Research article Directed evolution of acyl-CoA:diacylglycerol acyltransferase: Development and characterization of Brassica napus DGAT1 mutagenized libraries Rodrigo M.P. Siloto a,1 , Martin Truksa a,1 , Disa Brownfield a , Allen G. Good b , Randall J. Weselake a, * a Agricultural Lipid Biotechnology Program, Department of Agricultural, Food and Nutritional Science, University of Alberta, 4-10 Ag/For Centre, Edmonton, AB, Canada T6G 2E9 b Department of Biological Sciences, G-425, Biological Sciences Building, University of Alberta, Edmonton, AB, Canada T6G 2E9 article info Article history: Received 14 November 2008 Accepted 30 December 2008 Available online 9 January 2009 Keywords: Error-prone PCR High-throughput screening Membrane protein Nile red TAG synthase Triacylglycerol biosynthesis Saccharomyces cerevisiae abstract Metabolic flux to triacylglycerol (TAG) may be limited by the level of acyl-CoA:diacylglycerol acyl- transferase (DGAT, EC 2.3.1.20) activity. In some species, this enzyme also appears to play a role in the channeling of specific fatty acyl moieties into TAG. The objective of this work is to implement a directed evolution approach to enhance the catalytic efficiency of type-1 DGAT from Brassica napus (BnDGAT1). We generated randomly mutagenized libraries of BnDGAT1 in a yeast expression vector using error-prone PCR. The mutagenized libraries were used to transform a Saccharomyces cerevisiae strain devoid of neutral lipid biosynthesis and analyzed using a high-throughput screening (HTS) system. The HTS, recently developed for this purpose, consisted of a positive selection of clones expressing active DGAT mutants followed by quantification of DGAT activity by fluorescence detection of TAG in yeast cells. The initial results indicated that the positive selection system efficiently eliminated DGAT mutants lacking enzyme activity. Screening of 1528 selected mutants revealed that some DGAT clones had enhanced ability to synthesize TAG in yeast. This was confirmed by analysis of individual clones that could carry mutations resulting in an increased catalytic efficiency. The directed evolution approach could lead to the development of an improved plant DGAT1 for increasing seed oil content in oleaginous crops. Ó 2009 Elsevier Masson SAS. All rights reserved. 1. Introduction Triacylglycerol (TAG) biosynthesis in oilseeds is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) which utilizes sn-1,2-diacylglycerol (DAG) and acyl-CoA as substrates [24,34]. DGAT activity resides in at least two distinct membrane- bound polypeptides, referred to as DGAT1 [6,17] and DGAT2 [7,23,30]. The level of DGAT activity in the developing seed may have a substantial effect on the flow of carbon into TAG [5,27]. This hypothesis was confirmed by forward and reverse genetics, revealing that, in several plant species, mutations in DGAT1 directly affect oil content and quality [20,37,38]. Over-expression of plant DGAT1 has been used to stimulate oil deposition first in Arabidopsis thaliana [19] and then in Brassica napus [32] under both greenhouse and field conditions. Similar results were obtained with the heterologous expression of a fungal DGAT2 in soybean (Glycine max) resulting in an average increase of 1.5% (w/w) in seed oil over multi-seasonal field trials [22]. Potential biotechnological applica- tions of DGATs in the modification of the fatty acid composition of seed oil will probably be on the increase in the near future given the recent reports on the involvement of DGAT2 in channeling specific fatty acids into TAG [4,21,30]. To explore the full potential of DGAT in oilseed metabolic engineering, it is desirable to better understand the enzyme’s mechanism of action and regulation. Since DGAT1 is an integral membrane protein with multiple transmembrane domains, the resolution of its three-dimensional structure and the ensuing experiments that would shed light on the structure–function relationship are currently beyond reach. An alternative approach for gaining insight into structure–function relationships in DGAT is through directed evolution which forgoes rational design and relies instead on randomly generated modifications combined with a suitable method of selection. This approach could potentially result in DGAT variants with enhanced catalytic efficiency as demonstrated in numerous instances for other enzymes [36]. Examples pertaining directly to plant biotechnology include improvement of existing genes involved in insect and herbicide resistance [8,18]. Regardless of the method used to generate the random modi- fications, directed evolution requires a reliable high-throughput system of screening (HTS) capable of selecting variants with desired Abbreviations: DGAT, acyl-CoA:diacylglycerol acyltransferase; epPCR, error- prone PCR; HTS, high-throughput system; Kb, kilo base. * Corresponding author. Tel.: þ1 780 492 4401; fax: þ1 780 492 6739. E-mail address: randall.weselake@ualberta.ca (R.J. Weselake). 1 RMPS and MT have contributed equally to this work and should be both regarded as the first author. Contents lists available at ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy 0981-9428/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2008.12.019 Plant Physiology and Biochemistry 47 (2009) 456–461