Yeast Kluyveromyces Marxianus Research Articles

Identification of a novel gene required for competitive growth at high temperature in the thermotolerant yeast Kluyveromyces marxianus.

It is important to understand the basis of thermotolerance in yeasts to broaden their application in industrial biotechnology. The capacity to run bioprocesses at temperatures above 40 °C is of great interest but this is beyond the growth range of most of the commonly used yeast species. In contrast, some industrial yeasts such as Kluyveromyces marxianus can grow at temperatures of 45 °C or higher. Such species are valuable for direct use in industrial biotechnology and as a vehicle to study the genetic and physiological basis of yeast thermotolerance. In previous work, we reported that evolutionarily young genes disproportionately changed expression when yeast were growing under stressful conditions and postulated that such genes could be important for long-term adaptation to stress. Here, we tested this hypothesis in K. marxianus by identifying and studying species-specific genes that showed increased expression during high-temperature growth. Twelve such genes were identified and 11 were successfully inactivated using CRISPR-mediated mutagenesis. One gene, KLMX_70384, is required for competitive growth at high temperature, supporting the hypothesis that evolutionary young genes could play roles in adaptation to harsh environments. KLMX_70384 is predicted to encode an 83 aa peptide, and RNA sequencing and ribo-sequencing were used to confirm transcription and translation of the gene. The precise function of KLMX_70384 remains unknown but some features are suggestive of RNA-binding activity. The gene is located in what was previously considered an intergenic region of the genome, which lacks homologues in other yeasts or in databases. Overall, the data support the hypothesis that genes that arose de novo in K. marxianus after the speciation event that separated K. marxianus and K. lactis contribute to some of its unique traits.

Optimization of fermentation for γ-aminobutyric acid (GABA) production by yeast Kluyveromyces marxianus C21 in okara (soybean residue).

γ-Aminobutyric acid (GABA) is a non-protein amino acid with a variety of physiological functions. Recently, yeast Kluyveromyces marxianus strains involved in the catabolism and anabolism of GABA can be used as a microbial platform for GABA production. Okara, rich in nutrients, can be used as a low-cost fermentation substrate for the production of functional materials. This study first proved the advantages of the okara medium to produce GABA by K. marxianus C21 when L-glutamate (L-Glu) or monosodium glutamate (MSG) is the substrate. The highest production of GABA was obtained with 4.31g/L at optimization condition of culture temperature 35°C, fermentation time 60h, and initial pH 4.0. Furthermore, adding peptone significantly increased the GABA production while glucose and vitamin B6 had no positive impact on GABA production. This research provided a powerful new strategy of GABA production by K. marxianus C21 fermentation and is expected to be widely utilized in the functional foods industry to increase GABA content for consumers as a daily supplement as suggested.

Respiratory reoxidation of NADH is a key contributor to high oxygen requirements of oxygen-limited cultures of Ogataea parapolymorpha.

While thermotolerance is an attractive trait for yeasts used in industrial ethanol production, oxygen requirements of known thermotolerant species are incompatible with process requirements. Analysis of oxygen-sufficient and oxygen-limited chemostat cultures of the facultatively fermentative, thermotolerant species Ogataea parapolymorpha showed its minimum oxygen requirements to be an order of magnitude larger than those reported for the thermotolerant yeast Kluyveromyces marxianus. High oxygen requirements of O. parapolymorpha coincided with a near absence of glycerol, a key NADH/NAD+ redox-cofactor-balancing product in many other yeasts, in oxygen-limited cultures. Genome analysis indicated absence of orthologs of the Saccharomyces cerevisiae glycerol-3-phosphate-phosphatase genes GPP1 and GPP2. Co-feeding of acetoin, whose conversion to 2,3-butanediol enables reoxidation of cytosolic NADH, supported a 2.5-fold increase of the biomass concentration in oxygen-limited cultures. An O. parapolymorpha strain in which key genes involved in mitochondrial reoxidation of NADH were inactivated did produce glycerol, but transcriptome analysis did not reveal a clear candidate for a responsible phosphatase. Expression of S. cerevisiae GPD2, which encodes NAD+-dependent glycerol-3-phosphate dehydrogenase, and GPP1 supported increased glycerol production by oxygen-limited chemostat cultures of O. parapolymorpha. These results identify dependence on respiration for NADH reoxidation as a key contributor to unexpectedly high oxygen requirements of O. parapolymorpha.

