Thermotolerant Yeast Kluyveromyces Marxianus Research Articles

SUMO targeting of a stress-tolerant Ulp1 SUMO protease.

SUMO proteases of the SENP/Ulp family are master regulators of both sumoylation and desumoylation and regulate SUMO homeostasis in eukaryotic cells. SUMO conjugates rapidly increase in response to cellular stress, including nutrient starvation, hypoxia, osmotic stress, DNA damage, heat shock, and other proteotoxic stressors. Nevertheless, little is known about the regulation and targeting of SUMO proteases during stress. To this end we have undertaken a detailed comparison of the SUMO-binding activity of the budding yeast protein Ulp1 (ScUlp1) and its ortholog in the thermotolerant yeast Kluyveromyces marxianus, KmUlp1. We find that the catalytic UD domains of both ScUlp1 and KmUlp1 show a high degree of sequence conservation, complement a ulp1Δ mutant in vivo, and process a SUMO precursor in vitro. Next, to compare the SUMO-trapping features of both SUMO proteases we produced catalytically inactive recombinant fragments of the UD domains of ScUlp1 and KmUlp1, termed ScUTAG and KmUTAG respectively. Both ScUTAG and KmUTAG were able to efficiently bind a variety of purified SUMO isoforms and bound immobilized SUMO1 with nanomolar affinity. However, KmUTAG showed a greatly enhanced ability to bind SUMO and SUMO-modified proteins in the presence of oxidative, temperature and other stressors that induce protein misfolding. We also investigated whether a SUMO-interacting motif (SIM) in the UD domain of KmULP1 that is not conserved in ScUlp1 may contribute to the SUMO-binding properties of KmUTAG. In summary, our data reveal important details about how SUMO proteases target and bind their sumoylated substrates, especially under stress conditions. We also show that the robust pan-SUMO binding features of KmUTAG can be exploited to detect and study SUMO-modified proteins in cell culture systems.

Thermotolerant Yeast Kluyveromyces marxianus Reveals More Tolerance to Heat Shock than the Brewery Yeast Saccharomyces cerevisiae.

The thermotolerant yeast Kluyveromyces marxianus, growing at high temperature (45℃) , showed stronger survival under heat shock at 50℃ than the brewing yeast Saccharomyces cerevisiae, which was unable to grow at 45℃. The survival rate of K. marxianus decreased to 10% during heat shock at 50℃ for 20 min, and to less than 0.01% at 60℃ for 20 min. Cells with damaged cellular membranes were infrequently observed at 50℃ and had decreased significantly from heat shock at 60℃. The metabolic activity of K. marxianus was retained at 50℃, whereas that of S. cerevisiae was not. The trehalose content of K. marxianus was approximately two times that of S. cerevisiae. These results suggest that K. marxianus protects itself from heat shock-induced damage through the use of trehalose (a protective molecule in S. cerevisiae) as well as other different factors.

Evolutionary Adaptation of Kluyveromyces marxianus NIRE-K3 for Enhanced Xylose Utilization

The evolutionary adaptation was approached on the thermotolerant yeast Kluyveromyces marxianus NIRE-K3 at 45oC on xylose as a sole source of carbon for enhancement of xylose uptake. After 60 cycles, evolved strain K. marxianus NIRE-K3.1 showed comparatively 3.75-fold and 3.0-fold higher specific growth and xylose uptake rates, respectively than that of native strain. Moreover, the short lag phase was also observed on adapted strain. During batch fermentation with xylose concentration of 30 g l-1, K. marxianus NIRE-K3.1 could utilize about 96 % of xylose in 72 h and produced 4.67 and 15.7 g l-1 of ethanol and xylitol, respectively, which were 9.72-fold and 4.63-fold higher than that of native strain. Similarly, specific sugar consumption rate, xylitol and ethanol yields were 5.07-fold, 1.15-fold and 2.44-fold higher as compared to the native strain, respectively. The results obtained after evolutionary adaptation of K. marxianus NIRE-K3 show the significant improvement in the xylose utilization, ethanol and xylitol yields, and productivities. By understanding the results obtained, the significance of evolutionary adaptation has been rationalized, since the adapted culture could be more stable and could enhance the productivity.

