Mutation of Amino Acid Motif Involved in Stereoselectivity by Ketoreductases and Analysis of Its Product

doi:10.13560/j.cnki.biotech.bull.1985.2016-1195

Abstract

Abstract: In order to identify whether LDD motif in ketoreductase domain of polyketide synthase from Saccharopolyspora erythraea（EryKR）can account for the stereocontrol of 2-methylcyclohexanone reduction,we constructed the 4 recombinants,Escherichia coli BL21（pET28a-eryKR1）with heterologous expressing ketoreductase in the first module（EryKR1）of polyketide synthase, E.coli BL21（pET28a-eryKR2）with heterologous expressing ketoreductase in the second module（EryKR2）of polyketide synthase,E.coli BL21（pET28a-TeryKR1）of the site-mutated EryKR1 in which the nucleotide sequence coding for amino acid residues LDD replaced by PQQ,and E.coli BL21（pET28a-TeryKR2）of the site-mutated EryKR2 while PQQ replaced by LDD. SDS-PAGE demonstrated that ketoreductases were expressed in these 4 recombinants after induction by IPTG. Specific activity of crude enzyme was 1.49 U/mg,0.37 U/mg,0.94 U/mg and 0.31 U/mg,respectively. Gas chromatography analyses of 2-methylcyclohexanone reduction catalyzed by 4 recombinants showed that mutated recombinant E.coli BL21（pET28a-TeryKR1）mainly reduced 2-methylcyclohexanone to trans-2-methylcyclohexanol,unlike the main product of cis-2-methylcyclohexanol catalyzed by wild-type recombinant Escherichia coli BL21（pET28a-eryKR1）. Furthermore,main reduction product of mutant E.coli BL21（pET28a-TeryKR2）was cis-2-methylcyclohexanol,rather than the trans-2-methylcyclohexanol by wild-type recombinant E.coli BL21（pET28a-eryKR2）,confirming the key role of LDD motif in controlling the stereoselectivity of 2-methylcyclohexanone reduction.

Key words: polyketide synthase, ketoreductase, 2-methylcyclohexanone, mutation, stereoselectivity

LI Ling-ling, LÜ Zao-sheng, ZUO Zhen-yu, YANG Zhong-hua, LIU Yao-ning, SONG Cai-wei. Mutation of Amino Acid Motif Involved in Stereoselectivity by Ketoreductases and Analysis of Its Product[J]. Biotechnology Bulletin, 2017, 33(6): 214-222.

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