Enhanced production of medium-chain-length polyhydroxyalkanoates (PHA) by PHA depolymerase knockout mutant of Pseudomonas putida KT2442

Abstract

Pseudomonas putida KT2442 is a medium-chain-length polyhydroxyalkanoates (PHA) producer. One of the main shortages in the production of PHA has been the intracellular PHA degradation caused by its endogenous PHA depolymerase. The aim of this study was to improve PHA production via removing the PHA degradation mechanism. PHA depolymerase phaZ knockout mutant P. putida KTMQ01 was successfully constructed, which accumulated 86 wt% medium-chain-length PHA (mcl PHA) when cultured in mineral medium containing sodium octanoate as the carbon source compared with P. putida KT2442 which produced only 66 wt% of its cell dry weight (CDW). P. putida KTMQ01 cultured over a five-day period on sodium octanoate produced 4.5 g L(-1)-4.0 g L(-1) CDW containing approximately 80 wt% PHA without degradation. In contrast, P. putida KT2442 was observed with decreasing CDW and PHA from over 4 to less than 2 g L(-1) over the same period of time, indicating the function of PHA depolymerases which reduced the amount of PHA from around 50 wt% to none over the incubation period. RT-PCR analysis showed that phaC2 transcriptional level of P. putida KTMQ01 was higher than that of P. putida KT2442, indicating the possibility of relief on negative control of phaC2 transcription by the deletion of phaZ, which combined with the lack of in vivo PHA degradation, led to more PHA accumulation. P. putida KTMQ01 contained PHA granules with larger sizes and smaller numbers than those of P. putida KT2442.