Transcriptome analysis of the thermotolerant yeast Kluyveromyces marxianus CCT 7735 under ethanol stress.

The thermotolerant yeast Kluyveromyces marxianus displays a potential to be used for ethanol production from both whey and lignocellulosic biomass at elevated temperatures, which is highly alluring to reduce the cost of the bioprocess. Nevertheless, contrary to Saccharomyces cerevisiae, K. marxianus cannot tolerate high ethanol concentrations. We report the transcriptional profile alterations in K. marxianus under ethanol stress in order to gain insights about mechanisms involved with ethanol response. Time-dependent changes have been characterized under the exposure of 6% ethanol and compared with the unstressed cells prior to the ethanol addition. Our results reveal that the metabolic flow through the central metabolic pathways is impaired under the applied ethanol stress. Consistent with these results, we also observe that genes involved with ribosome biogenesis are downregulated and gene-encoding heat shock proteins are upregulated. Remarkably, the expression of some gene-encoding enzymes related to unsaturated fatty acid and ergosterol biosynthesis decreases upon ethanol exposure, and free fatty acid and ergosterol measurements demonstrate that their content in K. marxianus does not change under this stress. These results are in contrast to the increase previously reported with S. cerevisiae subjected to ethanol stress and suggest that the restructuration of K. marxianus membrane composition differs in the two yeasts which gives important clues to understand the low ethanol tolerance of K. marxianus compared to S. cerevisiae.

Rational design of Kluyveromyces marxianus ZJB14056 aldo-keto reductase KmAKR to enhance diastereoselectivity and activity.

t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-1b) is a valuable chiral synthon of atorvastatin calcium. A novel NADPH-specific aldo-keto reductase (AKR) was identified from a thermotolerant yeast Kluyveromyces marxianus ZJB14056 by genome database mining, displaying t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1a) reducing activity and moderate diastereoselectivity (dep∼80.5%). Molecular homology modeling and docking studies demonstrated that the side chain of Trp297 blocks binding of (5R)-1a to KmAKR. The mutation of Trp297 to His led to dramatic conformational changes and significant improvement in both diastereoselectivity and activity. In comparison with KmAKR, KmAKR-W297H displayed strict diastereoselectivity, and 2.8-fold, 3.9-fold improvement in kcat and kcat/Km toward (5R)-1a, which were 10.36s-1 and 6.56s-1·mM-1 respectively. Coupling KmAKR-W297H with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for coenzyme regeneration, 100mM (5R)-1a was completely reduced to (3R,5R)-1b within 12h, in a dep >99.5%.

CRISPR\u2013Cas9-enabled genetic disruptions for understanding ethanol and ethyl acetate biosynthesis in Kluyveromyces marxianus

BackgroundThe thermotolerant yeast Kluyveromyces marxianus shows promise as an industrial host for the biochemical production of fuels and chemicals. Wild-type strains are known to ferment high titers of ethanol and can effectively convert a wide range of C5, C6, and C12 sugars into the volatile short-chain ester ethyl acetate. Strain engineering, however, has been limited due to a lack of advanced genome-editing tools and an incomplete understanding of ester and ethanol biosynthesis.ResultsEnabled by the design of hybrid RNA polymerase III promoters, this work adapts the CRISPR–Cas9 system from Streptococcus pyogenes for use in K. marxianus. The system was used to rapidly create functional disruptions to alcohol dehydrogenase (ADH) and alcohol-O-acetyltransferase (ATF) genes with putative function in ethyl acetate and ethanol biosynthesis. Screening of the KmATF disrupted strain revealed that Atf activity contributes to ethyl acetate biosynthesis, but the knockout reduced ethyl acetate titers by only ~15%. Overexpression experiments revealed that KmAdh7 can catalyze the oxidation of hemiacetal to ethyl acetate. Finally, analysis of the KmADH2 disrupted strain showed that the knockout almost completely eliminated ethanol production and resulted in the accumulation of acetaldehyde.ConclusionsNewly designed RNA polymerase III promoters for sgRNA expression in K. marxianus enable a CRISPR–Cas9 genome-editing system for the thermotolerant yeast. This system was used to disrupt genes involved in ethyl acetate biosynthesis, specifically KmADH1–7 and KmATF. KmAdh2 was found to be critical for aerobic and anaerobic ethanol production. Aerobically produced ethanol supplies the biosynthesis of ethyl acetate catalyzed by KmAtf. KmAdh7 was found to exhibit activity toward the oxidation of hemiacetal, a possible alternative route for the synthesis of ethyl acetate.

