[Fermentations of xylose and arabinose by Kluyveromyces marxianus].

Kluyveromyces marxianus, as unconventional yeast, attracts more and more attention in the biofuel fermentation. Although this sort of yeasts can ferment pentose sugars, the fermentation capacity differs largely. Xylose and arabinose fermentation by three K. marxianus strains (K. m 9009, K. m 1911 and K. m 1727) were compared at different temperatures. The results showed that the fermentation performance of the three strains had significant difference under different fermentation temperatures. Especially, the sugar consumption rate and alcohol yield of K. m 9009 and K. m 1727 at 40 ℃ were better than 30 ℃. This results fully reflect the fermentation advantages of K. marxianus yeast under high-temperature. On this basis, five genes (XR, XDH, XK, AR and LAD) coding key metabolic enzymes in three different yeasts were amplified by PCR, and the sequence were compared by Clustalx 2.1. The results showed that the amino acid sequences coding key enzymes have similarity of over 98% with the reference sequences reported in the literature. Furthermore, the difference of amino acid was not at the key site of its enzyme, so the differences between three stains were not caused by the gene level, but by transcribed or translation regulation level. By real-time PCR experiment, we determined the gene expression levels of four key enzymes (XR, XDH, XK and ADH) in the xylose metabolism pathway of K. m 1727 and K. m 1911 at different fermentation time points. The results showed that, for thermotolerant yeast K. m 1727, the low expression level of XDH and XK genes was the main factors leading to accumulation of xylitol. In addition, according to the pathway of Zygosaccharomyces bailii, which have been reported in NCBI and KEGG, the xylose and arabinose metabolic pathways of K. marxianus were identified, which laid foundation for further improving the pentose fermentation ability by metabolic engineering.

Enhanced fermentative performance under stresses of multiple lignocellulose-derived inhibitors by overexpression of a typical 2-Cys peroxiredoxin from Kluyveromyces marxianus.

BACKGROUND: Bioethanol from lignocellulosic materials is of great significance to the production of renewable fuels due to its wide sources. However, multiple inhibitors generated from pretreatments represent great challenges for its industrial-scale fermentation. Despite the complex toxicity mechanisms, lignocellulose-derived inhibitors have been reported to be related to the levels of intracellular reactive oxygen species (ROS), which makes oxidoreductase a potential target for the enhancement of the tolerance of yeasts to these inhibitors. RESULTS: A typical 2-Cys peroxiredoxin from Kluyveromyces marxianus Y179 (KmTPX1) was identified, and its overexpression was achieved in Saccharomyces cerevisiae 280. Strain TPX1 with overexpressed KmTPX1 gene showed an enhanced tolerance to oxidative stresses. Serial dilution assay indicated that KmTPX1 gene contributed to a better cellular growth behavior, when the cells were exposed to multiple lignocellulose-derived inhibitors, such as formic acid, acetic acid, furfural, ethanol, and salt. In particular, KmTPX1 expression also possessed enhanced tolerance to a mixture of formic acid, acetic acid, and furfural (FAF) with a shorter lag period. The maximum glucose consumption rate and ethanol generation rate in KmTPX1-expressing strain were significantly improved, compared with the control. The mechanism of improved tolerance to FAF depends on the lower level of intracellular ROS for cell survival under stress. CONCLUSION: A new functional gene KmTPX1 from K. marxianus is firstly associated with the enhanced tolerance to multiple lignocellulose-derived inhibitors in S. cerevisiae. We provided a possible detoxification mechanism of the KmTPX1 for further theoretical research; meanwhile, we provided a powerful potential for application of the KmTPX1 overexpressing strain in ethanol production from lignocellulosic materials.

[Heterologous expression, purification and characterization of exo-inulinase from Kluyveromyces marxianus YX01].

To improve the inulinase application in biotechnology, the characteristic of inulinase from Kluyveromyces marxianus YX01 was investigated. The inu gene of K. marxianus YX01 was transformed into Pichiapastoris GS115 host cells with molecular biology techniques. Then we achieved the heterologous expression of exo-inulinase whose molecular mass was about 86.0 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Furthermore, six His-tag was added to the inulinase and a two-step method was applied in the purification of inulinase, including concentration via dialysis by polyethylene glycol 20 000 and metal Ni-NTA Agarose affinity adsorption. The purification factor of purified protein was 3.6 and the recovery rate of enzyme activity was 33.1%. We characterized the purified inulinase. The optimum temperature was 60 degrees C and pH was 4.62. When inulin and sucrose were used as substrates, the K(m) and V(max) values were 80.53 g/L vs 4.49 g/(L x min) and 183.10 g/L vs 20.20 g/(L x min), respectively. In addition, metal ions including Mn2+, Ca2+, Cu2+, Zn2+ and Fe2+ exhibited different degrees of inhibition on the enzyme activity, and Cu2+, Zn2+ and Fe2+ exhibited the most significant inhibition. Our findings might lay a good foundation for industrial application of inulinase.

Transcriptional analysis of Kluyveromyces marxianus for ethanol production from inulin using consolidated bioprocessing technology.

BACKGROUND: Ethanol production from non-crop materials, such as Jerusalem artichokes, would make a great contribution to the energy industry. The non-conventional yeast, Kluyveromyces marxianus, is able to carry out ethanol fermentation of sugar molecules obtained from inulin-containing materials by consolidated bioprocessing. Lower inulin concentrations and micro-aeration can lead to a relatively fast and ideal fermentation process; however, it is unclear what causes the inhibition of higher concentrations of inulin and the promotion effect of aeration. RESULTS: Next-generation sequencing technology was used to study the global transcriptional response of K. marxianus Y179 under three fermentation conditions, including 120 g/L inulin without aeration (120-N), 230 g/L inulin without aeration (230-N), 230 g/L inulin with aeration by ORP controlling at -130 mV (230-130mV). A total of 35.55 million clean reads were generated from three samples, of which 4,820 predicted that open reading frames were annotated. For differential expression analysis, 950 and 1,452 differentially expressed genes were discovered under the conditions of 230-130mV and 120-N, respectively, and the sample 230-N was used as the control. These genes are mainly associated with the pathways of central carbon metabolism and ethanol formation. Increased expression of inulinase and the low activity of the autophagy-related gene, ATG8, ensured fast and ideal fermentation processes. CONCLUSIONS: Despite being reported as the "crabtree-negative" species, K. marxianus Y179 could achieve favorable ethanol fermentation profiles under micro-aeration and high inulin concentrations. K. marxianus Y179 cells responded to inulin concentrations and micro-aeration that is involved in the whole ethanol metabolism network. These results will serve as an important foundation for further exploration of the regulatory mechanisms involved in ethanol fermentation from inulin by consolidated bioprocessing and also provide a valuable reference for future studies on optimization and reconstruction of the metabolism network in K. marxianus.