Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (11): 257-266.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1508

Previous Articles     Next Articles

Cloning,Expression and Characterization of a Novel Acyltransferase GPAT

LIU Shan1,2(), YE Wei2, ZHU Mu-zi2, LI Sai-ni2, DENG Zhang-shuang1(), ZHANG Wei-min2()   

  1. 1. College of Biology and Pharmacy,Three Gorges University,Yichang 443000
    2. Institute of Microbiology,Guangdong Academy of Sciences,State Key Laboratory of Applied Microbiology Southern China,Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application,Guangzhou 510070
  • Received:2020-12-13 Online:2021-11-26 Published:2021-12-03
  • Contact: DENG Zhang-shuang,ZHANG Wei-min E-mail:shanliu1995@126.com;dzs163@163.com;wmzhang@gdim.cn

Abstract:

Lithocarpins,a new polyketide skeleton compound isolated from marine fungus Phomopsis littocarpus,has the potential to be developed as lead compound of novel antitumor drugs. Based on genome sequencing and bioinformatics analysis,the biosynthetic gene cluster of lithocarpins was predicted. The unknown functional gene g7779 was amplified and successfully expressed in Escherichia coli. The recombinant protein was purified by Ni-NTA affinity chromatography. The function of the purified protein was analyzed by mass spectrometry,bioinformatics analysis combined with enzyme activity detection. The results showed that g7779 encoded a novel bi-functional enzyme(glutamic-pyruvic transaminase-acyltransferase,GPAT)with strong acyltransferase activity and certain glutamic pyruvic transaminase activity,its purity was 98.6%. Structural analysis showed that the protein was composed of 326 amino acids with a molecular weight of 36 kD. The secondary structure of GPAT consisted of 55.52% α-helix,29.45% random coil,9.82% extend strand and 5.21% β-turn. The activity of acyltransferase demonstrated that the optimal reaction temperature of GPAT for acetyl CoA was 35℃,and the highest enzyme activity was at pH 7.0. GPAT showed good thermal stability at 40℃,and the residual enzyme activity was > 80% after 2 h treatment at this temperature;but the thermal stability was poor at 50℃,and the residual enzyme activity was < 10% after 30 min. The values of Km and Vmax of GPAT towards acetyl CoA were 761.57 μmol/L and 29 370 μmol/(mg·min),respectively. The results may provide molecular biological basis for the elucidation of lithocarpins biosynthesis pathway.

Key words: marine fungus, Phomopsis lithocarpus, heterologous expression, acyltransferase, enzymatic properties, bioinformatics