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.

Transcriptomic Analysis Reveals the Detoxification Mechanism of Chilo suppressalis in Response to the Novel Pesticide Cyproflanilide.

Chilo suppressalis is one of the most damaging rice pests in China's rice-growing regions. Chemical pesticides are the primary method for pest control; the excessive use of insecticides has resulted in pesticide resistance. C. suppressalis is highly susceptible to cyproflanilide, a novel pesticide with high efficacy. However, the acute toxicity and detoxification mechanisms remain unclear. We carried out a bioassay experiment with C. suppressalis larvae and found that the LD10, LD30 and LD50 of cyproflanilide for 3rd instar larvae was 1.7 ng/per larvae, 6.62 ng/per larvae and 16.92 ng/per larvae, respectively. Moreover, our field trial results showed that cyproflanilide had a 91.24% control efficiency against C. suppressalis. We investigated the effect of cyproflanilide (LD30) treatment on the transcriptome profiles of C. suppressalis larvae and found that 483 genes were up-regulated and 305 genes were down-regulated in response to cyproflanilide exposure, with significantly higher CYP4G90 and CYP4AU10 expression in the treatment group. The RNA interference knockdown of CYP4G90 and CYP4AU10 increased mortality by 20% and 18%, respectively, compared to the control. Our results indicate that cyproflanilide has effective insecticidal toxicological activity, and that the CYP4G90 and CYP4AU10 genes are involved in detoxification metabolism. These findings provide an insight into the toxicological basis of cyproflanilide and the means to develop efficient resistance management tools for C. suppressalis.

Characterization and optimization of the LAC4 upstream region for low-leakage expression in Kluyveromyces marxianus.

Kluyveromyces marxianus is a promising host for the production of heterologous proteins, chemicals, and bioethanol. One superior feature of this species is its capacity to assimilate lactose, which is rendered by the LAC12-LAC4 gene pair encoding a lactose permease and a ß-galactosidase enzyme. Little is known about the regulation of LAC4 in K. marxianus. In this study, we showed the presence of weak glucose repression in the regulation of LAC4 and that might contribute to the leaky expression of LAC4 in the glucose medium. In a mutagenesis screen of 1000-bp LAC4 upstream region, one mutant region, named H1, drove low-leakage expression of a URA3 reporter gene in glucose medium. Two mutations inside a polyadenosine stretch (poly(A)) of 5' UTR were major contributors to the low-leakage phenotype of H1. H1 directed low-leakage expression of GFP on a plasmid and that of LAC4 in situ in the glucose medium, which was not due to the reduction of mRNA levels. Meanwhile, H1 did not affect the induction of GFP or LAC4 by lactose. Cre recombinase expressed by H1 caused lower toxicity in the repressive condition and achieved higher yield after induction, compared with that expressed by a wild-type LAC4 upstream region or a strong INU1 promoter. Our study suggested that poly(A) inside 5' UTR played a role in regulating the expression of LAC4 in the repressive condition. Meanwhile, H1 provided a base for the development of a strict inducible system for expressing industrial proteins, especially toxic proteins.

Investigation of Kluyveromyces marxianus as a novel host for large-scale production of porcine parvovirus virus-like particles.

BACKGROUND: Porcine Parvovirus (PPV) is a Parvovirinae virus that can cause embryonic and fetal loss and death and mummification in affected fetal pigs. Unlike conventional vaccines, virus-like particles (VLPs) inherit the natural structure of their authentic virions and highly immunostimulatory that can induce strong humoral immune and T cell responses with no risk of pathogenicity. The production of PPV VLPs is still a challenge based on traditional expression platforms due to their low yields and high culture costs. Kluyveromyces marxianus is a safe and fast-growing eukaryote that can get high biomass with low-cost cultures. In this study, we investigated the expression and downstream processes of PPV VLPs in K. marxianus, and the potential for effective stand-alone vaccines. RESULTS: After optimization according to the codon bias of K. marxianus, the VP2 protein from Kresse strain was highly expressed. In a 5 L fermentator, the yield of PPV VLPs reached 2.5 g/L, quantified by HPLC, using a defined mineral medium after 48 h fermentation. Two strategies were established to purify intracellular PPV VLPs: (i) Using the cation exchange chromatography coupled with Sephacryl® S-500 HR chromatography to purify VLPs from the supernatants of pH adjusted cell lysates. (ii) Using anion exchange chromatography followed by cross-flow diafiltration to recover the VLPs precipitated in pH adjusted cell lysates. The purity of PPV VLPs reached about 95%, and total recovery was more than 60%. Vaccination of mice with the purified PPV VLPs induced high titers of specific IgG antibodies in sera, and showed hemagglutination inhibitions on both swine and guinea pig erythrocytes. Spleen lymphocyte proliferation and cytokines detection suggested the PPV VLPs produced by K. marxianus provoked the cellular immune and humoral immunity responses in mice. CONCLUSIONS: This is the highest production of recombinant PPV VLPs achieved to date. The superiorities, Generally Recognized As Safe (GRAS), high production, short lead time, and low cost, make K. marxianus a greatly competitive platform for bioproduction of PPV VLPs vaccine.

