Role of Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase in Oleaginous Streptomyces sp. Strain G25

Abstract

Recently, we isolated a novel Streptomyces strain which can accumulate extraordinarily large amounts of triacylglycerol (TAG) and consists of 64% fatty acids (dry weight) when cultivated with glucose and 50% fatty acids (dry weight) when cultivated with cellobiose. To identify putative gene products responsible for lipid storage and cellobiose utilization, we analyzed its draft genome sequence. A single gene encoding a wax ester synthase/acyl coenzyme A (CoA):diacylglycerol acyltransferase (WS/DGAT) was identified and heterologously expressed in Escherichia coli The purified enzyme AtfG25 showed acyltransferase activity with C12- or C16-acyl-CoA, C12 to C18 alcohols, or dipalmitoyl glycerol. This acyltransferase exhibits 24% amino acid identity to the model enzyme AtfA from Acinetobacter baylyi but has high sequence similarities to WS/DGATs from other Streptomyces species. To investigate the impact of AtfG25 on lipid accumulation, the respective gene, atfG25, was inactivated in Streptomyces sp. strain G25. However, cells of the insertion mutant still exhibited DGAT activity and were able to store TAG, albeit in lower quantities and at lower rates than the wild-type strain. These findings clearly indicate that AtfG25 has an important, but not exclusive, role in TAG biosynthesis in the novel Streptomyces isolate and suggest the presence of alternative metabolic pathways for lipid accumulation which are discussed in the present study.

Importance: A novel Streptomyces strain was isolated from desert soil, which represents an extreme environment with high temperatures, frequent drought, and nutrient scarcity. We believe that these harsh conditions promoted the development of the capacity for this strain to accumulate extraordinarily large amounts of lipids. In this study, we present the analysis of its draft genome sequence with a special focus on enzymes potentially involved in its lipid storage. Furthermore, the activity and importance of the detected acyltransferase were studied. As discussed in this paper, and in contrast to many other bacteria, streptomycetes seem to possess a complex metabolic network to synthesize lipids, whereof crucial steps are still largely unknown. This paper therefore provides insights into a range of topics, including extremophile bacteria, the physiology of lipid accumulation, and the biotechnological production of bacterial lipids.

FIG 1

Putative metabolic pathways for de novo (branched-chain) triacylglycerol biosynthesis in Streptomyces sp. G25 from cellobiose. Abbreviations and enzyme names: ACC, acetyl-CoA carboxylase; (A)GPAT, (1-acyl)glycerol-3-phosphate acyltransferase; α-KG, α-ketoglutarate; BCAA, branched-chain amino acid; BCAT, BCAA transaminase; BCDHC, branched-chain α-keto acid dehydrogenase; BglC, β-glycosidase; CebEFG, cellobiose binding and transport proteins; DAG, diacylglycerol; DGAT, diacylglycerol acyltransferase; DHAP, dihydroxy-acetone phosphate; FabB, ketoacyl-ACP synthase I; FabD, malonyl-CoA:ACP transacylase; FabF, ketoacyl-ACP synthase II; FabG, 3-ketoacyl-ACP reductase; FabH, 3-ketoacyl-ACP synthase III; FabI, enoyl-ACP reductase; FabZ, 3-hydroxyacyl-ACP dehydrase; FadD, acyl-CoA synthetase; G3P DH, glycerol-3-phosphate dehydrogenase; Glu, glutamate; [H], reduction equivalent (NADH or NADPH); (HS-)ACP, acyl carrier protein (with a free sulfhydryl group); (HS-)CoA, coenzyme A (with a free sulfhydryl group); P, phosphate; PAP, phosphatidate phosphatase; PDH, pyruvate dehydrogenase.

FIG 2

Genomic regions showing genes located up- and downstream from diacylglycerol (DGAT) genes sco0958, sav7256, and atf_G25 in Streptomyces coelicolor (A), S. avermitilis (B), Streptomyces sp. G25 wild type (C), or atf_G25 mutant (D). Arrows indicate the primer binding sites used to verify the insertion mutant. Abbreviations: Ap^r, apramycin resistance; DH, (short-chain) dehydrogenase; G1P AT, glucose-1-phosphate adenylyltransferase; GH, glycosyl hydrolase family-like protein; GS, glycogen synthase; HD PH, HD phosphohydrolase; HP, hypothetical protein; Mg-T, magnesium transporter; α-1,2-M, α-1,2-mannosidase.

FIG 3

Specific enzyme activities of purified (His₁₀-)Atf_G25 from Streptomyces sp. G25 determined photometrically with acyl-CoAs (C₁₆-CoA, palmitoyl-CoA; C₁₂-CoA, lauryl-CoA) and alcohols (C₁₂, dodecanol; C₁₄, tetradecanol; C₁₆, hexadecanol; C_16:1, palmitoleyl alcohol; C₁₈, octadecanol; or DPG, 1,2-dipalmitoyl-sn-glycerol).

FIG 4

Separation of lipid extracts of Streptomyces sp. G25 wild type (WT) and atf_G25ΩAp^r mutant strain (Ω) by thin-layer chromatography (TLC). The strains were cultivated for 3, 6, 10, and 14 days in 30 ml MSM with 0.025% (wt/vol) NH₄Cl and 1% (wt/vol) glucose at 30°C. Three milligrams of dried cells was extracted with chloroform-methanol (2:1), and the extracts were separated in n-hexane–diethyl ether–acetic acid (80:20:1, vol/vol/vol). FFA, free fatty acid standard (66 μg of oleic acid); TAG, triacylglycerol standard (20 μg triolein); WE, wax ester standard (40 μg oleyl oleate).

FIG 5

Total fatty acid (TFA) contents of the cell dry weight (CDW) showing the fatty acid profiles of Streptomyces sp. G25 wild type (WT) and Streptomyces sp. G25 atf_G25ΩAp^r mutant (Ω), cultivated in triplicate in MSM with 0.025% (wt/vol) NH₄Cl and 1% (wt/vol) glucose for 3, 6, 10, and 14 days at 30°C. Mean values for each fatty acid with standard deviations are shown.