Heterologous expression of Spathaspora passalidarum xylose reductase and xylitol dehydrogenase genes improved xylose fermentation ability of Aureobasidium pullulans

Abstract Background Aureobasidium pullulans is a yeast-like fungus that can ferment xylose to generate high-value-added products, such as pullulan, heavy oil, and melanin. The combinatorial expression of two xylose reductase (XR) genes and two xylitol dehydrogenase (XDH) genes from Spathaspora passalidarum and the heterologous expression of the Piromyces sp. xylose isomerase (XI) gene were induced in A. pullulans to increase the consumption capability of A. pullulans on xylose. Results The overexpression of XYL1.2 (encoding XR) and XYL2.2 (encoding XDH) was the most beneficial for xylose utilization, resulting in a 17.76% increase in consumed xylose compared with the parent strain, whereas the introduction of the Piromyces sp. XI pathway failed to enhance xylose utilization efficiency. Mutants with superior xylose fermentation performance exhibited increased intracellular reducing equivalents. The fermentation performance of all recombinant strains was not affected when glucose or sucrose was utilized as the carbon source. The strain with overexpression of XYL1.2 and XYL2.2 exhibited excellent fermentation performance with mimicked hydrolysate, and pullulan production increased by 97.72% compared with that of the parent strain. Conclusions The present work indicates that the P4 mutant (using the XR/XDH pathway) with overexpressed XYL1.2 and XYL2.2 exhibited the best xylose fermentation performance. The P4 strain showed the highest intracellular reducing equivalents and XR and XDH activity, with consequently improved pullulan productivity and reduced melanin production. This valuable development in aerobic fermentation by the P4 strain may provide guidance for the biotransformation of xylose to high-value products by A. pullulans through genetic approach

A Seamless Gene Deletion Method and Its Application for Regulation of Higher Alcohols and Ester in Baijiu Saccharomyces cerevisiae

The security of engineering Saccharomyces cerevisiae is becoming more focused on industrial production in consideration of the public concern regarding genetically modified organisms. In this work, a rapid and highly efficient system for seamless gene deletion in S. cerevisiae was developed through two-step integration protocol combined with endonuclease I-SCEI expression. The factors affecting the frequency of the second homologous recombination were optimized, and studies indicated that the mutant strains with 500 bp direct repeats and that have been incubating in galactose (0.5 g/100 mL) medium at 30°C and 180 r/min for 24 h permit high frequency (6.86 × 10−4) of the second homologous recombination. Furthermore, DNA sequence assays showed only self-DNA in native location without any foreign genes after deletion using this method. The seamless gene deletion method was applied to the construction of the engineering strains with BAT2 (encoding aminotransferase) deletion and ATF1 (alcohol acetyltransferases) overexpression. The mutants exhibited significant effects on higher alcohol reduction and ester improvement after Baijiu fermentation. The engineered strains can be used in industrial production in security, thereby meeting the requirements of modern science and technology

Negative Roles of a Novel Nitrogen Metabolite Repression-Related Gene, TAR1, in Laccase Production and Nitrate Utilization by the Basidiomycete Cryptococcus neoformans▿

The multicopper oxidase laccase is widespread in fungi and has great industrial importance. One puzzle regarding laccase production in the basidiomycetous yeast Cryptococcus neoformans is that it is inhibited by high temperature (e.g., 37°C). In this paper, we report identification of a nitrogen metabolite repression-related gene, TAR1, which is responsible for laccase repression. Disruption of TAR1 results in a significant increase in the level of LAC1 mRNA at 37°C. The putative protein Tar1 shares a moderate level of similarity with the nitrogen metabolite repressors Nmr1 and NmrA from Neurospora crassa and Aspergillus nidulans, respectively. Likewise, Tar1 has a negative role in the utilization of nitrate. Furthermore, the structure of Tar1 is unique. Tar1 lacks the long C-terminal region of Nmr1 and NmrA. It contains the canonical Rossmann fold motif, GlyXXGlyXXGly, whereas Nmr1 and NmrA have variable residues at the Gly positions. Interestingly, the promoter region of TAR1 contains three TTC/GAA repeats which are likely the heat shock factor (Hsf) binding sites, implying that Hsf has a role in laccase inhibition. TAR1 mediation of temperature-associated repression of LAC1 suggests a novel mechanism of laccase regulation and a new function for Nmr proteins. Our work may be helpful for industry in terms of promotion of laccase activity

