Multiple Pathways for Triacylglycerol Biosynthesis in Streptomyces coelicolor

Abstract

The terminal reaction in triacylglyceride (TAG) biosynthesis is the esterification of diacylglycerol (DAG) with a fatty acid molecule. To study this reaction in Streptomyces coelicolor, we analyzed three candidate genes (sco0958, sco1280, and sco0123) whose products significantly resemble the recently identified wax ester synthase/acyl-coenzyme A (CoA):DAG acyltransferase (DGAT) from Acinetobacter baylyi. The deletion of either sco0123 or sco1280 resulted in no detectable decrease in TAG accumulation. In contrast, the deletion of sco0958 produced a dramatic reduction in neutral lipid production, whereas the overexpression of this gene yielded a significant increase in de novo TAG biosynthesis. In vitro activity assays showed that Sco0958 mediates the esterification of DAG using long-chain acyl-CoAs (C 14 to C 18 ) as acyl donors. The K m and V max values of this enzyme for myristoyl-CoA were 45 M and 822 nmol mg ؊1 min ؊1 , respectively. Significantly, the triple mutant strain was not completely devoid of storage lipids, indicating the existence of alternative TAG-biosynthetic routes. We present strong evidence demonstrating that the residual production of TAG in this mutant strain is mediated, at least in part, by an acyl-CoA-dependent pathway, since the triple mutant still exhibited DGAT activity. More importantly, there was substantial phospholipid:DGAT (PDAT) activity in the wild type and in the triple mutant. This is the first time that a PDAT activity has been reported for bacteria, highlighting the extreme metabolic diversity of this industrially important soil microorganism.Triacylglycerols (TAGs) are the most common lipid-based energy reserves in animals, plants, and eukaryotic microorganisms (3). In bacteria, the most abundant class of neutral lipids are polyhydroxyalkanoic acids (3), but a few examples of substantial TAG accumulation have been reported, mainly in the actinomycetes Mycobacterium (4), Nocardia (1), Rhodococcus (2), and Streptomyces (27). Furthermore, the biosynthesis of wax esters (WEs) (oxoesters of long-chain primary fatty alcohols and long-chain fatty acids) has been frequently reported for members of the genus Acinetobacter (23). Recently, storage lipid accumulation in the hydrocarbonoclastic marine bacterium Alcanivorax borkumensis was reported (19), representing the first example of significant TAG accumulation in a gramnegative prokaryote.Three different classes of enzymes are known to mediate TAG formation from diacylglycerol (DAG) (22). Acyl-coenzyme A (CoA):DAG acyltransferase (DGAT) catalyzes the acylation of DAG using acyl-CoAs as substrates. In eukaryotes, two DGAT families (DGAT1 and DGAT2) with no sequence resemblance to each other have been identified and characterized. Members of the DGAT1 gene family were found in animals and plants (7,16,31), whereas members of the DGAT2 gene family are found in animals (8), plants (5), and Saccharomyces cerevisiae (32). Acyl-CoA-independent TAG synthesis in yeast and plants is mediated by a phospholipid (PL):DGAT (PDAT) that uses PLs...

Acyltransferases in Bacteria

“…The finding that there was a residual DGAT activity and TAG synthesis left in the triple mutant (sco0958 sco1280 sco0123) of S. coelicolor again supported this hypothesis (75). In S. coelicolor, bacterial phospholipid:DGAT (PDAT) activity could be proven for the first time.…”

“…The authors further speculated that Sco1280 might have a very limited substrate range or that it might need an interaction with an oily surface or another protein for full activity. Sco0123, in contrast, seems to have a completely different physiological role (75).…”

“…In the genome of S. coelicolor, there are genes for three proteins with remote similarities to AtfA from A. baylyi: Sco0958, Sco0123, and Sco1280 (75). Only Sco0958, exhibiting 25% identity to AtfA, contributes to TAG biosynthesis in S. coelicolor strain A3(2): its deletion seriously reduced TAG accumulation to approximately 30% of the S. coelicolor wild-type TAG level, whereas a deletion of the other two candidates had no effect.…”

“…Thus, glycerophospholipids can act as acyl donors for TAG biosynthesis via the PDAT pathway in addition to (or as substitute for) the DGAT pathway catalyzed by AtfA-like acyltransferases. So far, the bacterial PDAT-mediating enzyme has not been identified, and the genome of S. coelicolor does not feature any obvious sequence similarity to characterized eukaryotic PDATs (e.g., Lro1 from S. cerevisiae) (75).…”

Metabolic engineering of microorganisms to produce omega-3 very long-chain polyunsaturated fatty acids

“…142 Subsequently, the same authors also described the successful 143 reconstruction of the EPA and DHA biosynthetic pathway in the 144 seeds of an oilseed crop, C. sativa. They successfully engineered this 145 species to accumulate high levels of x3-VLCPUFAs in its seed oils 146 and yielded two iterations, in which one accumulated EPA up to 147 31% of the seed oils and the other accumulated up to 12% EPA 148 and 14% DHA in the seed oils [25]. These research efforts demon- 149 strate the feasibility of large-scale production of x3-VLCPUFAs in 150 the oilseeds of transgenic crops, which have proved to be a prom- 151 ising alternative to fish oils.…”