Efficient production of porcine circovirus virus-like particles using the nonconventional yeast Kluyveromyces marxianus.

Porcine circovirus type 2 (PCV2) is a ubiquitous virus with high pathogenicity closely associated with the postweaning multisystemic wasting syndrome (PMWS) and porcine circovirus diseases (PCVDs), which caused significant economic losses in the swine industry worldwide every year. The PCV2 virus-like particles (VLPs) are a powerful subunit vaccine that can elicit high immune response due to its native PCV2 virus morphology. The baculovirus expression system is the widely used platform for producing commercial PCV2 VLP vaccines, but its yield and cost limited the development of low-cost vaccines for veterinary applications. Here, we applied a nonconventional yeast Kluyveromyces marxianus to enhance the production of PCV2 VLPs. After codon optimization, the PCV2 Cap protein was expressed in K. marxianus and assemble spontaneously into VLPs. Using a chemically defined medium, we achieved approximately 1.91 g/L of PCV2 VLP antigen in a 5-L bioreactor after high cell density fermentation for 72 h. That yield greatly exceeded to recently reported PCV2 VLPs obtained by baculovirus-insect cell, Escherichia coli and Pichia pastoris. By the means of two-step chromatography, 652.8 mg of PCV2 VLP antigen was obtained from 1 L of the recombinant K. marxianus cell culture. The PCV2 VLPs induced high level of anti-PCV2 IgG antibody in mice serums and decreased the virus titers in both livers and spleens of the challenged mice. These results illustrated that K. marxianus is a powerful yeast for cost-effective production of PCV2 VLP vaccines.

Mutational Mtc6p attenuates autophagy and improves secretory expression of heterologous proteins in Kluyveromyces marxianus.

BACKGROUND: The yeast Kluyveromyces marxianus is an emerging cell factory for heterologous protein biosynthesis and its use holds tremendous advantages for multiple applications. However, which genes influence the productivity of desired proteins in K. marxianus has so far been investigated by very few studies. RESULTS: In this study, we constructed a K. marxianus recombinant (FIM1/Est1E), which expressed the heterologous ruminal feruloyl esterase Est1E as reporter. UV-60Co-γ irradiation mutagenesis was performed on this recombinant, and one mutant (be termed as T1) was screened and reported, in which the productivity of heterologous Est1E was increased by at least tenfold compared to the parental FIM1/Est1E recombinant. Transcriptional perturbance was profiled and presented that the intracellular vesicle trafficking was enhanced while autophagy be weakened in the T1 mutant. Moreover, whole-genome sequencing combined with CRISPR/Cas9 mediated gene-editing identified a novel functional protein Mtc6p, which was prematurely terminated at Tyr251 by deletion of a single cytosine at 755 loci of its ORF in the T1 mutant. We found that deleting C755 of MTC6 in FIM1 led to 4.86-fold increase in the production of Est1E compared to FIM1, while the autophagy level decreased by 47%; on the contrary, when reinstating C755 of MTC6 in the T1 mutant, the production of Est1E decreased by 66% compared to T1, while the autophagy level increased by 124%. Additionally, in the recombinant with attenuated autophagy (i.e., FIM1 mtc6C755Δ and T1) or interdicted autophagy (i.e., FIM1 atg1Δ and T1 atg1Δ), the productivity of three other heterologous proteins was also increased, specifically the heterologous mannase Man330, the ß-1,4-endoxylanase XynCDBFV or the conventional EGFP. CONCLUSIONS: Our results demonstrated that Mtc6p was involved in regulating autophagy; attenuating or interdicting autophagy would dramatically improve the yields of desired proteins in K. marxianus, and this modulation could be achieved by focusing on the premature mutation of Mtc6p target.

