Fatty Acid and Alcohol Metabolism in Pseudomonas putida: Functional Analysis Using Random Barcode Transposon Sequencing

Abstract

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.

Towards robust Pseudomonas cell factories to harbour novel biosynthetic pathways

“…To achieve high throughput, recent studies combined the selection methodology of ALE with the 'targeting each gene' concept of transposon libraries and tracked the population dynamics in mixed cultures exposed to stress to quantify which mutant strains were enriched [15,61,115]. Conceptually similar, trackable multiplex recombineering (TRMR) of a barcoded cassette with a strong promoter and ribosome-binding site (RBS) upstream of virtually every gene allowed quantification of each mutant in mixed culture under stress conditions [116].…”

Production of l -Theanine by Escherichia coli in the Absence of Supplemental Ethylamine

“…In particular, v -transaminase transfers the amino group from a carbon atom without a carboxyl group and often exhibits broad substrate capacity (39,40), which raised the possibility of an acetaldehyde transaminase in this family. Pseudomonas putida, a Gram-negative bacterium, decomposes diverse molecules in its environment, indicating that P. putida promiscuously utilizes carbon, nitrogen, or both from various compounds (41,42). In nitrogen utilization, in particular, deamination of nitrogen sources catalyzed by transaminases may be involved, because the genome of P. putida encodes numerous transaminases.…”

REC protein family expansion by the emergence of a new signaling pathway

“…Duplicate 600 μL samples were then combined and BarSeq analysis was conducted as described previously(Wetmore et al 2015; Rand et al 2017; Incha et al 2020). Single carbon source fitness data is available at http://fit.genomics.lbl.gov (Thompson et al 2020).…”