Biosynthesis of Tropolones in Streptomyces spp.: Interweaving Biosynthesis and Degradation of Phenylacetic Acid and Hydroxylations on the Tropone Ring

Abstract

Tropolonoids are important natural products that contain a unique seven-membered aromatic tropolone core and exhibit remarkable biological activities. 3,7-Dihydroxytropolone (DHT) isolated from species is a multiply hydroxylated tropolone exhibiting antimicrobial, anticancer, and antiviral activities. Herein, we determined the DHT biosynthetic pathway by heterologous expression, gene deletion, and bioconversion. Nine genes and some of the aerobic phenylacetic acid degradation pathway genes () located outside of the biosynthetic gene cluster are required for the heterologous production of DHT. The gene encodes a single-domain protein homologous to the C-terminal enoyl-CoA hydratase domain of PaaZ. TrlA truncates the phenylacetic acid catabolic pathway and redirects it towards the formation of heptacyclic intermediates. TrlB is a 3-deoxy-D-arabino-heptulosonic acid-7-phosphate (DAHP) synthase homolog. TrlH is an unusual bifunctional protein bearing an N-terminal prephenate dehydratase domain and a C-terminal chorismate mutase domain. TrlB and TrlH enhanced biosynthesis of phenylpyruvate, thereby providing abundant precursor for the prolific production of DHT in Six seven-membered carbocyclic compounds were identified from the gene deletion mutants of ,, , and Four of these chemicals, including 1,4,6-cycloheptatriene-1-carboxylic acid, tropone, tropolone and 7-hydroxytropolone, were verified as key biosynthetic intermediates. TrlF is required for the conversion of 1,4,6-cycloheptatriene-1-carboxylic acid into tropone. Monooxygenases TrlE and TrlCD catalyze the regioselective hydroxylations of tropone to afford DHT. This study reveals a natural association of anabolism of chorismate and phenylpyruvate, catabolism of phenylacetic acid, and biosynthesis of tropolones in spp.Tropolonoids are promising drug lead compounds because of their versatile bioactivities attributed to their highly oxidized seven-membered aromatic ring scaffolds. Our study here provides a clear insight into the biosynthesis of 3,7-dihydroxytropolone (DHT) through the identification of key genes responsible for the formation and modification of the seven-membered aromatic core. We also reveal the intrinsic mechanism of elevated production of DHT and related tropolonoids in spp. The study on DHT biosynthesis in exhibits a good example of antibiotic production in which both anabolic and catabolic pathways of primary metabolism are interwoven into the biosynthesis of secondary metabolites. Furthermore, our study sets the stage for metabolic engineering of tropolonoid natural products biosynthetic pathway and provides alternative synthetic biology tools for engineering novel tropolonoids.

Bacterial Tropone Natural Products and Derivatives: Overview of their Biosynthesis, Bioactivities, Ecological Role and Biotechnological Potential

“…These Gram-positive bacteria generate 13 via a dedicated gene cluster (named trl) that promotes the shunting of the 1 catabolic pathway. [40] In total, nine additional genes (trlA-trlH and trlR) organized in three operons were found in this gene cluster. Heterologous production and gene deletion experiments confirmed 9, as well as 11, and 12 as intermediates in 13 biosynthesis.…”

“…were also reported as producers of tropones such as 13 first isolated from S. tropolofaciens. [39] More recently, S. cyaneofuscatus Soc7 and S. luteogriseus Slg41 were also shown to produce 13 at high titers in addition to other tropolones such as 12 or 5-hydroxytropolone (14; [40] Figures 1 and 7). These Gram-positive bacteria generate 13 via a dedicated gene cluster (named trl) that promotes the shunting of the 1 catabolic pathway.…”

“…Precursor supply possibly also involves TrlG, which was hypothesized to catalyze the conversion of phenylpyruvate into 1. [40] Notably, the two crucial enzymes for redirecting the metabolic flow of the 1 catabolic pathway are TrlA and TrlF.…”

“…Hence, these compounds might also be of interest for further evaluation and development as potential drugs. Com-pared to S. tropolofaciens K611-97, from which 13 was first isolated, [40] S. cyaneofuscatus Soc7 as well as S. luteogriseus Slg41 produced 13 without genetic modification with substantially higher yields (ca. 10,000 times) of 380 and 230 mg/L, respectively.…”

“…The data suggested that shikimate pathway enzymes (TrlB, TrlH, possibly TrlG) and a transcriptional regulator (TrlR) increased the production of 13 by promoting the de novo synthesis of aromatic pathway precursors. [40] Genetic manipulation of these producers might possibly further improve the yield. Other obvious starting points for bioengineering efforts could be the proposed ring hydroxylases TrlE and TrlCD.…”