploids and triploids contain long genomic tracts showing loss of heterozygosity (LOH). All six isolates from dairy environments were diploid or triploid, whereas 6 out 7 isolates from non-dairy environment were haploid. This also correlated with the presence of functional LAC12 alleles only in dairy haplotypes. The diploids were hybrids between a non-dairy and a dairy haplotype, whereas triploids included three copies of a dairy haplotype.

Kluyveromyces marxianus is a thermotolerant yeast with multiple biotechnological potentials for industrial applications, which can metabolize a broad range of carbon sources, including less conventional sugars like lactose, xylose, arabinose and inulin. These phenotypic traits are sustained even up to 45 °C, what makes it a relevant candidate for industrial biotechnology applications, such as ethanol production. It is therefore of much interest to get more insight into the metabolism of this yeast. Recent studies suggested, that thermotolerance is achieved by reducing the number of growth-determining proteins or suppressing oxidative phosphorylation. Here we aimed to find related factors contributing to the thermotolerance of K. marxianus.

Various carbon sources were evaluated for production of inulinase by yeast, Kluyveromyces marxianus MTCC 3995. Highest inulinase activity was observed with Dahlia extract (25.3 nkat mL(-1)) as carbon source. The enzyme activity was 1.4 folds higher than that observed in media containing pure chicory inulin (17.8 nkat mL(-1)). The yeast showed good growth on a simple medium containing dahlia extract (20% w/v) and yeast extract (2%w/v) as carbon and nitrogen source respectively, in 96 h. at 28°C and 120 rpm. Lowest inulinase yield (4.8 nkat mL(-1)) was seen in the medium containing glucose as C-source. Although varied inulinase levels were noticed on different C- sources, Inulinase: Sucrase (I/S) ratios were noticed to be similar. Among various protein sources tested, yeast extract was found to be the best source followed by beef extract (17.9 nkat mL(-1)) and peptone (13.8 nkat mL(-1)). The enzyme was optimally active at pH (4.0) and 50°C. TLC analysis of end product revealed that inulinase hydrolyzed inulin exclusively into fructose. Results suggest that the dahlia extract induced exoinulinase synthesis in Kluyveromyces marxianus and can be utilized as a potential substrate for inulinase production.

Throughout history, the yeast Saccharomyces cerevisiae has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However, S. cerevisiae has proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineered the thermotolerant yeast Kluyveromyces marxianus to create a new synthetic biology platform. Using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats with Cas9)-mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traits-thermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.IMPORTANCE The yeast Kluyveromyces marxianus grows at high temperatures and on a wide range of carbon sources, making it a promising host for industrial biotechnology to produce renewable chemicals from plant biomass feedstocks. However, major genetic engineering limitations have kept this yeast from replacing the commonly used yeast Saccharomyces cerevisiae in industrial applications. Here, we describe genetic tools for genome editing and breeding K. marxianus strains, which we use to create a new thermotolerant strain with promising fatty acid production. These results open the door to using K. marxianus as a versatile synthetic biology platform organism for industrial applications.