Enhancement of fengycin production in Bacillus amyloliquefaciens by genome shuffling and relative gene expression analysis using RT-PCR

Genome shuffling is an efficient approach for the rapid engineering of microbial strains with desirable industrial phenotypes. In this study, we used genome shuffling in an attempt to improve fengycin production of the wild-type strain Bacillus amyloliquefaciens ES-2-4. After 2 rounds of genome shuffling, a high-yield recombinant F2-72 (FMB72) strain that exhibited 8.30-fold increases in fengycin production was obtained. Comparative analysis of synthetase gene (fenA) expression was conducted between the initial and shuffled strains using fluorescent quantitation RT-PCR. Delta CT (threshold cycle) relative quantitation analysis revealed that fengycin synthetase gene (fenA) expression at the transcriptional level in the FMB72 strain was 12.77-fold greater than in the ES-2-4 wild type. The shuffled strain has a potential application in food and pharmaceutical industries. At the same time, the analysis of improved phenotypes will provide more valuable data for inverse metabolic engineering.

Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of theWHI2Gene Identified through Inverse Metabolic Engineering

ABSTRACTDevelopment of acetic acid-resistantSaccharomyces cerevisiaeis important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge due to limited information on effective genetic perturbation targets for improving acetic acid resistance in the yeast. This study employed a genomic-library-based inverse metabolic engineering approach to successfully identify a novel gene target,WHI2(encoding a cytoplasmatic globular scaffold protein), which elicited improved acetic acid resistance inS. cerevisiae. Overexpression ofWHI2significantly improved glucose and/or xylose fermentation under acetic acid stress in engineered yeast. TheWHI2-overexpressing strain had 5-times-higher specific ethanol productivity than the control in glucose fermentation with acetic acid. Analysis of the expression ofWHI2gene products (including protein and transcript) determined that acetic acid induced endogenous expression of Whi2 inS. cerevisiae. Meanwhile, thewhi2Δ mutant strain had substantially higher susceptibility to acetic acid than the wild type, suggesting the important role of Whi2 in the acetic acid response inS. cerevisiae. Additionally, overexpression ofWHI2and of a cognate phosphatase gene,PSR1, had a synergistic effect in improving acetic acid resistance, suggesting that Whi2 might function in combination with Psr1 to elicit the acetic acid resistance mechanism. These results improve our understanding of the yeast response to acetic acid stress and provide a new strategy to breed acetic acid-resistant yeast strains for renewable biofuel production.

Activating and Elucidating Metabolism of Complex Sugars in Yarrowia lipolytica

ABSTRACTThe oleaginous yeastYarrowia lipolyticais an industrially important host for production of organic acids, oleochemicals, lipids, and proteins with broad biotechnological applications. Albeit known for decades, the unique native metabolism ofY. lipolyticafor using complex fermentable sugars, which are abundant in lignocellulosic biomass, is poorly understood. In this study, we activated and elucidated the native sugar metabolism inY. lipolyticafor cell growth on xylose and cellobiose as well as their mixtures with glucose through comprehensive metabolic and transcriptomic analyses. We identified 7 putative glucose-specific transporters, 16 putative xylose-specific transporters, and 4 putative cellobiose-specific transporters that are transcriptionally upregulated for growth on respective single sugars.Y. lipolyticais capable of using xylose as a carbon source, but xylose dehydrogenase is the key bottleneck of xylose assimilation and is transcriptionally repressed by glucose.Y. lipolyticahas a set of 5 extracellular and 6 intracellular β-glucosidases and is capable of assimilating cellobiose via extra- and intracellular mechanisms, the latter being dominant for growth on cellobiose as a sole carbon source. Strikingly,Y. lipolyticaexhibited enhanced sugar utilization for growth in mixed sugars, with strong carbon catabolite activation for growth on the mixture of xylose and cellobiose and with mild carbon catabolite repression of glucose on xylose and cellobiose. The results of this study shed light on fundamental understanding of the complex native sugar metabolism ofY. lipolyticaand will help guide inverse metabolic engineering ofY. lipolyticafor enhanced conversion of biomass-derived fermentable sugars to chemicals and fuels.

Improved Production of a Heterologous Amylase in Saccharomyces cerevisiae by Inverse Metabolic Engineering

ABSTRACTThe increasing demand for industrial enzymes and biopharmaceutical proteins relies on robust production hosts with high protein yield and productivity. Being one of the best-studied model organisms and capable of performing posttranslational modifications, the yeastSaccharomyces cerevisiaeis widely used as a cell factory for recombinant protein production. However, many recombinant proteins are produced at only 1% (or less) of the theoretical capacity due to the complexity of the secretory pathway, which has not been fully exploited. In this study, we applied the concept of inverse metabolic engineering to identify novel targets for improving protein secretion. Screening that combined UV-random mutagenesis and selection for growth on starch was performed to find mutant strains producing heterologous amylase 5-fold above the level produced by the reference strain. Genomic mutations that could be associated with higher amylase secretion were identified through whole-genome sequencing. Several single-point mutations, including an S196I point mutation in theVTA1gene coding for a protein involved in vacuolar sorting, were evaluated by introducing these to the starting strain. By applying this modification alone, the amylase secretion could be improved by 35%. As a complement to the identification of genomic variants, transcriptome analysis was also performed in order to understand on a global level the transcriptional changes associated with the improved amylase production caused by UV mutagenesis.