Optimization of 2-Phenylethanol Production from Sweet Whey Fermentation Using Kluyveromyces marxianus

The growing demand for natural products benefits the development of bioprocesses to obtain value-added compounds using residues such as sweet whey, which is rich in lactose. The yeast Kluyveromyces marxianus can ferment sweet whey to obtain 2-phenylethanol (2-PhEtOH), which is a superior alcohol with a rose aroma. Such fermentation only requires the addition of L-phenylalanine (precursor) and (NH4)2SO4 (salt). Therefore, it was sought to improve the fermentation conditions to produce 2-PhEtOH, which, in turn, would achieve the maximum decrease in the Chemical Oxygen Demand (COD) of the fermentation medium. With the use of the Response Surface Methodology and the application of a Central Composite Design for optimization, two parameters were evaluated as a function of time: salt concentration and precursor. The experimental data were adjusted to a second order polynomial, identifying that the precursor concentration presents a statistically significant effect. The best conditions were: 4.50 g/L of precursor and 0.76 g/L of salt, with a maximum production of 1.2 g/L (2-PhEtOH) at 48 h and achieving a maximum percentage of COD removal of 76% at 96 h. Finally, the optimal conditions were experimentally validated, recommending the use of the model.

Two Biotechnological Approaches to the Preparative Synthesis of Natural Dihydrocoumarin

In this work, we describe two different biotechnological processes that provide the natural flavour dihydrocoumarin in preparative scale. Both the presented approaches are based on the enzyme-mediated reduction of natural coumarin. The first one is a whole-cell process exploiting the reductive activity of the yeast Kluyveromyces marxianus, a Generally Recognized As Safe (GRAS) microorganism that possesses high resistance to the substrate toxicity. Differently, the second is based on the reduction of natural coumarin by nicotinamide adenine dinucleotide phosphate (NADPH) and using the Old Yellow Enzyme reductase OYE2 as catalyst. NADPH is used in catalytic amount since the co-factor regeneration is warranted employing an enzymatic system based on glucose oxidation, in turn catalysed by a further enzyme, namely glucose dehydrogenase (GDH). Both processes compare favourably over the previously reported industrial method as they work with higher coumarin concentration (up to 3 g/L for the enzymatic process) yet allowing the complete conversion of the substrate. Furthermore, the two approaches have significant differences. The microbial reduction is experimentally simple but the isolated dihydrocoumarin yield does not exceed 60%. On the contrary, the enzymatic approach requires the use of two specially prepared recombinant enzymes, however, it is more efficient, affording the product in 90% of isolated yield.

Protocols for marker-free gene knock-out and knock-down in Kluyveromyces marxianus using CRISPR/Cas9.

ABSTRACTThere is increased interest in strain engineering in the food and industrial yeast Kluyveromyces marxianus and a number of CRISPR/Cas9 systems have been described and used by different groups. The methods that we developed allow for very rapid and efficient inactivation of target genes using the endogenous DNA repair mechanisms of the cell. The strains and plasmids that we use are freely available, and here we provide a set of integrated protocols to easily inactivate genes and to precisely integrate DNA fragments into the genome, for example for promoter replacement, allelic swaps or introduction of point mutations. The protocols use the Cas9/gRNA expression plasmid pUCC001 and Golden Gate assembly for molecular cloning of targeting sequences. A genome-wide set of target sequences is provided. Using these plasmids in wild-type strains or in strains lacking non-homologous end-joining (NHEJ) DNA repair, the first set of protocols explain how to introduce indels (NHEJ-mediated) or precise deletions (homology-dependent repair (HDR)-mediated) at precise targets. The second set of protocols describe how to swap a promoter or coding sequence to yield a reprogrammed gene. The methods do not require the use of dominant or auxotrophic marker genes and thus the strains generated are marker-free. The protocols have been tested in multiple K. marxianus strains, are straightforward and can be carried out in any molecular biology laboratory without specialized equipment.

