Lactococcus lactis fabH, encoding beta-ketoacyl-acyl carrier protein synthase, can be functionally replaced by the Plasmodium falciparum congener.
Plasmodium falciparum, in addition to scavenging essential fatty acids from its intra- and intercellular environments, possesses a functional complement of type II fatty acid synthase (FAS) enzymes targeted to the apicoplast organelle. Recent evidence suggests that products of the plasmodial FAS II system may be critical for the parasite's liver-to-blood cycle transition, and it has been speculated that endogenously generated fatty acids may be precursors for essential cofactors, such as lipoate, in the apicoplast. beta-Ketoacyl-acyl carrier protein (ACP) synthase III (pfKASIII or FabH) is one of the key enzymes in the initiating steps of the FAS II pathway, possessing two functions in P. falciparum: the decarboxylative thio-Claisen condensation of malonyl-ACP and various acyl coenzymes A (acyl-CoAs; KAS activity) and the acetyl-CoA:ACP transacylase reaction (ACAT). Here, we report the generation and characterization of a hybrid Lactococcus lactis strain that translates pfKASIII instead of L. lactis fabH to initiate fatty acid biosynthesis. The L. lactis expression vector pMG36e was modified for the efficient overexpression of the plasmodial gene in L. lactis. Transcriptional analysis indicated high-efficiency overexpression, and biochemical KAS and ACAT assays confirm these activities in cell extracts. Phenotypically, the L. lactis strain expressing pfKASIII has a growth rate and fatty acid profiles that are comparable to those of the strain complemented with its endogenous gene, suggesting that pfKASIII can use L. lactis ACP as substrate and perform near-normal function in L. lactis cells. This strain may have potential application as a bacterial model for pfKASIII inhibitor prescreening.
Du
Yu
Y
Vanderbilt University, Department of Chemistry, 7300 Stevenson Center, Nashville, TN 37232, USA.
Gisselberg
Jolyn E
JE
Johnson
Jacob D
JD
Lee
Patricia J
PJ
Prigge
Sean T
ST
Bachmann
Brian O
BO
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D053898:Biosynthetic Pathways / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005227:Fatty Acids / Q000096:biosynthesis; D017353:Gene Deletion; D015870:Gene Expression; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D013294:Lactococcus lactis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D010957:Plasmids; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D005227:Fatty Acids; D011994:Recombinant Proteins; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase