# The link of carbon catabolite repression elements, small RNAs CrcY and CrcZ and polyhydroxyalkanoate metabolism in Pseudomonas putida KT2440

## TL;DR

This paper explores how small RNAs CrcY and CrcZ in Pseudomonas putida KT2440 influence PHA production and polymer properties, offering insights for improving bioplastic manufacturing.

## Contribution

The study reveals the regulatory roles of CrcY and CrcZ in PHA metabolism and demonstrates their impact on polymer properties and production efficiency.

## Key findings

- Overexpression of CrcY and CrcZ increases PHA titre by 1.3- to 3.5-fold and reduces polymer molecular weight.
- CrcY and CrcZ compensate for each other, and their removal decreases PHA production by 2.5-fold.
- Overexpression of CrcY and CrcZ affects mcl-PHA accumulation and molecular weight, useful for producing polymer additives.

## Abstract

Polyhydroxyalkanoates (PHAs), biodegradable polymers, can be synthesised and degraded by a number of bacteria. With a range of monomer composition and molecular weight, these polymers can be used for packaging to medical applications. However, the production cost, inadequate mechanical properties, and challenging melt processing properties are major impediments.

Understanding and harnessing the regulatory networks underpinning PHA production in a model organism Pseudomonas putida KT2440 is an invaluable tool to increase PHA production and alter polymer properties for specific applications.

The small RNAs CrcY and CrcZ, key components of the carbon catabolite repression (CCR) system, are implicated in PHA metabolism in P. putida KT2440. Their in trans overexpression in P. putida KT2440 shows a 1.3- to 3.5-fold increase in PHA titre (g/L), using glucose or octanoate as feedstocks. This is accompanied by a decrease in the Mw of the synthesised polymer. Among the proteins showing differential expression in response to CrcY and CrcZ overexpression, glutaryl-CoA dehydrogenase GcdH, involved in the catabolism of lysine, hydroxylysine, and tryptophan, and gamma-glutamyl transpeptidase GGT, involved in glutathione metabolism, showed a consistent increase in abundance across different conditions. It also appears that CrcY and CrcZ can compensate for each other, as only when both sRNAs are removed is a 2.5-fold decrease in PHA observed. We also show that these sRNAs require other CCR elements, Hfq and Crc, for their role in PHA metabolism.

One strategy to overcome poor mechanical properties of PHAs is to blend them with a second polymer. Medium chain length (mcl)-PHA acts as a plasticiser when blended with poly-3-hydroxybutyrate (PHB), the most widespread used PHA resin. Here we show a clear effect of the overexpression of CCR elements CrcY and CrcZ in P. putida KT2440 on the amount of the accumulated mcl-PHA and its Mw, making this tool valuable to produce mcl-PHA-based additives.

These findings highlight the complementary regulatory roles of CrcY and CrcZ in modulating CCR to optimise PHA production. This study provides insights into leveraging CCR elements to enhance the efficiency of PHA biosynthesis, contributing to the development of sustainable bioplastic production.

The online version contains supplementary material available at 10.1186/s13068-025-02707-5.