Establishment of Kluyveromyces marxianus as a Microbial Cell Factory for Lignocellulosic Processes: Production of High Value Furan Derivatives.

The establishment of lignocellulosic biorefineries is dependent on microorganisms being able to cope with the stressful conditions resulting from the release of inhibitory compounds during biomass processing. The yeast Kluyveromyces marxianus has been explored as an alternative microbial factory due to its thermotolerance and ability to natively metabolize xylose. The lignocellulose-derived inhibitors furfural and 5-hydroxymethylfurfural (HMF) are considered promising building-block platforms that can be converted into a wide variety of high-value derivatives. Here, several K. marxianus strains, isolated from cocoa fermentation, were evaluated for xylose consumption and tolerance towards acetic acid, furfural, and HMF. The potential of this yeast to reduce furfural and HMF at high inhibitory loads was disclosed and characterized. Our results associated HMF reduction with NADPH while furfural-reducing activity was higher with NADH. In addition, furans’ inhibitory effect was higher when combined with xylose consumption. The furan derivatives produced by K. marxianus in different conditions were identified. Furthermore, one selected isolate was efficiently used as a whole-cell biocatalyst to convert furfural and HMF into their derivatives, furfuryl alcohol and 2,5-bis(hydroxymethyl)furan (BHMF), with high yields and productivities. These results validate K. marxianus as a promising microbial platform in lignocellulosic biorefineries.

Development of a Continuous System for 2-Phenylethanol Bioproduction by Yeast on Whey Permeate-Based Medium.

2-Phenylethanol (2-PE) is an alcohol with a rosy scent and antimicrobial activity, and therefore, it is widely used in the food and cosmetic industries as an aroma and preservative. This work was aimed to draw up a technology for 2-PE bioproduction on whey permeate, which is waste produced by the dairy industry, rich in lactase and proteins. Its composition makes it a harmful waste to dispose of; however, with a properly selected microorganism, it could be converted to a value-added product. Herein, two yeast Kluyveromyces marxianus strains and one Kluyveromyces lactis, isolated from dairy products, were tested for 2-PE production, firstly on standard media and then on whey permeate based media in batch cultures. Thereafter, the 2-PE bioproduction in a continuous system in a 4.8 L bioreactor was developed, and subsequently, the final product was recovered from culture broth. The results showed that the yield of 2-PE production increased by 60% in the continuous culture compared to batch culture. Together with a notable reduction of chemical oxygen demand for whey permeate, the present study reports a complete, effective, and environmentally friendly strategy for 2-PE bioproduction with a space-time yield of 57.5 mg L−1 h−1.

CRECIMIENTO Y CARACTERÍSTICAS FERMENTATIVAS DE Kluyveromyces marxianus EN MATRICES DERIVADAS DE LACTOSUERO