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.

Transfer of disulfide bond formation modules via yeast artificial chromosomes promotes the expression of heterologous proteins in Kluyveromyces marxianus.

Kluyveromyces marxianus is a food-safe yeast with great potential for producing heterologous proteins. Improving the yield in K. marxianus remains a challenge and incorporating large-scale functional modules poses a technical obstacle in engineering. To address these issues, linear and circular yeast artificial chromosomes of K. marxianus (KmYACs) were constructed and loaded with disulfide bond formation modules from Pichia pastoris or K. marxianus. These modules contained up to seven genes with a maximum size of 15 kb. KmYACs carried telomeres either from K. marxianus or Tetrahymena. KmYACs were transferred successfully into K. marxianus and stably propagated without affecting the normal growth of the host, regardless of the type of telomeres and configurations of KmYACs. KmYACs increased the overall expression levels of disulfide bond formation genes and significantly enhanced the yield of various heterologous proteins. In high-density fermentation, the use of KmYACs resulted in a glucoamylase yield of 16.8 g/l, the highest reported level to date in K. marxianus. Transcriptomic and metabolomic analysis of cells containing KmYACs suggested increased flavin adenine dinucleotide biosynthesis, enhanced flux entering the tricarboxylic acid cycle, and a preferred demand for lysine and arginine as features of cells overexpressing heterologous proteins. Consistently, supplementing lysine or arginine further improved the yield. Therefore, KmYAC provides a powerful platform for manipulating large modules with enormous potential for industrial applications and fundamental research. Transferring the disulfide bond formation module via YACs proves to be an efficient strategy for improving the yield of heterologous proteins, and this strategy may be applied to optimize other microbial cell factories.

Characterization of a new alginate lyase from newly isolated Flavobacterium sp. S20.

Alginate lyase is a promising biocatalyst because of its application in saccharification of alginate for the production of biochemicals and renewable biofuels. This study described the isolation of a new alginate metabolizing bacterium, Flavobacterium sp. S20, from sludge samples and the characterization of its alginate lyase Alg2A. The alginate lyase gene, alg2A, was obtained by constructing and screening the genomic library of the strain S20 and overexpressed in Escherichia coli. Substrate specificity assays indicated Alg2A preferred poly-α-L-guluronate as a substrate over poly-ß-D-mannuronate. In the saccharification process of a high content (10 %, w/v) of sodium alginate, the recombinant alginate lyase Alg2A yielded 152 of mM the reducing sugars after 69 h of reaction, and the amounts of oligosaccharides with a different degree of polymerization (DP) generated by Alg2A gradually accumulated without significant variation in the distribution of oligosaccharide compositions. These results indicated that Alg2A possessed high enzymatic capability for saccharifying the alginate, which could be used in saccharifying the alginate biomass prior to the main fermentation process for biofuels. In addition, Alg2A had a different endolytic reaction mode from both the two commercial alginate lyases and other alginate lyases from polysaccharide lyase family 7 owing to high yields of penta-, hex-, and hepta-saccharides in the hydrolysis products of Alg2A. Thus, Alg2A could be a good tool for the large-scale preparation of alginate oligosaccharides with high DP.

High-level expression of leghemoglobin in Kluyveromyces marxianus by remodeling the heme metabolism pathway.

Soy leghemoglobin, when bound to heme, imparts a meat-like color and flavor and can serve as a substitute for animal-derived proteins. Enhancing cellular heme synthesis improves the recombinant expression of leghemoglobin in yeast. To achieve high-level expression of leghemoglobin A (LBA) in Kluyveromyces marxianus, a food-safe yeast, large-scale heme synthesis modules were transferred into K. marxianus using yeast artificial chromosomes (KmYACs). These modules contained up to 8 native and heterologous genes to promote the supply of heme precursors and downstream synthesis. Next, eight genes inhibiting heme or LBA synthesis were individually or combinatorially deleted, with the lsc1Δssn3Δ mutant yielding the best results. Subsequently, heme synthesis modules were combined with the lsc1Δssn3Δ mutant. In the resulting strains, the module genes were all actively expressed. Among these module genes, heterologous S. cerevisiae genes in the downstream heme synthesis pathway significantly enhanced the expression of their counterparts in K. marxianus, resulting in high heme content and LBA yield. After optimizing the medium recipe by adjusting the concentrations of glucose, glycine, and FeSO4·7H2O, a heme content of 66.32 mg/L and an intracellular LBA titer of 7.27 g/L were achieved in the engineered strain in a 5 L fermentor. This represents the highest intracellular expression of leghemoglobin in microorganisms to date. The leghemoglobin produced by K. marxianus can be utilized as a safe ingredient for plant-based protein products.

