Abstract
BackgroundTaking into account its thermotolerance, high growth rate, and broad substrate spectrum, Kluyveromyces marxianus can be considered an ideal consolidated bioprocessing (CBP). A major obstacle to ethanol production using K. marxianus is the low production of lignocellulolytic enzymes, which reduces the cellulose hydrolysis and ethanol production. Thus, further improvement of enzyme expression and secretion is essential.ResultsTo improve the expression of lignocellulolytic enzymes, the inulinase promoter and signal sequence from K. marxianus was optimized through mutagenesis. A T(-361)A mutation inside the promoter, a deletion of AT-rich region inside 5′UTR (UTR∆A), and a P10L substitution in the signal sequence increased the secretory expression of the feruloyl esterase Est1E by up to sixfold. T(-361)A and UTR∆A increased the mRNA expression, while the P10L substitution extended the hydrophobic core of signal sequence and promoted secretion of mature protein. P10L and T(-361)A mutations increased secretory expressions of other types of lignocellulolytic enzymes by up to threefold, including endo-1,4-β-glucanase RuCelA, endo-1,4-β-endoxylanase Xyn-CDBFV, and endo-1,4-β-mannanase MAN330. During the fed-batch fermentation of strains carrying optimized modules, the peak activities of RuCelA, Xyn-CDBFV, MAN330, and Est1E reached 24 U/mL, 25,600 U/mL, 10,200 U/mL, and 1220 U/mL, respectively. Importantly, higher yield of enzymes by optimized promoter and signal sequence were achieved in all tested carbon sources, including the major end products of lignocellulose saccharification and fermentation, with growth on xylose resulting in the highest production.ConclusionsThe engineered promoter and signal sequence presented increased secretory expressions of different lignocellulolytic enzymes in K. marxianus by means of various carbon resources. Activities of lignocellulolytic enzymes in fed-batch fermentation were the highest activities reported for K. marxianus so far. Our engineered modules are valuable in producing lignocellulolytic enzymes by K. marxianus and in constructing efficient CBP strains for cellulosic ethanol production.
Highlights
Taking into account its thermotolerance, high growth rate, and broad substrate spectrum, Kluyveromyces marxianus can be considered an ideal consolidated bioprocessing (CBP)
Characterization of mutations that improve secreted protein production The longest inulinase promoter characterized so far started from 1053 bp upstream of the start codon [31], which is corresponding to − 1058 bp in the strain used in this study
To explore potential regions that contribute to the activity of the inulinase promoter, we used a region starting at 1136 bp 5′ of the start codon (Fig. 1a)
Summary
Taking into account its thermotolerance, high growth rate, and broad substrate spectrum, Kluyveromyces marxianus can be considered an ideal consolidated bioprocessing (CBP). Kluyveromyces marxianus is a homothallic hemiascomycetous yeast species commonly isolated in dairy products, grape, and henequen [2] It is an aerobic, Crabtree negative yeast, and generates energy from both respiratory metabolism and ethanol fermentation [3, 4]. High growth rate, and the capacity to assimilate inulin, lactose, and pentose sugars like xylose and arabinose, K. marxianus can be considered to be a potential alternative to Saccharomyces cerevisiae for the production of ethanol from cellulosic feedstocks [5]. E.g., C. bescii, can directly convert cellulosic and hemicellulosic compositions to ethanol without chemical pretreatment of the feedstock [10]. None of these cellulolytic microbes can tolerate high concentrations of ethanol (> 10%). A S. cerevisiae strain engineered to use hemicellulose via coexpression of the Trichoderma reesei xylanase (xyn2), the Aspergillus niger xylosidase (xlnD), the Scheffersomyces stipitis xylulose kinase (xyl3), and Bacteroides thetaiotaomicron isomerase (xylA), produced 9 g/L of ethanol after 40 days under anaerobic fermentation [14]
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