Milk whey has a high content of organic matter; therefore, uncontrolled discharge can cause environmental problems. An alternative for whey utilization is the production of fermented alcoholic beverages by means of the yeast Kluyveromyces marxianus, able to use lactose as a carbon source. Four different liquid growth media were prepared: whey powder (SP) and demineralized whey powder (SPD) (65 g/L) dissolved in sterilized distilled water; SPCal, similar to SP in composition, but sterilized and centrifuged to remove proteins, and milk permeate (PER). All media were fermented by 24 hours at 30°C. Samples were taken every 8 hours, for pH, biomass, ethanol and lactose determination. K. marxianus (LFIQK1 strain) growth was similar in SP, SPD and SP Cal (biomass between 2 – 3 g/L) and lower in PER. Moderate pH decline was observed, final values being between 4,7 and 5,4. Significant lactose consumption was observed in every growth media, with final values around 12 g/L and good ethanol production, especially for SP, SPD, and PER mediums (17,0±0,7; 17,6±0,5; 16,3±0,2 g/L respectively). These results are encouraging for the production of alcoholic beverages from the studied media using K. marxianus as the fermentation agent.

Preparation and Characterization of Pickering Emulsions with Modified Okara Insoluble Dietary Fiber.

Modified okara insoluble dietary fiber (OIDF) has attracted great interest as a promising Pickering emulsifier. At present, the modification methods are mainly physicochemical methods, and the research on microbial modified OIDF as stabilizer is not clear. In this work, modified OIDF was prepared by yeast Kluyveromyces marxianus fermentation. The potential of modified OIDF as a Pickering emulsifier and the formation and stability of OIDF-Pickering emulsions stabilized by modified OIDF were characterized, respectively. The results showed that the specific surface area, hydrophilicity, and electronegativity of the modified OIDF were all enhanced compared with the unmodified OIDF. The existence of the network structure between droplets is the key to maintain the stability of the emulsions, as indicated by Croy-Scanning Electron Microscope (Croy-SEM) and rheological properties measurements. The stability of OIDF-Pickering emulsions was evaluated in terms of storage time, centrifugal force, pH value, and ionic strength (NaCl). Moreover, the OIDF-Pickering emulsions stabilized by modified OIDF showed better stability. These results will contribute to the development of efficient OIDF-based emulsifiers, expand the application of emulsions in more fields, and will greatly improve the high-value utilization of okara by-products.

Screening of the freeze‐drying protective agent for high‐quality milk beer yeast ( Kluyveromyces marxianus ) and optimization of freeze‐drying process conditions

Milk beer is a new type of milk beverage produced by two-step fermentation of lactic acid bacteria and yeast and created first in China. Yeast is the key strain of milk beer fermentation, which determines the flavor and quality of milk beer. In this study, the preparation process of high-quality yeast strain MJ1 active freeze-dried powder suitable for milk beer fermentation was optimized. Results showed that the optimal freeze-dried protective agent was used: sucrose 11.27%, mannitol 4.88%, maltodextrin 15.43%, skimmed milk powder 7.82%, and applying the optimal freeze-drying conditions: rotation speed 6,000 rpm, centrifugation time 15 min, the mixing ratio of centrifugal bacterial sludge and the protective agent is 1:1.5, the mixing equilibrium time is 30 min, the cell survival rate of strain MJ1 can reach 90.21% ± 1.04%. It laid the foundation for the preparation of active freeze-dried yeast powder and the industrial production of milk beer. Novelty impact statement (1) The formulation and conditions of the freeze-dried protectant of Kluyveromyces marxianus yeast strain MJ1 were optimized to improve the cell survival rate of the freeze-dried yeast powder, (2) the optimal storage conditions of the freeze-dried yeast powder of strain MJ1 were screened to provide theoretical reference for the long-term storage of freeze-dried yeast powder, and (3) the fermentation performance of the freeze-dried yeast powder of strain MJ1 was verified, which provided a reliable basis for the industrial production of milk beer.

Diploid genome assembly of Kluyveromyces marxianus NRRL Y-50883 (SLP1).

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.

