Random mutagenesis of atfA and screening for Acinetobacter baylyi mutants with an altered lipid accumulation

Abstract

Acinetobacter baylyi synthesizes significant amounts of wax esters (WE) and triacylglycerols (TAG) catalyzed by wax ester synthase/acyl‐CoA:diacylglycerol acyltransferase (WS/DGAT or AtfA), representing the key enzyme for bacterial lipid accumulation. However, the structure and exact biochemical mechanism of AtfA could not be elucidated, yet. Therefore, a combination of random mutagenesis, screening and sequencing of atfA gene variants was conducted to gain further insights into the relationship between sequence and function of the enzyme. Several mutations could be detected which seriously diminished lipid accumulation in A. baylyi as well as AtfA activity in recombinant E. coli strains, such as Glu15Lys, Trp67Gly, Ala126Asp, Ser374Pro, or Gly378Ser/Asp. The affected residues are more or less conserved among a wide range of AtfA homologs. Especially the highly conserved pattern SNVPGP seems to be crucial, as mutations inside this pattern drastically impair enzyme activity. Furthermore, it became obvious that the C‐terminal part of AtfA is indispensable for activity, although the catalytic core is located in the N‐terminal half of the enzyme. In silico studies suggest that the C‐terminus might form a coiled‐coil fold which could putatively represent the dimerization domain. Practical applications: WE, composed of a long‐chain acyl moiety and a fatty alcohol residue, are valuable ingredients of many commercial products like cosmetics, medical products or lubricants. However, natural sources for high‐quality WE are currently mainly restricted to the expensive oil of the jojoba plant or to carnauba wax. As A. baylyi naturally accumulates WE with a similar composition to jojoba‐oil, this organisms and the responsible acyltransferase AtfA, are of great interest regarding a sustainable biotechnological WE production. However, a lack of structural knowledge about this enzyme family currently constricts promising enzyme optimization approaches. The insights gained by random mutagenesis of AtfA can build a basis for further site‐directed mutagenesis approaches in order to optimize its activity, stability, and/or substrate range.

Use of Limited Proteolysis and Mutagenesis To Identify Folding Domains and Sequence Motifs Critical for Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase Activity

“…Thus, S388 and N389 could be the residues interacting with the acyl-CoA oxyanion for a proper binding of acyl-CoA. In fact, a point mutation in the equivalent serine of WS/DGAT AtfA (S374P) resulted in significant reduction of activity in A. baylyi (51). The essential role of R305 and the residues forming motifs I and II is consistent with the acyl-CoA-dependent acyltransferase fold.…”

Heterologous expression of a thermophilic diacylglycerol acyltransferase triggers triglyceride accumulation in Escherichia coli

“…The protein sequence of tDGAT was aligned with representative members of the WS/DGAT family ( S1 Fig ) through the software T-Coffee [ 31 ]. tDGAT contains all the conserved motifs characteristic of WS/DGAT [ 8 , 32 ]: mainly the catalytic site 140 HHaavDG 146 , motif I 118 PLW 120 and motif II 281 ND 282 . Like in other acyl-CoA-dependent acyltransferases [ 33 ] the catalytic motif 140 HHaavDG 146 , in the N-terminal domain of tDGAT, is predicted to be located in the hydrophobic pocket or channel that restricts the accessibility of hydrophilic substrates.…”

Genetic Regulation of Virulence and Antibiotic Resistance in Acinetobacter baumannii

“…is a small protein (67–68 amino acids) that was first functionally described in A. baylyi ADP1 [ 71 ]. The gene has been unfortunately termed atfA , which is a name shared with the wax ester synthase/acyl-CoA:diacylglycerol acetyltransferase enzyme in A. baylyi ADP1 [ 117 , 118 ]. Deletion of atfA transcription factor resulted in gene expression changes of over 500 genes including several virulence-associated traits.…”