Abstract
Cyanobacteria are potential hosts for the biosynthesis of oleochemical compounds. The metabolic precursors for such compounds are fatty acids and their derivatives, which require chemical activation to become substrates in further conversion steps. We characterized the acyl activating enzyme AAE15 of Arabidopsis encoded by At4g14070, which is a homologue of a cyanobacterial acyl-ACP synthetase (AAS). We expressed AAE15 in insect cells and demonstrated its AAS activity with medium chain fatty acid (C10–C14) substrates in vitro. Furthermore, we used AAE15 to complement a Synechocystisaas deletion mutant and showed that the new strain preferentially incorporates supplied medium chain fatty acids into internal lipid molecules. Based on this data we propose that AAE15 can be utilized in metabolic engineering strategies for cyanobacteria that aim to produce compounds based on medium chain fatty acids.
Highlights
In recent years metabolic engineering has benefited from advances in gene synthesis and assembly that allow the implementation of complex biosynthetic pathways into a variety of microorganisms (Keasling 2012; Yadav et al 2012; Seo et al 2013)
In Arabidopsis enzymes capable of activating fatty acids belong to a superfamily of acyl-activating enzymes (AAEs), which consists of 63 members, and is divided into seven clades based on sequence similarities (Shockey et al 2003)
AAE15 has ACP synthetase (AAS) activity in vitro, with specificity for medium chain fatty acids To determine the in vitro enzymatic activity of AAE15, the protein fused to an N-terminal polyhistidine-tag was expressed in a Baculovirus system
Summary
In recent years metabolic engineering has benefited from advances in gene synthesis and assembly that allow the implementation of complex biosynthetic pathways into a variety of microorganisms (Keasling 2012; Yadav et al 2012; Seo et al 2013). One focus of current research is the establishment of biosynthetic pathways for production a variety of oleo compounds such as fatty acids, alcohols, and alkanes in hosts such as yeast, Escherichia coli, and cyanobacteria (Steen et al 2010; Lennen and Pfleger 2013; Pfleger et al 2015; Savakis and Hellingwerf 2015). Clade I AAEs differ from all other AAEs by the presence of an amino acid stretch separating two highly conserved sequence motifs This amino acid linker is Kaczmarzyk et al AMB Expr (2016) 6:7 remarkably longer in the two remaining proteins of clade I, for which initial tests were unable to proof LACS activity (Shockey et al 2002). These proteins called AAE15 and AAE16 and encoded by At4g14070 and At3g23790, respectively, include an amino acid linker of approximately 70 amino acid residues, compared to about 40 amino acids found in eukaryotic LACSs (Shockey et al 2002)
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