Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida

Abstract

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...

Pseudomonas as Versatile Aromatics Cell Factory

“…The deletion of aromatics catabolic pathways is an important aspect in this. The much‐heralded metabolic versatility of pseudomonads [ 19–21 ] is a double‐edged blade. Their degradative power is often a benefit in transformation and funneling approaches.…”

“…[ 105 ] It has been used to determine the MaCoA availability in a heterologous host to assess the potential for polyketide synthesis. [ 21,106 ] In early attempts to produce flaviolin in pseudomonads, ≈6 mg L −1 were achieved in P. putida KT2440. [ 107 ] In recent attempts, testing RppA variants and different concentrations of supplemented glucose to complex medium, 65 mg L −1 were produced with a truncated enzyme variant.…”

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

“…The plate was shaken (700 rpm) in an INFORS HT Multitron (Infors USA Inc.) at 30 °C for 24 hrs. Duplicate 600 μL samples were then combined and BarSeq analysis was conducted as described previously [48][49][50] .…”

Functional analysis of the fatty acid and alcohol metabolism ofPseudomonas putidausing RB-TnSeq

“…Its upper glycolytic pathway architecture enables P. putida to natively generate large amounts of reducing equivalent (8), and it more robustly withstands metabolic burdens than many other frequently used host organisms (9). To date, a wide variety of products have been produced through metabolic engineering of P. putida, including valerolactam (10), curcuminoids (11), diacids (12), methyl-ketones (13), rhamnolipids (14), cis,cis-muconic acid (15), and many others (16). Recent advances in genome-scale metabolic modeling of P. putida make engineering efforts more efficient (7,17).…”

“…An approach that has proven to be particularly powerful is Random Barcode Transposon Sequencing (RB-TnSeq) (40,41). RB-TnSeq allows rapid and inexpensive genomewide profiling of individual gene fitness in various conditions, and has been used in P. putida to identify numerous novel metabolic pathways and aid in increasing titers of the polymer precursor valerolactam (10,11,(18)(19)(20). Here, we leverage RB-TnSeq to interrogate the genetic basis for the catabolism of multiple fatty acids and alcohols to develop an evidence-based understanding of the enzymes and pathways utilized in these metabolisms.…”