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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Caprolactam is an important polymer precursor to nylon traditionally derived from petroleum and produced on a scale of 5 million tons per year. Current biological pathways for the production of caprolactam are inefficient with titers not exceeding 2 mg/L, necessitating novel pathways for its production. As development of novel metabolic routes often require The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, expressed or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan).

Pseudomonas putida is a promising bacterial chassis for metabolic engineering given its ability to metabolize a wide array of carbon sources, especially aromatic compounds derived from lignin. However, this omnivorous metabolism can also be a hindrance when it can naturally metabolize products produced from engineered pathways. Herein we show that P. putida is able to use valerolactam as a sole carbon source, as well as degrade caprolactam. Lactams represent important nylon precursors, and are produced in quantities exceeding one million tons per year (Zhang et al., 2017). To better understand this metabolism we use a combination of Random Barcode Transposon Sequencing (RB-TnSeq) and shotgun proteomics to identify the oplBA locus as the likely responsible amide hydrolase that initiates valerolactam catabolism. Deletion of the oplBA genes prevented P. putida from growing on valerolactam, prevented the degradation of valerolactam in rich media, and dramatically reduced caprolactam degradation under the same conditions. Deletion of oplBA, as well as pathways that compete for precursors L-lysine or 5-aminovalerate, increased the titer of valerolactam from undetectable after 48 h of production to ~90 mg/L. This work may serve as a template to rapidly eliminate undesirable metabolism in non-model hosts in future metabolic engineering efforts.

Understanding the genetic basis of P. putida ’s diverse metabolism is imperative for us to reach its full potential as a host for metabolic engineering. Many target molecules of the bioeconomy and their precursors contain nitrogen.

31Pseudomonas putida is a saprophytic bacterium with robust carbon metabolisms and 32 strong solvent tolerance making it an attractive host for metabolic engineering and 33 bioremediation. Due to its diverse carbon metabolisms, its genome encodes an array of proteins 34 and enzymes that can be readily applied to produce valuable products. In this work we sought to 35 identify design principles and bottlenecks in the production of type III polyketide synthase 36 (T3PKS)-derived compounds in P. putida. T3PKS products are widely used as nutraceuticals and 37 medicines and often require aromatic starter units, such as coumaroyl-CoA, which is also an 38 intermediate in the native coumarate catabolic pathway of P. putida. Using a randomly barcoded 39 transposon mutant (RB-TnSeq) library, we assayed gene functions for a large portion of aromatic 40 catabolism, confirmed known pathways, and proposed new annotations for two aromatic 41 transporters. The tetrahydroxynapthalene synthase of Streptomyces coelicolor (RppA), a 42 microbial T3PKS, was then used to rapidly assay growth conditions for increased T3PKS 43 product accumulation. The feruloyl/coumaroyl CoA synthetase (Fcs) of P. putida was used to 44 supply coumaroyl-CoA for the curcuminoid synthase (CUS) of Oryza sativa, a plant T3PKS. We 45 identified that accumulation of coumaroyl-CoA in this pathway results in extended growth lag 46 times in P. putida. Deletion of the second step in coumarate catabolism, the enoyl-CoA 47 hydratase lyase (Ech), resulted in 'drop-in' production of the type III polyketide 48 bisdemethoxycurcumin. 49 1 INTRODUCTION 50 Secondary metabolites of fungi, plants, and bacteria have been used as medicines and 51 supplements for millennia [1]. Compounds such as naringenin, raspberry ketone, resveratrol, and 52 curcumin are widely used as nutraceuticals and are biosynthesized through similar pathways 53 3 [2,3]. Commercially, these chemicals are either extracted directly from plants or produced 54 synthetically, as in the case of raspberry ketone [4]. Renewable microbial production of these 55 compounds will decrease reliance on agriculture and fossil fuel-derived chemical synthesis. The 56 biosynthesis of these compounds (naringenin, raspberry ketone, resveratrol, and curcumin) relies 57 on a class of enzymes called type III polyketide synthases (T3PKSs). T3PKSs carry out iterative 58 Claisen condensation reactions typically with coenzyme A (CoA)-based starter and extender 59 units, both of which vary widely among these enzymes [5]. In the case of the 60 tetrahydroxynapthalene synthase of Streptomyces coelicolor (RppA) the starter and extender 61 units are simply malonyl-CoA, while in many plant T3PKSs the starter unit is a 62 phenylpropenoyl-CoA thioester, usually derived from ferulate, coumarate, or cinnamate [6,7]. 63Coumarate and ferulate are components of lignin found in lignocellulosic hydrolysate 64 (LH), which has been proposed for use as a renewable feedstock for biocatalysis [8,9]. However, 65 the limited capabilities of commonl...

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ABSTRACT Agrobacterium fabrum ARqua1 is a hybrid of Agrobacterium fabrum C58C bearing the megaplasmid pRiA4b. ARqua1 is used by many plant researchers to generate transgenic roots. The draft genome of ARqua1 includes a 249,350-bp contig that likely covers all of pRiA4b, and it will be a valuable resource to plant biologists.