Metabolic engineering of the thermophilic filamentous fungus Myceliophthora thermophila to produce fumaric acid

Abstract Background Fumaric acid is widely used in food and pharmaceutical industries and is recognized as a versatile industrial chemical feedstock. Increasing concerns about energy and environmental problems have resulted in a focus on fumaric acid production by microbial fermentation via bioconversion of renewable feedstocks. Filamentous fungi are the predominant microorganisms used to produce organic acids, including fumaric acid, and most studies to date have focused on Rhizopus species. Thermophilic filamentous fungi have many advantages for the production of compounds by industrial fermentation. However, no previous studies have focused on fumaric acid production by thermophilic fungi. Results We explored the feasibility of producing fumarate by metabolically engineering Myceliophthora thermophila using the CRISPR/Cas9 system. Screening of fumarases suggested that the fumarase from Candida krusei was the most suitable for efficient production of fumaric acid in M. thermophila. Introducing the C. krusei fumarase into M. thermophila increased the titer of fumaric acid by threefold. To further increase fumarate production, the intracellular fumarate digestion pathway was disrupted. After deletion of the two fumarate reductase and the mitochondrial fumarase genes of M. thermophila, the resulting strain exhibited a 2.33-fold increase in fumarate titer. Increasing the pool size of malate, the precursor of fumaric acid, significantly increased the final fumaric acid titer. Finally, disruption of the malate–aspartate shuttle increased the intracellular malate content by 2.16-fold and extracellular fumaric acid titer by 42%, compared with that of the parental strain. The strategic metabolic engineering of multiple genes resulted in a final strain that could produce up to 17 g/L fumaric acid from glucose in a fed-batch fermentation process. Conclusions This is the first metabolic engineering study on the production of fumaric acid by the thermophilic filamentous fungus M. thermophila. This cellulolytic fungal platform provides a promising method for the sustainable and efficient-cost production of fumaric acid from lignocellulose-derived carbon sources in the future

MOESM1 of Heterologous expression of Spathaspora passalidarum xylose reductase and xylitol dehydrogenase genes improved xylose fermentation ability of Aureobasidium pullulans

Additional file 1: Table S1. Primers used for genetic manipulation of A. pullulans var. melanogenum CBS 110374

Isolation and Characterization of a Marine Microalga for Biofuel Production with Astaxanthin as a Co-Product

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Reduced Production of Higher Alcohols by <i>Saccharomyces cerevisiae</i> in Red Wine Fermentation by Simultaneously Overexpressing <i>BAT1</i> and Deleting <i>BAT2</i>

In red wine, the contents of higher alcohols and ethyl carbamate (EC) are two significant health concerns. To reduce the production of higher alcohols by wine yeast YZ22 with low production of EC, one <i>BAT2</i> was replaced by a <i>BAT1</i> expression cassette first and then another <i>BAT2</i> was deleted to obtain the mutant SYBB3. Real-time quantitative PCR showed that the relative expression level of <i>BAT1</i> in SYBB3 improved 28 times compared with that in YZ22. The yields of isobutanol and 3-methyl-1-butanol produced by mutant SYBB3 reduced by 39.41% and 37.18% compared to those by the original strain YZ22, and the total production of higher alcohols decreased from 463.82 mg/L to 292.83 mg/L in must fermentation of Cabernet Sauvignon. Meanwhile, there were no obvious differences on fermentation characteristics of the mutant and parental strain. This research has suggested an effective strategy for decreasing production of higher alcohols in <i>Saccharomyces cerevisiae</i>