Host engineering for improved valerolactam production inPseudomonas putida

Abstract

25Pseudomonas putida is a promising bacterial chassis for metabolic engineering given its 26 ability to metabolize a wide array of carbon sources, especially aromatic compounds derived 27 from lignin. However, this omnivorous metabolism can also be a hindrance when it can naturally 28 metabolize products produced from engineered pathways. Herein we show that P. putida is able 29 to use valerolactam as a sole carbon source, as well as degrade caprolactam. Lactams represent 30 important nylon precursors, and are produced in quantities exceeding one million tons per 31 year[1]. To better understand this metabolism we use a combination of Random Barcode 32 Transposon Sequencing (RB-TnSeq) and shotgun proteomics to identify the oplBA locus as the 33 likely responsible amide hydrolase that initiates valerolactam catabolism. Deletion of the oplBA 34 genes prevented P. putida from growing on valerolactam, prevented the degradation of 35 valerolactam in rich media, and dramatically reduced caprolactam degradation under the same 36 conditions. Deletion of oplBA, as well as pathways that compete for precursors L-lysine or 5-37 aminovalerate, increased the titer of valerolactam from undetectable after 48 hours of production 38 to ~90 mg/L. This work may serve as a template to rapidly eliminate undesirable metabolism in 39 non-model hosts in future metabolic engineering efforts. 40 1 INTRODUCTION 41 Pseudomonas putida has attracted great attention as a potential chassis organism for 42 metabolic engineering due in large part to its ability to metabolize a wide variety of carbon 43 sources, particularly aromatic compounds that can be derived from lignin [2,3]. To more fully 44 realize this vision, much effort has been put forth recently to better characterize the central 45 metabolism of P. putida with updated genome-scale models [4], C 13 flux experiments [5,6], and 46high-throughput fitness assays, which have all contributed to a more complete understanding of 47 48 fully understood. 49 One consequence of this omnivorous metabolism is that P. putida possesses the ability to 50 degrade or fully catabolize chemicals metabolic engineers seek to produce in the host. An 51 ongoing challenge for P. putida host engineering will be to rapidly identify catabolic pathways 52 of important target molecules and eliminate them from the genome. The recent report of a novel 53 pathway for levulinic acid catabolism in P. putida KT2440 underscores the catabolic flexibility 54 of the host, and an additional obstacle towards producing high product titer [8]. While 55 challenging, this is not surprising; as a genus, Pseudomonads are well known for their ability to 56 degrade a wide range of naturally occurring or xenobiotic chemicals [9,10]. 57 Caprolactam and valerolactam are both important commodity chemicals used in the 58 synthesis of nylon polymers, with global production of caprolactam reaching over four million 59 metric tons [1]. Multiple efforts have been made to produce these chemicals biologically, with 60the titers of valerolactam...

Identification, Characterization, and Application of a Highly Sensitive Lactam Biosensor from Pseudomonas putida

“…Previously, we have leveraged shotgun proteomics to infer local regulation within the lysine catabolism of P. putida 16,[21][22][23] . From these data, multiple glutarate biosensors were engineered and used to measure relative metabolite amounts in the native host 21 .…”

“…To find the Hill fit to the two-plasmid system, EZ-RICH medium containing kanamycin, carbenicillin, and 0.0125 w/v% arabinose was inoculated with an overnight culture of the two- Valerolactam and caprolactam were measured via LC-QTOF-MS as described previously 16 . Liquid chromatographic separation was conducted at 20°C with a Kinetex HILIC column (50-mm length, 4.6-mm internal diameter, 2.6-µm particle size; Phenomenex, Torrance, CA) using a 1260 Series HPLC system (Agilent Technologies, Santa Clara, CA, USA).…”

Identification, characterization, and application of a highly sensitive lactam biosensor fromPseudomonas putida

“…From these data, multiple glutarate biosensors were engineered and used to measure relative metabolite amounts in the native host 21 . Here we again leveraged previously published proteomics data that showed OplBA to be specifically upregulated in the presence of valerolactam to develop a biosensor 16 .…”

Catalytic and structural properties of ATP‐dependent caprolactamase from Pseudomonas jessenii

“…Since for many of these the sequence similarity to the P. jessenii caprolactamase is much higher than to confirmed oxoprolinases or hydantoinases, the genes more likely encode lactamases. However, the function of these genes or proteins has not been established, with the exception of the oplAB genes of P. putida KT2440 35 . These oplAB genes were upregulated in cultures growing in the presence of valerolactam and caprolactam, and gene knockouts confirmed their role in lactam utilization.…”