In this report, we identify the relevant factors to increase production of medium chain n-alcohols through an expanded view of the reverse β-oxidation pathway. We began by creating a base strain capable of producing medium chain n-alcohols from glucose using a redox-balanced and growth-coupled metabolic engineering strategy. By dividing the heterologous enzymes in the pathway into different modules, we were able to identify and evaluate homologs of each enzyme within the pathway and identify several capable of enhancing medium chain alcohol titers and/or selectivity. In general, the identity of the trans-2-enoyl-CoA reductase (TER) and the direct overexpression of the thiolase (FadA) and β-hydroxy-acyl-CoA reductase (FadB) improved alcohol titer and the identity of the FadBA complex influenced the dominant chain length. Next, we linked the anaerobically induced VHb promoter from Vitreoscilla hemoglobin to each gene to remove the need for chemical inducers and ensure robust expression. The highest performing strain with the autoinduced reverse β-oxidation pathway produced n-alcohols at titers of 1.8 g/L with an apparent molar yield of 0.2 on glucose consumed in rich medium (52% of theoretical yield).

With its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, Pseudomonas putida KT2440 is emerging as an important chassis organism for metabolic engineering. Despite advances in our understanding of this organism, many gaps remain in our knowledge of the genetic basis of its metabolic capabilities. These gaps are particularly noticeable in our understanding of both fatty acid and alcohol catabolism, where many paralogs putatively coding for similar enzymes co-exist making biochemical assignment via sequence homology difficult. To rapidly assign function to the enzymes responsible for these metabolisms, we leveraged Random Barcode Transposon Sequencing (RB-TnSeq). Global fitness analyses of transposon libraries grown on 13 fatty acids and 10 alcohols produced strong phenotypes for hundreds of genes. Fitness data from mutant pools grown on varying chain length fatty acids indicated specific enzyme substrate preferences, and enabled us to hypothesize that DUF1302/DUF1329 family proteins potentially function as esterases. From the data we also postulate catabolic routes for the two biogasoline molecules isoprenol and isopentanol, which are catabolized via leucine metabolism after initial oxidation and activation with CoA. Because fatty acids and alcohols may serve as both feedstocks or final products of metabolic engineering efforts, the fitness data presented here will help guide future genomic modifications towards higher titers, rates, and yields. IMPORTANCE To engineer novel metabolic pathways into P. putida, a comprehensive understanding of the genetic basis of its versatile metabolism is essential. Here we provide functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of this bacterium. These data provide a framework facilitating precise genetic changes to prevent product degradation and channel the flux of specific pathway intermediates as desired.

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