Batch and fed-batch simultaneous saccharification and fermentation of primary sludge from pulp and paper mills

ABSTRACTPrimary sludge from a Portuguese pulp and paper mill, containing 60% of carbohydrates, and unbleached pulp (as reference material), with 93% of carbohydrates, were used to produce ethanol by simultaneous saccharification and fermentation (SSF). SSF was performed in batch or fed-batch conditions without the need of a pretreatment. Cellic® CTec2 was the cellulolytic enzymatic complex used and Saccharomyces cerevisiae (baker's yeast or ATCC 26602 strain) or the thermotolerant yeast Kluyveromyces marxianus NCYC 1426 were employed. Primary sludge was successfully converted to ethanol and the best results in SSF efficiency were obtained with S. cerevisiae. An ethanol concentration of 22.7 g L−1 was produced using a content of 50 g L−1 of carbohydrates from primary sludge, in batch conditions, with a global conversion yield of 81% and a production rate of 0.94 g L−1 h−1. Fed-batch operation enabled higher solids content (total carbohydrate concentration of 200 g L−1, equivalent to a consistency of 33%) and a reduction of three-quarters of cellulolytic enzyme load, leading to an ethanol concentration of 40.7 g L−1, although with lower yield and productivity. Xylitol with a concentration up to 7 g L−1 was also identified as by-product in the primary sludge bioconversion process.

Kluyveromyces marxianus as a host for heterologous protein synthesis.

The preferentially respiring and thermotolerant yeast Kluyveromyces marxianus is an emerging host for heterologous protein synthesis, surpassing the traditional preferentially fermenting yeast Saccharomyces cerevisiae in some important aspects: K . marxianus can grow at temperatures 10°C higher than S. cerevisiae, which may result in decreased costs for cooling bioreactors and reduced contamination risk; has ability to metabolize a wider variety of sugars, such as lactose and xylose; is the fastest growing eukaryote described so far; and does not require special cultivation techniques (such as fed-batch) to avoid fermentative metabolism. All these advantages exist together with a high secretory capacity, performance of eukaryotic post-translational modifications, and with a generally regarded as safe (GRAS) status. In the last years, replication origins from several Kluyveromyces spp. have been used for the construction of episomal vectors, and also integrative strategies have been developed based on the tendency for non-homologous recombination displayed by K. marxianus. The recessive URA3 auxotrophic marker and the dominant Kan(R) are mostly used for selection of transformed cells, but other markers have been made available. Homologous and heterologous promoters and secretion signals have been characterized, with the K. marxianus INU1 expression and secretion system being of remarkable functionality. The efficient synthesis of roughly 50 heterologous proteins has been demonstrated, including one thermophilic enzyme. In this mini-review, we summarize the physiological characteristics of K. marxianus relevant for its use in the efficient synthesis of heterologous proteins, the efforts performed hitherto in the development of a molecular toolbox for this purpose, and some successful examples.

A Bacterial Laccase for Enhancing Saccharification and Ethanol Fermentation of Steam-Pretreated Biomass

Different biological approaches, highlighting the use of laccases, have been developed as environmentally friendly alternatives for improving the saccharification and fermentation stages of steam-pretreated lignocellulosic biomass. This work evaluates the use of a novel bacterial laccase (MetZyme) for enhancing the hydrolysability and fermentability of steam-exploded wheat straw. When the water insoluble solids (WIS) fraction was treated with laccase or alkali alone, a modest increase of about 5% in the sugar recovery yield (glucose and xylose) was observed in both treatments. Interestingly, the combination of alkali extraction and laccase treatment boosted enzymatic hydrolysis, increasing the glucose and xylose concentration in the hydrolysate by 21% and 30%, respectively. With regards to the fermentation stage, the whole pretreated slurry was subjected to laccase treatment, lowering the phenol content by up to 21%. This reduction allowed us to improve the fermentation performance of the thermotolerant yeast Kluyveromyces marxianus CECT 10875 during a simultaneous saccharification and fermentation (SSF) process. Hence, a shorter adaptation period and an increase in the cell viability—measured in terms of colony forming units (CFU/mL)—could be observed in laccase-treated slurries. These differences were even more evident when a presaccharification step was performed prior to SSF. Novel biocatalysts such as the bacterial laccase presented in this work could play a key role in the implementation of a cost-effective technology in future biorefineries.

