Final Report Summary - BIOTEROL (Biodegradation of xenobiotic tertiary alcohols) The main objective of the planned study was to initiate lab evolution of an efficient degradation pathway for xenobiotic tertiary C6 to C10 alcohols (as the selective pressure) in single bacterial strains or consortia. The project focused on tertiary alcohol metabolites. The starting point was bacterial strains already known to be involved in tertiary alcohol degradation (e.g. strains Methylibium petroleiphilum PM1, Aquincola tertiaricarbonis L108, Castellaniella defragrans 65Phen and Pseudomonas aeruginosa PAO1). Evolutionary mechanisms such as recombination by horizontal gene transfer and point mutations in key enzymes will be investigated in detail. Since the beginning of the study, different scientific methods used in the genetic and proteomic fields have been used to point towards the elucidation of the C5 tertiary alcohol (tert-amyl alcohol, TAA, fuel oxygenate metabolite) pathway. Both direct and indirect mutagenesis were performed on A. tertiaricarbonis L108. Proteome expression profiles were compared under different carbon sources, and clear up-regulation and down-regulation of gene clusters were observed. Five gene candidates (with redundant function) were chosen as being involved in tertiary alcohol degradation, and these were tested in heterologous expression systems. Degradation of TAA is known to entail the metabolites MBO→Prenol→Prenal→3-methyl-crotonyl-CoA. (Figure 1). One of the candidates, labelled as an aldehyde dehydrogenase, was confirmed as being involved in the conversion of Prenal to MCro-CoA, via positive enzymatic activity assays. The prenal dehydrogenase enzyme was NAD and Coenzyme A (CoA) dependent. Testing of the substrate specificity across a range of aldehydeswith different structure and chain length(linear/iso-branched, saturated/unsaturated,C2-C7) (Figure 2) resulted in higher affinity and activity for saturated C4 and C5 aldehydes compared to the unsaturated prenal itself. Under optimal NAD and CoA (2mM and 5mM respectively) conditions, assays were repeated resulting in the highest activity for the linear C5 aldehyde, i.e. pentanal. Thus we can infer that the genetic makeup of the TAA degradation enzymatic pathway had not adapted solely to degrade TAA, thus scope for rate improvement of the TAA pathway is possible. Additionally, structural analysis of the "prenal" dehydrogenase will be analyzed via mutations at active site or nearby to obtain more information on their effect on substrate specificity, and more importantly there effect on adaptation to smaller/larger tertiary alcohol metabolites. Can the prenal dehydrogenase then be used for higher compounds than the C5 prenal, and will mutations in the corresponding gene occur to adapt to higher compounds when selective pressure is enforced? Growth of A. tertiaricarbonisL108 on C6, C7 and C8 TAA homologues were tested, however, generation times increased with increasing compound size, with no growth observed on C8. Thus preliminary tests were performed on different strains for 3-methyl-3-pentenal (3M3P, C6 homolog compound of TAA) and its MBO homologue (3M1P3ol, 3-methyl-1-penten-3-ol). Growth on 3M1P3ol was exceptional in all cases, including consortia of PM1 and 65Phen, but growth and regulation was hindered when grown on 3M3P. Observation of adaption towards 3M3P was chosen for further study. Evolution lines of strain L108 with the C6 compound was serially transferred in batch cultures. Serial transfers were stopped at 100 generations. Genetic and physiological and chemical analysis of the initial culture and the evolved cultures were performed. Generation times of L108 were reduced by 40%, i.e. from 34h to 20h. Testing of the prenal dehydrogenase gene at the initial and final stages did not prove different. Thus the reduction in generation time could be due to factors in gene regulation or to the increase performance in the other gene candidates. The work will help to better assess the fate of such tertiary intermediates of higher oxygenate ether degradation. Basically, performance of the resulting cultures degrading C6 to C10 tertiary alcohols will provide us with kinetic parameters for modeling in situ bioremediation potential. In addition, as the degrading cultures are not the result of targeted genetic manipulations, they may also be employed for the removal of pollutants at contaminated sites. Furthermore, the prenal dehydrogenase capacity for a reverse reaction was tested and confirmed with pentanoyl-CoA reduction. Due to the demonstrated specificity for medium-chain substrates, this enzyme might be employed in combination with a suitable aldehyde reductase/reverse alcohol dehydrogenase reaction for the biotechnological production of fuels (i.e. C5 to C6 alcohols) from carboxylic acids and sugars. Abbreviations - TAA: tert-amyl alcohol, MBO: 2-methyl-3-buten-2-ol, Prenol: 3-methyl-but-2-en-1-ol, Prenal: 3-methylbut-2-enal, MCro-CoA: 3-methylcrotonyl-CoA, 3M3P:3-methyl - 3- pentanal.3M1P3ol: 3-methyl-1-penten-3-ol, TBA - tert-butyl alcohol Related documents final1-final-report-figure-file.pdf
