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In order to use Acinetobacter baylyi ADP1 as a host for molecularly enhanced triacylglyceride (TAG) production, it is important to understand key enzymatic steps involved in TAG biosynthesis and use. Four different thioesterase genes were cloned from Acinetobacter baylyi ADP1 and expressed in E. coli to investigate their contribution to the free fatty acid (FFAs) accumulation. Overexpression of the genes tesA? (a leaderless form of the gene tesA) and tesC resulted in increased accumulation of FFAs when compared with the host strain. The TesA? thioesterase tended to produce shorter chain and unsaturated FFAs, myristic acid and oleic acid, respectively, compared to TesC and other investigated thioesterases. We identified four crucial one-step enzymatic reactions encoded by the genes lip1, lip2, acr1 and fadE, mutations that were predicted to have an effect on the TAG accumulation. Among a series of generated strains, a strain that had the lip1, lip2 and fadE mutations was constructed showing a 2.44-fold increase in the TAG production compare to the wild type. To achieve high yields of microbial biofuel production using Acinetobacter baylyi ADP1, we constructed and expressed two three-gene operons in E. coli K19 and A. baylyi strains having inactivated ?-oxidation pathway to observe operon effects on FFAs and overall lipid productions. E. coli RU35 produced 0.04 ?g of TAG/mg of dry biomass and E. coli RU14 produced 0.03 ?g of TAG/mg of dry biomass. E. coli K19 by itself does not possess any DGAT activity, hence the production of TAGs in the engineered strains is mediated by enzyme WS/DGAT encoded by the two plasmids. In all A. baylyi strains the introduction of the operon decreased TAGs accumulation by 16-63%. The decrease in accumulation of TAGs in the A. baylyi strains is predicted to be due to the action of the TesA? thioesterase, which is apparently very efficient in competing with WS/DGAT for acyl-CoA. This study demonstrates that finding of a thioesterase which has a low affinity to acyl-CoA and a high affinity to acyl-ACP is critical for the successful increase of TAGs in A. baylyi. |
