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Developing a broad-range promoter set for metabolic engineering in the thermotolerant yeast Kluyveromyces marxianus.

ABSTRACT: Kluyveromyces marxianus is an emerging host for metabolic engineering. This thermotolerant yeast is the fastest growing eukaryote, has high flux through the TCA cycle, and can metabolize a broad range of C5, C6, and C12 carbon sources. In comparison to the common host Saccharomyces cerevisiae, this non-conventional yeast suffers from a lack of metabolic engineering tools to control gene expression over a wide transcriptional range. To address this issue, we designed a library of 25 native-derived promoters from K. marxanius CBS6556 that spans 87-fold transcriptional strength under glucose metabolism. Six promoters from the library were further characterized in both glucose and xylose as well as across various temperatures from 30 to 45 °C. The temperature study revealed that in most cases EGFP expression decreased with elevating temperature; however, two promoters, P _SSA3 and P _ADH1 , increased expression above 40 °C in both xylose and glucose. The six-promoter set was also validated in xylose for triacetic acid lactone (TAL) production. By controlling the expression level of heterologous 2-pyrone synthase (2-PS), the specific TAL titer increased over 8-fold at 37 °C. Cultures at 41 °C exhibited a similar TAL biosynthesis capability, while at 30 °C TAL levels were lower. Taken together, these results advance the metabolic engineering tool set in K. marxianus and further develop this new host for chemical biosynthesis.

SUBMITTER: Lang X

PROVIDER: S-EPMC7508702 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background:Taking 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. Results:To 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-?-glucanase RuCelA, endo-1,4-?-endoxylanase Xyn-CDBFV, and endo-1,4-?-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. Conclusions:The 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.

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Developing a broad-range promoter set for metabolic engineering in the thermotolerant yeast Kluyveromyces marxianus.

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