Biochemical and kinetic characterization of GH43 ß-D-xylosidase/α-L-arabinofuranosidase and GH30 α-L-arabinofuranosidase/ß-D -xylosidase from rumen metagenome.

The present study focuses on characterization of two hemicellulases, RuXyn1 and RuXyn2, from rumen bacterial metagenome and their capabilities for degradation of xylans. Glycosyl hydrolase (GH) family 43 ß-D -xylosidase/α-L -arabinofuranosidase RuXyn1 can hydrolyze p-nitrophenyl-ß-D -xylopyranoside (pNPX), p-nitrophenyl-α-L -arabinofuranoside (pNPA), and xylo-oligosaccharide substrates, while GH30 1,5-α-L -arabinofuranosidase/ß-D -xylosidase RuXyn2, the first α-L -arabinofuranosidase assigned to this GH family, shows activities towards 1,5-α-L -arabinobiose and pNPX substrates but no activity for pNPA. Kinetic analysis for aryl-glycosides revealed that RuXyn2 had higher catalytic efficiency than RuXyn1 toward pNPX substrate. RuXyn1 shows high synergism with endoxylanase, elevating by 73% the reducing sugars released from brichwood xylans, and converted most intermediate xylo-oligosaccharide hydrolysate into xylose. The high xylose conversion capability of RuXyn1 suggests it has potential applications in enzymatic production of xylose and improvement of hemicellulose saccharification for production of biofuels. RuXyn2 shows no obviously synergistic effect in the endoxylanase-coupled assay for enzymatic saccharification of xylan. Further cosmid DNA sequencing revealed a neighboring putative GH43 α-L -arabinofuranosidase RuAra1 and two putative GH3 ß-xylosidase/arabinosidases, RuXyn3 and RuXyn5, downstream of RuXyn2, indicating that this hemicellulase gene cluster may be responsible for production of end-product, xylose and arabinose, from hemicellulose biomass.

Amino acid substitutions in the N-terminus, cord and α-helix domains improved the thermostability of a family 11 xylanase XynR8.

The thermostability of xylanase XynR8 from uncultured Neocallimastigales rumen fungal was improved by combining random point mutagenesis with site-directed mutagenesis guided by rational design, and a thermostable variant, XynR8_VNE, was identified. This variant contained three amino acid substitutions, I38V, D137N and G151E, and showed an increased melting temperature of 8.8 °C in comparison with the wild type. At 65 °C the wild-type enzyme lost all of its activity after treatment for 30 min, but XynR8_VNE retained about 65 % activity. To elucidate the mechanism of thermal stabilization, three-dimensional structures were predicted for XynR8 and its variant. We found that the tight packing density and new salt bridge caused by the substitutions may be responsible for the improved thermostability. These three substitutions are located in the N-terminus, cord and α-helix domains, respectively. Hence, the stability of these three domains may be crucial for the thermostability of family 11 xylanases.

[Isolation and characterization of an alkaline xylanasefrom a newly isolated Bacillus sp. QH14].

Twenty-fivealkaline xylanase producing strains were isolated from Qinghai Lake side soil samples. Among these strains, QH14 produced 648.79 U/mLxylanase, and the enzymatic specific activity was 1148.56 U/mg after purification. This alkaline xylanase producing strain belongs to genus Bacillus based on16S rDNA sequencing analysis and then was designated as Bacillus sp. QH14. The alkalinexylanaseencoding gene, XynQH14, was cloned from Bacillus sp. QH14 and expressed in Escherichiacoli BL21 (DE3). The specific activity of the recombinant xylanase XynQH14 was 700.47 Umg-1 after purification by Ni-NTA affinity chromatography. The optimal temperature and pH of XynQH14 were 60℃ and pH9.2, respectively. Its activity was 50% of initial activity after incubation at 55 ℃ for 1h, 80% at pH7-11 at 37 ℃ for 24 h, and 31.02% at pH11 at 50℃ after 24 h, indicating that XynQH14 isthermostable and alkali-stable. These properties ofXynQH14 suggest its favorable potential applications in pulp and paper industries.

