Fermentations Of Pentoses By Kluyveromyces Marxianus

Construction of strains that are capable of efficiently utilizing pentoses represents great challenges for the development of lignocellulosic ethanol.In this paper,the fermentative performance of pentoses by six thermotolerant Kluyveromyces marxianus strains were studied.On this basis,three strains with significant differences in fermentative performance were selected to find out the key points that affected their pentose fermentation,and then the genetically modified K.marxianus for possible enhanced pentose utilization were further evaluated.Firstly,we compared the fermentation profiles of six different K.marxianus strains at 30 oC and 40 oC using low concentrations of xylose and arabinose as the sole carbon source,among which three strains(K.m 9009,K.m 1911 and K.m 1727)were selected to investigate their pentose consumption at higher concentrations.K.m 1727 is the prominent strain in xylose fermentation at both 30 oC and 40 oC,which consumed 32.94 g/L and 34.67 g/L xylose to produce 9.22 g/L and 19.25 g/L xylitol within five days,respectively,and almost no ethanol was detected;in contrast the slowest K.m 1911 fermented 29.29 g/L and 15.81 g/L xylose to produce both xylitol(0.95 g/L and 0.25 g/L)and ethanol(2.68 g/L and 0.92 g/L)within six days.As for arabinose fermentation,K.m 9009 showed an obvious advantage in sugar consumption at 30 oC,which consumed 30.70 g/L arabinose,and produced 13.85 g/L arabinitol within 7 days;while at 40 oC,the fastest sugar consumption was achieved by K.m 1727,which could consume 33.54 g/L arabinose,and produce 25.86 g/L arabinitol within 6 days.K.m 1911 has been still proved to be a poor arabinose consumer at both 30 oC and 40 oC,which manifested as both sugar utilization(7.17 g/L and 23.55 g/L)and arabinitol production(6.06 g/L and 17.96 g/L).In a word,these three strains displayed a significant differences in the fermentation of both xylose and arabinose.Secondly,we analyzed the differences of the fermentation performance between different strains at gene level,transcriptional level and intracellular metabolite level.At gene level,five genes(XR,XDH,XK and AR,LAD),encoding the key metabolic enzymes in three different yeasts,were sequenced,and results showed that the amino acid sequences have similarities of over 98% with the reference sequences reported in the literature.Hence,the differences between the three stains were not caused by mutated amino acids that lie in non-essential sites.Besides,at transcriptional level,we determined the gene expression levels of four key enzymes(XR,XDH,XK,ADH)in the xylose metabolism pathway of K.m 1727 and K.m 1911 at different fermentation time points by real-time PCR experiments.The results showed that,for thermotolerant yeast K.m 1727,the low expression levels of XDH and XK genes were the main factors leading to accumulation of xylitol.Finally,at the level of intracellular metabolites,we measured 13 intracellular metabolites of K.m 1727 and K.m 1911 in the xylose fermentation cycles.The results showed that xylose metabolism in K.m 1727 remained at the first step,and most of the xylose was converted to xylitol.Finally,K.m 1727-5,which showed the most promising potential to be applied in lignocellulosic ethanol,was chosen for genetic modification.A single overexpression of XDH and XK genes was achieved to promote the xylose metabolic pathway.XDH gene overexpression has been proved to significantly enhance the xylose fermentation,but the XK gene overexpression showed few advantages compared with the original strain.