Protoplast transformation of Kluyveromyces marxianus

The dairy yeast Kluyveromyces marxianus is a promising cell factory for producing bioethanol and heterologous proteins, as well as a robust synthetic biology platform host, due to its safe status and beneficial traits, including fast growth and thermotolerance. However, the lack of high-efficiency transformation methods hampers the fundamental research and industrial application of this yeast. Protoplast transformation is one of the most commonly used fungal transformation methods, but it yet remains unexplored in K. marxianus. Here, we established the protoplast transformation method of K. marxianus for the first time. A series of parameters on the transformation efficiency were optimized: cells were collected in the late-log phase and treated with zymolyase for protoplasting; the transformation was performed at 0°C with carrier DNA, CaCl2 , and PEG; after transformation, protoplasts were recovered in a solid regeneration medium containing 3-4% agar and 0.8m sorbitol. By using the optimized method, plasmids of 10, 24, and 58kb were successfully transformed into K. marxianus. The highest efficiency reached 1.8×104 transformants per μg DNA, which is 18-fold higher than the lithium acetate method. This protoplast transformation method will promote the genetic engineering of K. marxianus that requires high-efficiency transformation or the introduction of large DNA fragments.

Enhanced recovery of astaxanthin from recombinant Kluyveromyces marxianus with ultrasonication-assisted alcohol/salt aqueous biphasic system

Microbial astaxanthin with strong antioxidant activity is greatly demanded for diverse applications. Extractive disruption in aqueous biphasic system (ABS) integrates the cells disruption and biomolecules recovery processes in one-step operation, allowing the direct recovery of intracellular biomolecules with biphasic system upon released from cells. In this study, astaxanthin was recovered from recombinant Kluyveromyces marxianus yeast cells via extractive disruption using alcohol/salt ABS. Recombinant K.marxianus yeast is engineered to produce high concentration of free form astaxanthin. Highest partition coefficient (K= 90.02 ± 2.25) and yield (Y= 96.80% ± 0.05) of astaxanthin were obtained with ABS composed of 20% (w/w) 1-propanol and 20% (w/w) sodium citrate of pH 5, 0.5% (w/w) yeast cells loading and additional of 1% (w/w) 1-butyl-3-methylimidazolium tetrafluoroborate (Bmim)BF4 to improve the migration of astaxanthin to alcohol-rich top phase. The incorporation of 2.5h of ultrasonication to the biphasic system further enhanced the astaxanthin recovery in ABS. The direct recovery of astaxanthin from recombinant K.marxianus cells was demonstrated with the ultrasonication-assisted alcohol/salt ABS which integrates the extraction and concentration of astaxanthin in a single-step operation.

Various short autonomously replicating sequences from the yeast Kluyveromyces marxianus seemingly without canonical consensus.

Eukaryotic autonomously replicating sequences (ARSs) are composed of three domains, A, B, and C. Domain A is comprised of an ARS consensus sequence (ACS), while the B domain has the DNA unwinding element and the C domain is important for DNA-protein interactions. In Saccharomyces cerevisiae and Kluyveromyces lactis ARS101, the ACS is commonly composed of 11 bp, 5ˊ-(A/T)AAA(C/T)ATAAA(A/T)-3ˊ. This core sequence is essential for S. cerevisiae and K. lactis ARS activity. In this study, we identified ARS-containing sequences from genomic libraries of the yeast Kluyveromyces marxianus DMKU3-1042 and validated their replication activities. The identified K. marxianus DMKU3-1042 ARSs (KmARSs) have very effective replication ability but their sequences are divergent and share no common consensus. We have carried out point mutations, deletions, and base pairs substitutions within the sequences of some of the KmARSs to identify the sequence(s) that influence the replication activity. Consensus sequences same as the 11 bp ACS of S. cerevisiae and K. lactis were not found in all minimum functional KmARSs reported here except KmARS7. Moreover, partial sequences from different KmARSs are interchangeable among each other to retain the ARS activity. We have also specifically identified the essential nucleotides, which are indispensable for replication, within some of the KmARSs. Our deletions analysis revealed that only 21 bp in KmARS18 could retain the ARS activity. The identified KmARSs in this study are unique compared to other yeasts’ ARSs, do not share common ACS, and are interchangeable.

