Cloning of a novel levoglucosan kinase gene from Lipomyces starkeyi and its expression in Escherichia coli |
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Authors: | Jianghong Dai Zhisheng Yu Yongzhi He Ling Zhang Zhihui Bai Zhiyang Dong Yuguo Du Hongxun Zhang |
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Institution: | (1) College of Resources & Environment, Graduate University of the Chinese Academy of Sciences, 100049 Beijing, People’s Republic of China;(2) Institute of Microbiology, Chinese Academy of Sciences, 100101 Beijing, People’s Republic of China;(3) Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 100085 Beijing, People’s Republic of China |
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Abstract: | Levoglucosan, cellulosic pyrolysate, is converted to glucose-6-phosphate by a specific levoglucosan kinase in fungi. A novel
cDNA of levoglucosan kinase gene (lgk) from yeast Lipomyces starkeyi YZ-215 was isolated by RACE method. The 1,445 bp cDNA fragment (lgk) harbouring the kinase gene exhibited one open reading frame (ORF) composed of 1,317 bp flanked by a 14 bp 5′-UTR and a 114 bp
3′-UTR, including a 25 bp poly(A) tail. The ORF encoded a 439 amino acid polypeptide with a 48.4 kDa predicted molecular mass.
Analysis of amino sequence revealed that the kinase belonged to the bacterial anhydro-N-acetylmuramic acid kinase (AnmK) family, and kinase-like proteins existed in some fungi, especially in filamentous fungi
such as Aspergillus. The kinase gene was transformed into Escherichia coli BL21 (DE3), recombinant E. coli could grow in M9 minimal medium with levoglucosan as a sole carbon source when induced by IPTG. In addition, the recombinant
kinase was overexpressed, purified and characterized. The kinase was stable at pH 7–10 and showed maximum activity at 30°C
and pH 9.0 as natural kinase, but presented higher thermostability. Kinetic constants (apparent K
m values) for LG and ATP were 105.3 ± 12.5 and 0.20 ± 0.02 mM, respectively. Furthermore, the kinase showed substrate specificity
for LG. This novel levoglucosan kinase gene would be useful in constructing recombinant microbial strains for the efficient
bioconversion of cellulosic pyrolysate to ethanol.
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Keywords: | Levoglucosan kinase gene Lipomyces starkeyi YZ-215 RACE Cellulose pyrolysis |
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