Enhancement in xylose utilization using Kluyveromyces marxianus NIRE-K1 through evolutionary adaptation approach.

The evolutionary adaptation was carried out on the thermotolerant yeast Kluyveromyces marxianus NIRE-K1 at 45 °C up to 60 batches to enhance its xylose utilization capability. The adapted strain showed higher specific growth rate and 3-fold xylose uptake rate and short lag phase as compared to the native strain. During aerobic growth adapted yeast showed 2.81-fold higher xylose utilization than that of native. In anaerobic batch fermentation, adapted yeast utilized about 91% of xylose in 72 h and produced 2.88 and 18.75 g l⁻¹ of ethanol and xylitol, respectively, which were 5.11 and 5.71-fold higher than that of native. Ethanol yield, xylitol yield and specific sugar consumption rate obtained by the adapted cells were found to be 1.57, 1.65 and 4.84-fold higher than that of native yeast, respectively. Aforesaid results suggested that the evolutionary adaptation will be a very effective strategy in the near future for economic lignocellulosic ethanol production.

A Kluyveromyces marxianus 2-deoxyglucose-resistant mutant with enhanced activity of xylose utilization.

Thermotolerant ethanologenic yeast Kluyveromyces marxianus is capable of fermenting various sugars including xylose but glucose represses to hamper the utilization of other sugars. To acquire glucose repression-defective strains, 33 isolates as 2-deoxyglucose (2-DOG)-resistant mutants were acquired from about 100 colonies grown on plates containing 2-DOG, which were derived from an efficient strain DMKU 3-1042. According to the characteristics of sugar consumption abilities and cell growth and ethanol accumulation along with cultivation time, they were classified into three groups. The first group (3 isolates) utilized glucose and xylose in similar patterns along with cultivation to those of the parental strain, presumably due to reduction of the uptake of 2-DOG or enhancement of its export. The second group (29 isolates) showed greatly delayed utilization of glucose, presumably by reduction of the uptake or initial catabolism of glucose. The last group, only one isolate, showed enhanced utilization ability of xylose in the presence of glucose. Further analysis revealed that the isolate had a single nucleotide mutation to cause amino acid substitution (G270S) in RAG5 encoding hexokinase and exhibited very low activity of the enzyme. The possible mechanism of defectiveness of glucose repression in the mutant is discussed in this paper. [Int Microbiol 18(4):235-244 (2015)].

Rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP(H)-preferring xylose reductase-xylitol dehydrogenase pathway

Conversion of xylose to ethanol by yeasts is a challenge because of the redox imbalances under oxygen-limited conditions. The thermotolerant yeast Kluyveromyces marxianus grows well with xylose as a carbon source at elevated temperatures, but its xylose fermentation ability is weak. In this study, a combination of the NADPH-preferring xylose reductase (XR) from Neurospora crassa and the NADP+-preferring xylitol dehydrogenase (XDH) mutant from Scheffersomyces stipitis (Pichia stipitis) was constructed. The xylose fermentation ability and redox balance of the recombinant strains were improved significantly by over-expression of several downstream genes. The intracellular concentrations of coenzymes and the reduced coenzyme/oxidized coenzyme ratio increased significantly in these metabolic strains. The byproducts, such as glycerol and acetic acid, were significantly reduced by the disruption of glycerol-3-phosphate dehydrogenase (GPD1). The resulting engineered K. marxianus YZJ088 strain produced 44.95g/L ethanol from 118.39g/L xylose with a productivity of 2.49g/L/h at 42°C. Additionally, YZJ088 realized glucose and xylose co-fermentation and produced 51.43g/L ethanol from a mixture of 103.97g/L xylose and 40.96g/L glucose with a productivity of 2.14g/L/h at 42°C. These promising results validate the YZJ088 strain as an excellent producer of ethanol from xylose through the synthetic xylose assimilation pathway.

The Application of Thermotolerant Yeast Kluyveromyces marxianus as a Potential Industrial Workhorse for Biofuel Production

Kluyveromyces marxianus is a well-known thermotolerant yeast. Although Saccharomyces cerevisiae is the most commonly used yeast species for ethanol production, the thermotolerant K. marxianus is more suitable for simultaneous saccharification and fermentation (SSF) processes. This is because enzymatic saccharification usually requires a higher temperature than that needed for the optimum growth of S. cerevisiae. In this study, we compared the fermentation patterns of S. cerevisiae and K. marxianus under various temperatures of fermentation. The results show that at a fermentation temperature of 45°C, K. marxianus exhibited more than two fold higher growth rate and ethanol production rate in comparison to S. cerevisiae. For SSF using starch or corn stover as the sole carbon source by K. marxianus, the high temperature (45°C) fermentations showed higher enzymatic activities and ethanol production compared to SSF at 30°C. These results demonstrate the potential of the thermotolerant yeast K. marxianus for SSF in the industrial production of biofuels.

Enhanced Xylitol Production by Mutant Kluyveromyces marxianus 36907-FMEL1 Due to Improved Xylose Reductase Activity.

A directed evolution and random mutagenesis were carried out with thermotolerant yeast Kluyveromyces marxianus ATCC 36907 for efficient xylitol production. The final selected strain, K. marxianus 36907-FMEL1, exhibited 120 and 39 % improvements of xylitol concentration and xylitol yield, respectively, as compared to the parental strain, K. marxianus ATCC 36907. According to enzymatic assays for xylose reductase (XR) activities, XR activity from K. marxianus 36907-FMEL1 was around twofold higher than that from the parental strain. Interestingly, the ratios of NADH-linked and NADPH-linked XR activities were highly changed from 1.92 to 1.30 when K. marxianus ATCC 36907 and K. marxianus 36907-FMEL1 were compared. As results of KmXYL1 genes sequencing, it was found that cysteine was substituted to tyrosine at position 36 after strain development which might cause enhanced XR activity from K. marxianus 36907-FMEL1.

Ethanol Production from Sugarcane Bagasse Using SSF Process and Thermotolerant Yeast

Abstract. Sugarcane bagasse (SCB) pretreated by hydrothermolysis at 195°C for 10 min was used as a substrate for ethanol production in a simultaneous saccharification and fermentation (SSF) process with the thermotolerant yeast Kluyveromyces marxianus IMB3 at 45°C. SSF was carried out for 7 days using 15 and 30 filter paper units (FPU) cellulase g -1 treated SCB (Accellerase 1500) to determine the effect of enzyme loading on ethanol production. Different pretreated dry solids loadings of 10% and 15% (w/w) were investigated in this work during enzymatic hydrolysis. Results showed 89.7% hemicellulose removal during pretreatment and glucan recovery of 97.8%. The highest ethanol concentration of 29.2 g L -1 was obtained with 30 FPU cellulase g -1 treated SCB within 72 h, which was equivalent to 58.9% of the maximum theoretical ethanol yield.

Second generation bioethanol production at high gravity of pilot-scale pretreated wheat straw employing newly isolated thermotolerant yeast Kluyveromyces marxianus DBTIOC-35

Application of thermotolerant yeastKluyveromyces marxianusDBTIOC-35 in SSF decreases overall process time, and increases productivity and yield by allowing elimination of presaccharification step and use of high biomass concentration, respectively.

Deletion of a KU80 homolog enhances homologous recombination in the thermotolerant yeast Kluyveromyces marxianus.

Targeted gene replacement in the thermotolerant yeast Kluyveromyces marxianus KCTC 17555 has been hampered by its propensity to non-homologous end joining (NHEJ). To enhance homologous recombination (HR) by blocking NHEJ, we identified and disrupted the K. marxianus KU80 gene. The ku80 deletion mutant strain (Kmku80∆) of K. marxianus KCTC 17555 did not show apparent growth defects under several conditions with the exception of exposure to tunicamycin. The targeted disruption of the three model genes, KmLEU2, KmPDC1, and KmPDC5, was increased by 13-70 % in Kmku80∆, although the efficiency was greatly affected by the length of the homologous flanking fragments. In contrast, the double HR frequency was 0-13.7 % in the wild-type strain even with flanking fragments 1 kb long. Therefore, Kmku80∆ promises to be a useful recipient strain for targeted gene manipulation.