Hydrolysis of lignocellulose by anaerobic fungi produces free sugars and organic acids for two-stage fine chemical production with Kluyveromyces marxianus.

Development of the bioeconomy is driven by our ability to access the energy-rich carbon trapped in recalcitrant plant materials. Current strategies to release this carbon rely on expensive enzyme cocktails and physicochemical pretreatment, producing inhibitory compounds that hinder subsequent microbial bioproduction. Anaerobic fungi are an appealing solution as they hydrolyze crude, untreated biomass at ambient conditions into sugars that can be converted into value-added products by partner organisms. However, some carbon is lost to anaerobic fungal fermentation products. To improve efficiency and recapture this lost carbon, we built a two-stage bioprocessing system pairing the anaerobic fungus Piromyces indianae with the yeast Kluyveromyces marxianus, which grows on a wide range of sugars and fermentation products. In doing so we produce fine and commodity chemicals directly from untreated lignocellulose. P. indianae efficiently hydrolyzed substrates such as corn stover and poplar to generate sugars, fermentation acids, and ethanol, which K. marxianus consumed while producing 2.4g/L ethyl acetate. An engineered strain of K. marxianus was also able to produce 550 mg/L 2-phenylethanol and 150 mg/L isoamyl alcohol from P. indianae hydrolyzed lignocellulosic biomass. Despite the use of crude untreated plant material, production yields were comparable to optimized rich yeast media due to the use of all available carbon including organic acids, which formed up to 97% of free carbon in the fungal hydrolysate. This work demonstrates that anaerobic fungal pretreatment of lignocellulose can sustain the production of fine chemicals at high efficiency by partnering organisms with broad substrate versatility.

Production of aroma and flavor‐rich fusel alcohols by cheese whey fermentation using the Kluyveromyces marxianus and Debaryomyces hansenii yeasts in monoculture and co‐culture modes

AbstractBACKGROUNDWhey is one of the main agro‐industrial by‐products generated in the production of cheese. It is a residue with low pH and high content of organic and inorganic compounds, such as lactose, proteins, and minerals that can be used as nutrients in fermentation. The aim of the present study was to produce biomolecules with aroma and flavor properties by whey fermentation using Kluyveromyces marxianus (KM) and Debaryomyces hansenii (DH).RESULTSThese yeasts produced and accumulated in the culture broth aroma and flavor compounds, the main ones being ethanol, glycerol, propanoic acid, dihydroxyacetone, methionol, isopentanol, and 2‐phenylethanol (2PE). This last one was retained as the target biomolecule, because of its potential to be commercialized industrially. Both yeasts were able to metabolize L‐phenylalanine (Lphe) to produce 2PE, in monoculture and co‐culture modes. When yeasts were used under monoculture mode, KM produced the highest 2PE concentration 82 ± 28 mg/L under aerobic fermentation, with yield of 0.16 ± 0.08 g2PE/gLphe and a productivity of 0.86 ± 0.18 mg2PE/L*h at a fermentation time of 96 h. Whereas in co‐culture mode the 2PE yield was 0.38 g2PE/gLphe, twice as high as the maximum yield for monocultures. This yield corresponded to a productivity of 1.93 ± 0.02 mg2PE/L*h.CONCLUSIONThe whey fermentation using KM and DH in co‐culture mode is technically feasible. The KM yeast is apparently dominant and the co‐culture of both yeasts led to increase in the 2PE yield and the productivity. The faster kinetics of KM quickly induced substrate starvation triggering early production and accumulation of 2PE. © 2021 Society of Chemical Industry (SCI).

Uncoupling glucose sensing from GAL metabolism for heterologous lactose fermentation in Saccharomyces cerevisiae.

