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Engineering Starch Kinases For Increased Biofuel Production Efficiency
Author(s) -
Rondon Alberto,
Emanuelle Shane
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.798.21
Subject(s) - starch , biochemistry , chemistry , kinase , biophysics , biology
The growth of the bioethanol industry over the last decade has been a result of the growing demand for gasoline fuels, which in the United States constitutes at least 10% by volume, and the implementation of higher Renewable Fuel Standards (RFS). Bioethanol is produced by the fermentation of simple sugars by yeasts and as a result starch feedstocks must undergo pretreatment steps. The various mechanical, thermal and chemical treatments used to degrade starch are both expensive and energy intensive. Alternatively, plants use a reversible phosphorylation mechanism to solubilize starch and access stored glucose for nocturnal metabolism. Starch is formed by linear α‐1,4 linked glucose units and branching glucose via α‐1,6 bonds which form insoluble helical structures. Starch kinases phosphorylate the crystalline layers, disrupting the helical structures, and exposing the polymer to hydrolysis by amylases. This mechanism could be adopted during biofuels production to decrease the need for solubilizing treatments. Starch kinases are characterized by two distinct domains: carbohydrate binding modules (CBMs) and a dikinase domain connected via a peptide linker. The CBM functions to locate the kinase on the starch substrate, which is subsequently phosphorylated by the kinase domain. In order to expand the substrate affinity of a kinase, we fused the CBM from a Neurospora crassa starch‐active polysaccharide monooxygenase to multiple dikinase domains. We defined the melting temperature of the chimeric proteins using differential scanning fluorimetry and found that the chimeric proteins had increased stability. Using [33P‐β‐]ATP, we radiolabeled starch with the chimeric proteins and found that they exhibited at least 7‐fold greater activity than wild type dikinase using maize, barley, and potato starch as the substrate. Support or Funding Information NSF IIA‐1355438 (R. Andrews), NSF MCB‐1252345 (M. Gentry) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .