Identification of the phd gene cluster responsible for phenylpropanoid utilization in Corynebacterium glutamicum

Abstract

Phenylpropanoids as abundant, lignin-derived compounds represent sustainable feedstocks for biotechnological production processes. We found that the biotechnologically important soil bacterium Corynebacterium glutamicum is able to grow on phenylpropanoids such as p-coumaric acid, ferulic acid, caffeic acid, and 3-(4-hydroxyphenyl)propionic acid as sole carbon and energy sources. Global gene expression analyses identified a gene cluster (cg0340-cg0341 and cg0344-cg0347), which showed increased transcription levels in response to phenylpropanoids. The gene cg0340 (designated phdT) encodes for a putative transporter protein, whereas cg0341 and cg0344-cg0347 (phdA-E) encode enzymes involved in the β-oxidation of phenylpropanoids. The phd gene cluster is transcriptionally controlled by a MarR-type repressor encoded by cg0343 (phdR). Cultivation experiments conducted with C. glutamicum strains carrying single-gene deletions showed that loss of phdA, phdB, phdC, or phdE abolished growth of C. glutamicum with all phenylpropanoid substrates tested. The deletion of phdD (encoding for putative acyl-CoA dehydrogenase) additionally abolished growth with the α,β-saturated phenylpropanoid 3-(4-hydroxyphenyl)propionic acid. However, the observed growth defect of all constructed single-gene deletion strains could be abolished through plasmid-borne expression of the respective genes. These results and the intracellular accumulation of pathway intermediates determined via LC-ESI-MS/MS in single-gene deletion mutants showed that the phd gene cluster encodes for a CoA-dependent, β-oxidative deacetylation pathway, which is essential for the utilization of phenylpropanoids in C. glutamicum.

Biotransformation of ferulic acid to protocatechuic acid by Corynebacterium glutamicum ATCC 21420 engineered to express vanillate O-demethylase

“…C. glutamicum converts FA to PCA via the proposed pathways as follows: (I) non-β-oxidative, CoA-dependent (Merkens et al 2005; Brinkrolf et al 2006) and (II) β-oxidative, CoA-dependent degradation of phenylpropanoid ( phd ) (Kallscheuer et al 2016) (Fig. 1).…”

“…Fcs feruloyl-CoA synthase, Ech enoyl-CoA hydratase/aldolase, Vdh vanillin dehydrogenase, and Van vanillate 3- O -demethylase. II β-oxidative, CoA-dependent phenylpropanoid degradation pathway (Kallscheuer et al 2016). PhdA acyl-CoA ligase, PhdE enoyl-CoA hydratase, PhdB 3-hydroxyacyl-CoA dehydrogenase, PhdC 3-oxoacyl-CoA ketohydrolase, and Van …”

BacHBerry: BACterial Hosts for production of Bioactive phenolics from bERRY fruits

“…Global gene expression analyses were conducted, which led to the identification of a gene cluster consisting of seven genes (designated phdT, phdA, phdR, phdB, phdC, phdD and phdE). In response to feeding of the above-mentioned phenylpropanoids these genes were upregulated up to 100-fold (Kallscheuer et al 2016a). In subsequent studies, it turned out that the genes in this cluster code for the enzymes of the phenylpropanoid catabolic pathway in C. glutamicum.…”

“…The maximum growth rates on the selected phenylpropanoids ranged from 0.15 to 0.23 h -1 corresponding to doubling times of 3-4.5 h (Kallscheuer et al 2016a). We suspected that the activity of this pathway would come into conflict with any attempt to produce polyphenols as both pathways compete for phenylpropanoids.…”

“…Unexpectedly, C. glutamicum failed to grow on the phenylpropanoid, cinnamic acid, which lacks ring para-hydroxylation. Astonishingly, a C. glutamicum strain with a single deletion of the gene phdR coding for the identified transcriptional repressor was able to grow with cinnamic acid as sole carbon and energy source (Kallscheuer et al 2016a). This indicated that PhdR has a pronounced specificity for hydroxylated phenylpropanoids, especially when hydroxylated in para-position of the aromatic ring.…”