Sulfuric acid hydrolysis and detoxification of red alga Pterocladiella capillacea for bioethanol fermentation with thermotolerant yeast Kluyveromyces marxianus.

One-step sulfuric acid saccharification of the red alga Pterocladiella capillacea was optimized, and various detoxification methods (neutralization, overliming, and electrodialysis) of the acid hydrolysate were evaluated for fermentation with the thermotolerant yeast Kluyveromyces marxianus. A proximate composition analysis indicated that P. capillacea was rich in carbohydrates. A significant galactose recovery of 81.1 ± 5% was also achieved under the conditions of a 12% (w/v) biomass load, 5% (v/v) sulfuric acid, 121°C, and hydrolysis for 30 min. Among the various detoxification methods, electrodialysis was identified as the most suitable for fermentable sugar recovery and organic acid removal (100% reduction of formic and levulinic acids), even though it failed to reduce the amount of the inhibitor 5-HMF. As a result, K. marxianus fermentation with the electrodialyzed acid hydrolysate of P. capillacea resulted in the best ethanol levels and fermentation efficiency.

Direct fermentation of raw starch using a Kluyveromyces marxianus strain that expresses glucoamylase and Alpha‐amylase to produce ethanol

Raw starch and raw cassava tuber powder were directly and efficiently fermented at elevated temperatures to produce ethanol using the thermotolerant yeast Kluyveromyces marxianus that expresses α-amylase from Aspergillus oryzae as well as α-amylase and glucoamylase from Debaryomyces occidentalis. Among the constructed K. marxianus strains, YRL 009 had the highest efficiency in direct starch fermentation. Raw starch from corn, potato, cassava, or wheat can be fermented at temperatures higher than 40°C. At the optimal fermentation temperature 42°C, YRL 009 produced 66.52 g/L ethanol from 200 g/L cassava starch, which was the highest production among the selected raw starches. This production increased to 79.75 g/L ethanol with a 78.3% theoretical yield (with all cassava starch were consumed) from raw cassava starch at higher initial cell densities. Fermentation was also carried out at 45 and 48°C. By using 200 g/L raw cassava starch, 137.11 and 87.71 g/L sugar were consumed with 55.36 and 32.16 g/L ethanol produced, respectively. Furthermore, this strain could directly ferment 200 g/L nonsterile raw cassava tuber powder (containing 178.52 g/L cassava starch) without additional nutritional supplements to produce 69.73 g/L ethanol by consuming 166.07 g/L sugar at 42°C. YRL 009, which has consolidated bioprocessing ability, is the best strain for fermenting starches at elevated temperatures that has been reported to date.

Ethanol from laccase-detoxified lignocellulose by the thermotolerant yeast Kluyveromyces marxianus—Effects of steam pretreatment conditions, process configurations and substrate loadings

In our previous work, the ability of laccase enzymes to improve the fermentation performance of the thermotolerant yeast Kluyveromyces marxianus CECT 10875 on steam-exploded wheat straw slurry was demonstrated. As a continuation of this study, the present research evaluates different aspects, including pretreatment conditions, process configurations and substrate loadings, with the aim to proceed towards the use of K. marxianus and laccases for second generation ethanol production. For it, two wheat straw slurries resulting from different steam explosion pretreatment conditions (200°C, 2.5min and 220°C, 2.5min) were employed at various substrate loadings [5–14% (w/v)] under two process configurations: SSF (simultaneous saccharification and fermentation) and PSSF (presaccharification and simultaneous saccharification and fermentation). The better performance of K. marxianus was observed on the slurry produced at softer conditions. Its lower inhibitors content allowed to increase the total solids loading up to 10% (w/v) in both process configurations, reaching higher ethanol concentrations (12g/L). Moreover, laccase detoxification improved these results, particularly in SSF processes, increasing the substrate loading up to 12% (w/v) and, consequently, obtaining the highest ethanol concentration (16.7g/L).