Development of a system for direct lactose to ethanol fermentation provides a market for the massive amounts of underutilized whey permeate made by the dairy industry. For this system, glucose and galactose metabolism were uncoupled in Saccharomyces cerevisiae by deleting two negative regulatory genes, GAL80 and MIG1, and introducing the essential lactose hydrolase LAC4 and lactose transporter LAC12, from the native but inefficient lactose fermenting yeast Kluyveromyces marxianus. Previously, integration of the LAC4 and LAC12 genes into the MIG1 and NTH1 loci was achieved to construct strain AY-51024M. Low rates of lactose conversion led us to generate the Δmig1Δgal80 diploid mutant strain AY-GM from AY-5, which exhibited loss of diauxic growth and glucose repression, subsequently taking up galactose for consumption at a significantly higher rate and yielding higher ethanol concentrations than strain AY-51024M. Similarly, in cheese whey permeate powder solution (CWPS) during three, repeated, batch processes in a 5L bioreactor containing either 100g/L or 150g/L lactose, the lactose uptake and ethanol productivity rates were both significantly greater than that of AY-51024M, while the overall fermentation times were considerably lower. Using the Cre-loxp system for deletion of the MIG1 and GAL80 genes to relieve glucose repression, and LAC4 and LAC12 overexpression to increase lactose uptake and conversion provides an efficient basis for yeast fermentation of whey permeate by-product into ethanol.

A comparative study on bioethanol production from rice straw and banana pseudostem through simultaneous saccharification and fermentation using Kluyveromyces marxianus

To improve the efficiency of second-generation bioethanol production, simultaneous Saccharification and Fermentation (SSF) of top agricultural wastes in the Philippines such as rice straw and banana pseudostem was conducted using commercial enzymes and the thermotolerant yeast Kluyveromyces marxianus. Residues were initially subjected to chemical treatment prior to SSF. A constant inoculum loading of 10% vv -1 was introduced during SSF, and mixed with a nutrient supplemented solution. Aside from the enzyme loading, the effect of reaction temperature and reaction time on ethanol concentration was assessed. Among the tested parameters, only reaction time had a significant effect on the bioethanol concentration from both biomasses. For a pre-treated rice straw with 70.93% ww -1 holocellulose, highest ethanol concentration obtained was 6.30+0.44 gL -1 at 45°C, reaction time of 48 h and with enzyme loading of 30 FPUg -1. On the other hand, 5.35+0.29 gL -1 ethanol was achieved from SSF of banana pseudostem with 67.75% ww -1 holocellulose, also at 35°C, at a lower reaction time of 24 h and same enzyme loading of 30 FPUg -1. This study also proves that the thermotolerant K. marxianus was capable of producing bioethanol from lignocellulosics through SSF, thereby considering it as a potential alternative to Saccharomyces cerevisiae for bioethanol production.

Identification of novel pentose transporters in Kluyveromyces marxianus using a new screening platform.

ABSTRACTThe capacity of yeasts to assimilate xylose or arabinose is strongly dependent on plasma membrane transport proteins. Because pentoses comprise a substantial proportion of available sugars in lignocellulosic hydrolysates, their utilisation is centrally important for the development of second generation biorefineries. Relatively few native pentose transporters have been studied and there is intense interest in expanding the repertoire. To aid the identification of novel transporters, we developed a screening platform in the native pentose-utilising yeast Kluyveromyces marxianus. This involved the targeted deletion of twelve transporters of the major facilitator superfamily (MFS) and application of a synthetic biology pipeline for rapid testing of candidate pentose transporters. Using this K. marxianus ΔPT platform, we identified several K. marxianus putative xylose or arabinose transporter proteins that recovered a null strain's ability to growth on these pentoses. Four proteins of the HGT-family were able to support growth in media with high or low concentrations of either xylose or arabinose, while six HXT-like proteins displayed growth only at high xylose concentrations, indicating solely low affinity transport activity. The study offers new insights into the evolution of sugar transporters in yeast and expands the set of native pentose transporters for future functional and biotechnological studies.