Sneha Srikrishnan

University of California, Irvine, Irvine, California, United States

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Publications (3)10.79 Total impact

  • Sneha Srikrishnan, Wilfred Chen, Nancy A Da Silva
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    ABSTRACT: Five trimeric xylanosomes were successfully assembled on the cell surface of Saccharomyces cerevisiae. Three dockerin-tagged fungal enzymes, an endoxylanase (XynAc) from Thermomyces lanuginosus, a β-xylosidase (XlnDt) from Aspergillus niger and an acetylxylan esterase (AwAXEf) from Aspergillus awamori, were displayed for the synergistic saccharification of birchwood xylan. The surface-expression scaffoldins were modular constructs with or without carbohydrate binding modules from Thermotoga maritima (family 22) or Clostridium thermocellum (family 3). The synergy due to enzyme-enzyme and enzyme-substrate proximity, and the effects of binding domain choice and position on xylan hydrolysis were determined. The scaffoldin-based enzymes (with no binding domain) showed a 1.6-fold increase in hydrolytic activity over free enzymes; this can be attributed to enzyme-enzyme proximity within the scaffoldin. The addition of a xylan binding domain from T. maritima improved hydrolysis by 2.1-fold relative to the scaffoldin without a binding domain (signifying enzyme-substrate synergy), and 3.3-fold over free enzymes, with a xylose productivity of 105 mg g(-1) substrate after 72 h hydrolysis. This system was also superior to the xylanosome carrying the cellulose binding module from C. thermocellum by 1.4-fold. Furthermore, swapping the xylan binding module position within the scaffoldin resulted in 1.5-fold more hydrolysis when the binding domain was adjacent to the endoxylanase. These results demonstrate the applicability of designer xylanosomes toward hemicellulose saccharification in yeast, and the importance of the choice and position of the carbohydrate binding module for enhanced synergy. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc.
    Biotechnology and Bioengineering 07/2012; · 4.16 Impact Factor
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    ABSTRACT: Variants of the Thermoascus aurantiacus Eg1 enzyme with higher catalytic efficiency than wild-type were obtained via site-directed mutagenesis. Using a rational mutagenesis approach based on structural bioinformatics and evolutionary analysis, two positions (F16S and Y95F) were identified as priority sites for mutagenesis. The mutant and parent enzymes were expressed and secreted from Pichia pastoris and the single site mutants F16S and Y95F showed 1.7- and 4.0-fold increases in k(cat) and 1.5- and 2.5-fold improvements in hydrolytic activity on cellulosic substrates, respectively, while maintaining thermostability. Similar to the parent enzyme, the two variants were active between pH 4.0 and 8.0 and showed optimal activity at temperature 70°C at pH 5.0. The purified enzymes were active at 50°C for over 12 h and retained at least 80% of initial activity for 2 h at 70°C. In contrast to the improved hydrolysis seen with the single mutation enzymes, no improvement was observed with a third variant carrying a combination of both mutations, which instead showed a 60% reduction in catalytic efficiency. This work further demonstrates that non-catalytic amino acid residues can be engineered to enhance catalytic efficiency in pretreatment enzymes of interest.
    Biotechnology and Bioengineering 12/2011; 109(6):1595-9. · 4.16 Impact Factor
  • Nancy A Da Silva, Sneha Srikrishnan
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    ABSTRACT: Metabolic pathway engineering in the yeast Saccharomyces cerevisiae leads to improved production of a wide range of compounds, ranging from ethanol (from biomass) to natural products such as sesquiterpenes. The introduction of multienzyme pathways requires precise control over the level and timing of expression of the associated genes. Gene number and promoter strength/regulation are two critical control points, and multiple studies have focused on modulating these in yeast. This MiniReview focuses on methods for introducing genes and controlling their copy number and on the many promoters (both constitutive and inducible) that have been successfully employed. The advantages and disadvantages of the methods will be presented, and applications to pathway engineering will be highlighted.
    FEMS Yeast Research 11/2011; 12(2):197-214. · 2.46 Impact Factor