Characterization of essential elements for improved episomal expressions in Kluyveromyces marxianus.

Kluyveromyces marxianus is a promising cell factory for producing heterologous proteins. Essential components including partition element, copy number control element, promoter, and terminator in multicopy plasmids applicable for K. marxianus are not extensively characterized. The pKD1 is an endogenous multicopy plasmid identified in K. lactis, and the pKD1-based plasmid is the only multicopy plasmid successfully applied in K. marxianus. The circular plasmid pKD1 contains three major open reading frames, namely A, B, and C. Here, we showed that the A gene was responsible for maintaining the high-copy number of pKD1-based plasmid in K. marxianus. Deletion of the B or C gene impaired the stable propagation of pKD1-based plasmid in K. marxianus, and this defect could not be rescued by the trans expression of B and C genes. In a quantitative analysis of a series of promoters and terminators, AFT1 promoter from Pichia pastoris, OM45 promoter and INU1 terminator from K. marxianus, and a set of synthetic terminators, supported high-level expressions. A combination of AFT1 or OM45 promoter with INU1 terminator in a pKD1-based plasmid achieved high-level expressions of four different heterologous proteins. Our study provides valuable elements for high-level episomal expressions in K. marxianus.

Correlation Between Improved Mating Efficiency and Weakened Scaffold-Kinase Interaction in the Mating Pheromone Response Pathway Revealed by Interspecies Complementation.

Scaffold protein Ste5 and associated kinases, including Ste11, Ste7, and Fus3, are core components of the mating pheromone pathway, which is required to induce a mating response. Orthologs of these proteins are widely present in fungi, but to which extent one protein can be replaced by its ortholog is less well understood. Here, interspecies complementation was carried out to evaluate the functional homology of Ste5 and associated kinases in Kluyveromyces lactis, K. marxianus, and Saccharomyces cerevisiae. These three species occupy important positions in the evolution of hemiascomycetes. Results indicated that Ste5 and associated kinases in K. lactis and K. marxianus could be functionally replaced by their orthologs to different extents. However, the extent of sequence identity, either between full-length proteins or between domains, did not necessarily indicate the extent of functional replaceability. For example, Ste5, the most unconserved protein in sequence, achieved the highest average functional replaceability. Notably, swapping Ste5 between K. lactis and K. marxianus significantly promoted mating in both species and the weakened interaction between the Ste5 and Ste7 might contribute to this phenotype. Consistently, chimeric Ste5 displaying a higher affinity for Ste7 decreased the mating efficiency, while chimeric Ste5 displaying a lower affinity for Ste7 improved the mating efficiency. Furthermore, the length of a negatively charged segment in the Ste7-binding domain of Ste5 was negatively correlated with the mating efficiency in K. lactis and K. marxianus. Extending the length of the segment in KlSte5 improved its interaction with Ste7 and that might contribute to the reduced mating efficiency. Our study suggested a novel role of Ste5-Ste7 interaction in the negative regulation of the pheromone pathway. Meanwhile, Ste5 mutants displaying improved mating efficiency facilitated the breeding and selection of Kluyveromyces strains for industrial applications.

Downregulation of ammonium uptake improves the growth and tolerance of Kluyveromyces marxianus at high temperature.

The growth and tolerance of Kluyveromyces marxianus at high temperatures decreased significantly in the synthetic medium (SM), which is commonly used in industrial fermentations. After 100 days of adaptive laboratory evolution, a strain named KM234 exhibited excellent tolerance at a high temperature, without loss of its growth ability at a moderate temperature. Transcriptomic analysis revealed that the KM234 strain decreased the expression of the ammonium (NH4+ ) transporter gene MEP3 and increased the synthesis of the amino acid carbon backbone, which may contribute greatly to the high-temperature growth phenotype. High NH4+ content in SM significantly increased the reactive oxygen species (ROS) production at high temperatures and thus caused toxicity to yeast cells. Replacing NH4+ with organic nitrogen sources or increasing the concentration of potassium ions (K+ ) in the medium restored the growth of the wild-type K. marxianus at a high temperature in SM. We also showed that the NH4+ toxicity mitigated by K+ might closely depend on the KIN1 gene. Our results provide a practical solution to industrial fermentation under high-temperature conditions.

