Identification and physiological characterization of phosphatidic acid phosphatase enzymes involved in triacylglycerol biosynthesis in Streptomyces coelicolor.
BACKGROUND
Phosphatidic acid phosphatase (PAP, EC 3.1.3.4) catalyzes the dephosphorylation of phosphatidate yielding diacylglycerol (DAG), the lipid precursor for triacylglycerol (TAG) biosynthesis. Despite the importance of PAP activity in TAG producing bacteria, studies to establish its role in lipid metabolism have been so far restricted only to eukaryotes. Considering the increasing interest of bacterial TAG as a potential source of raw material for biofuel production, we have focused our studies on the identification and physiological characterization of the putative PAP present in the TAG producing bacterium Streptomyces coelicolor.


RESULTS
We have identified two S. coelicolor genes, named lppα (SCO1102) and lppβ (SCO1753), encoding for functional PAP proteins. Both enzymes mediate, at least in part, the formation of DAG for neutral lipid biosynthesis. Heterologous expression of lppα and lppβ genes in E. coli resulted in enhanced PAP activity in the membrane fractions of the recombinant strains and concomitantly in higher levels of DAG. In addition, the expression of these genes in yeast complemented the temperature-sensitive growth phenotype of the PAP deficient strain GHY58 (dpp1lpp1pah1). In S. coelicolor, disruption of either lppα or lppβ had no effect on TAG accumulation; however, the simultaneous mutation of both genes provoked a drastic reduction in de novo TAG biosynthesis as well as in total TAG content. Consistently, overexpression of Lppα and Lppβ in the wild type strain of S. coelicolor led to a significant increase in TAG production.


CONCLUSIONS
The present study describes the identification of PAP enzymes in bacteria and provides further insights on the genetic basis for prokaryotic oiliness. Furthermore, this finding completes the whole set of enzymes required for de novo TAG biosynthesis pathway in S. coelicolor. Remarkably, the overexpression of these PAPs in Streptomyces bacteria contributes to a higher productivity of this single cell oil. Altogether, these results provide new elements and tools for future cell engineering for next-generation biofuels production.
Microbiology Division, IBR (Instituto de Biología Molecular y Celular de Rosario), Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK, Rosario, Argentina.
Santiago
S
Comba
Simón
S
Menendez-Bravo
Ana
A
Arabolaza
Hugo
H
Gramajo
D001426:Bacterial Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D010711:Phosphatidate Phosphatase / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000201:enzymology*; D014280:Triglycerides / Q000096:biosynthesis*
C011439:1,2-diacylglycerol; D001426:Bacterial Proteins; D004075:Diglycerides; D011994:Recombinant Proteins; D014280:Triglycerides; D010711:Phosphatidate Phosphatase