Characterization and reconstitution of a new fungal type III polyketide synthase from Aspergillus oryzae

• Published in: Enzyme and microbial technology Vol. 46; no. 7; pp. 575 - 580
• Main Authors: Yu, Dayu, Zeng, Jia, Chen, Dong, Zhan, Jixun
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Inc 01-06-2010; Elsevier
• Subjects: Aspergillus oryzae; Biological and medical sciences; Biotechnology; CsyA; Enzymes; Fundamental and applied biological sciences. Psychology; In vivo biosynthesis; Pyrone synthase; Starter units; Type III polyketide synthase; CsyA; Pyrone synthase; Starter units; Aspergillus oryzae; Type III polyketide synthase; In vivo biosynthesis; Enzyme; Biosynthesis; Synthase; Characterization; Fungi; In vivo; Reconstitution; Fungi Imperfecti
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2010.02.011

A putative type III polyketide synthase (PKS) gene,
csyA, was cloned from
Aspergillus oryzae. It was ligated into pET28a and expressed in
Escherichia coli BL21-CodonPlus (DE3)-RIL. CsyA was identified as a pyrone synthase which has displayed broad substrate specificity toward fatty acyl-CoA starter units to yield triketide and tetraketide pyrones. It synthesizes triketide pyrones by adding two units of malonyl-CoA to the short chain starter units (C4–C8 CoAs), while yields both triketide and tetraketide pyrones from longer fatty acyl starters (C10–C18 CoAs). A series of fatty acyl N-acetylcysteamine thioesters (SNACs) (C5–C9) including odd-numbered fatty acyl starters were synthesized, all of which can be accepted by CsyA as the starter units to synthesize pyrones, indicating that this enzyme has broad substrate specificity toward fatty acyl starters and can be used to synthesize some unnatural pyrones. The optimal temperature and pH for CsyA are 55
°C and 7.5, respectively. Under the optimal enzymatic conditions, CsyA exhibits high activity toward C6 and C7 starter units, and the
k
cat/
K
m
values for hexanoyl- and heptanoyl-SNAC are 291.21 and 1615.58
s
−1
M
−1, respectively. In addition to its
in vitro activity, CsyA can also take fatty acid biosynthetic intermediates to synthesize a series of fatty acyl-primed pyrones in
E. coli cells.