Comparative metabolome and transcriptome analyses of the properties of Kluyveromyces marxianus and Saccharomyces yeasts in apple cider fermentation.

This study explored the application of Kluyveromyces marxianus and Saccharomyces cerevisiae (commercial and wild type) in the alcoholic fermentation of Fuji apple juice under static conditions. Metabolome analyses revealed that ethyl esters, including ethyl hexanoate, ethyl decanoate, ethyl octanoate, octanoic acid and decanoic acid, were the dominant components in ciders fermented by the Saccharomyces yeasts. In the K. marxianus ciders, ethyl acetate, hexyl acetate, propyl acetate and acetic acid were the most abundant volatiles, suggesting that the cider fermented by K. marxianus might have a fruitier smell. Transcriptome analyses were adapted to gain insight into the differential metabolite patterns between K. marxianus and S. cerevisiae during cider fermentation. GO and KEGG enrichments revealed that the metabolic pathways of glucose, organic acids and amino acids during cider fermentation were quite different between these two yeasts. The K. marxianus strain exhibited a higher rate of glycolysis and ethanol fermentation than did Saccharomyces yeasts under oxygen-limited conditions. It also reduced the metabolic flux of acetate into acetyl-CoA and then into the TCA cycle, increasing the syntheses of ethyl acetate and relevant esters, which may affect its cell growth under anaerobic conditions but enriched the taste and variety of aromas in apple cider.

Establishment of a Cre-loxP System Based on a Leaky LAC4 Promoter and an Unstable panARS Element in Kluyveromyces marxianus.

The Cre-loxP system produces structural variations, such as deletion, duplication, inversion and translocation, at specific loci and induces chromosomal rearrangements in the genome. To achieve chromosomal rearrangements in Kluyveromyces marxianus, the positions and sequences of centromeres were identified in this species for the first time. Next, a Cre-loxP system was established in K. marxianus. In this system, the Cre recombinase was expressed from a leaky LAC4 promoter in a plasmid to alleviate the cytotoxicity of Cre, and the unstable plasmid contained a panARS element to facilitate the clearance of the plasmid from the cells. By using LAC4 as a reporter gene, the recombination frequencies between loxP sites or loxPsym sites were 99% and 73%, respectively. A K. marxianus strain containing 16 loxPsym sites in the genome was constructed. The recombination frequency of large-scale chromosomal rearrangements between 16 loxPsym sites was up to 38.9%. Our study provides valuable information and tools for studying chromosomal structures and functions in K. marxianus.

Characterization of a bifunctional xylanase/endoglucanase from yak rumen microorganisms.

A new gene, RuCelA, encoding a bifunctional xylanase/endoglucanase, was cloned from a metagenomic library of yak rumen microorganisms. RuCelA showed activity against xylan and carboxymethylcellulose (CMC), suggesting bifunctional xylanase/endoglucanase activity. The optimal conditions for xylanase and endoglucanase activities were 65°C, pH 7.0 and 50°C, pH 5.0, respectively. In addition, the presence of Co(+) and Co(2+) can greatly improve RuCelA's endoglucanase activity, while inhibits its xylanase activity. Further examination of substrate preference showed a higher activity against barley glucan and lichenin than against xylan and CMC. Using xylan and barley glucan as substrates, RuCelA displayed obvious synergistic effects with ß-1,4-xylosidase and ß-1,4-glucosidase. Generation of soluble oligosaccharides from lignocellulose is the key step in bioethanol production, and it is greatly notable that RuCelA can produce xylo-oligosaccharides and cello-oligosaccharides in the continuous saccharification of pretreated rice straw, which can be further degraded into fermentable sugars. Therefore, the bifunctional RuCelA distinguishes itself as an ideal candidate for industrial applications.

High level expression of a truncated ß-mannanase from alkaliphilic Bacillus sp. N16-5 in Kluyveromyces cicerisporus.

A truncated alkaline ß-mannanase from alkaliphilic Bacillus sp. N16-5 (MAN330) was expressed and secreted in Kluyveromyces cicerisporus. The recombinant engineered strain for MAN330 production was stable during 80 generations, and the maximum yield of MAN330 reached 3,795 U/ml in 15 l fermenter. MAN330 exhibited similar pH optima, temperature optima, and substrate specificities to its full-length protein (MAN493). However, stability of MAN330 was about 7% higher than that of MAN493 from pH 9-11. MAN330 had about 10% higher stability than MAN493 from 60°C to 80°C.