Maximising the efficiency of Terephthalic acid and Ethylene glycol metabolism in Pseudomonas umsongensis GO16

Polyethylene terephthalate (PET) is a plastic widely used for packaging material and synthetic fibres. Previously, upcycling PET into valuable products using micro-organism was demonstrated as a valuable approach to address plastic waste. PET can be enzymatically depolymerised into terephthalic acid (TA) and ethylene glycol (EG), followed by the conversion of the hydrolysate into bacterial polyesters, polyhydroxyalkanoates (PHAs), using bacteria such as Pseudomonas umsongensis GO16. PHAs are bacterial carbon and energy storage polyesters usually accumulated under an inorganic nutrient limitation stress. PHAs can be used in various applications due to their biodegradable, thermoplastic, and biocompatibility properties. While the proof-of-concept for the biotechnological strategy to upcycle PET was demonstrated, the efficiency of the metabolism of PET hydrolysate remains a challenge. Consequently, the aim of this project is to enhance the metabolism of TA and EG from PET hydrolysate in P. umsongensis GO16 by employing molecular biology tools. Regarding TA metabolism in P. umsongensis GO16, TA can be degraded by tph catabolic genes into protocatechuate (PCA). PCA is metabolised into TCA cycle intermediates through β-ketoadipate pathway. Depending on which position of aromatic ring PCA is cleaved, the final products entering the TCA cycle and the generated reduced energy equivalents can be varied. In this project, we deleted pcaGH genes to disable native ortho-cleavage pathway and introduced PCA para- or meta-cleavage pathway. It was confirmed that GO16 did not grow with TA as a sole carbon source when pcaGH genes were deleted and it recovered its growth when para- or meta-cleavage pathway was adopted. Furthermore, having both ortho- and meta-cleavage pathway in GO16 shortened the lag phase 2.7-fold compared to the control strain. A LysR-type transcriptional regulator (LTTRs) ttdR is located 5'- of the gene for glyoxylate carboligase (gcl) on the chromosome of P. umsongensis GO16. GO16 wildtype (WT) can utilise EG as a sole carbon and energy source, but ttdR knockout strain (ΔttdR) cannot grow on EG, demonstrating its role in EG metabolism. Conversely, when TtdR was overexpressed, this promoted EG consumption by 2-fold and increased biomass by 1.4-fold compared to the WT when 120 mM of EG was supplied as a substrate, further confirming its role as an activator of EG metabolism. GO16 natively produces both short-chain length (scl) PHA and medium-chain length (mcl) PHA from various carbon sources. Interestingly, ΔttdR strain produced sclPHA monomer 5.4-fold and 1.4-fold increased than WT when TA and glucose were used respectively. The deletion of ttdR also affected fatty acid metabolism as the mutant strain showed a very lag phase when grown with fatty acids compared to GO16 WT. This indicates a more complex role of ttdR than simply activating EG metabolism in GO16. We conducted proteomic analysis to identify the proteins affected by the TtdR in GO16. As expected, the proteins of the EG oxidation pathway were downregulated when TtdR was deleted. On the other hand, proteins involved in PHA synthesis, L-valine degradation, and propionate metabolism were upregulated in the ttdR KO strain. Moreover, proteins related to cellular regulations and signal transduction also appeared to be influenced by TtdR.
The findings of this study provide significant insights into the metabolism of the PET-upcycling model strain P. umsongensis GO16, highlighting its potential for metabolic engineering. By utilising the gained knowledge, the strain's metabolism can be further enhanced and exploited to expand its potential to upcycle PET to valuable products other than PHA.