Improved Production of Medium-Chain-Length Polyhydroxyalkanoates in Glucose-Based Fed-Batch Cultivations of Metabolically Engineered Pseudomonas putida Strains

Abstract

One of the major challenges in metabolic engineering for enhanced synthesis of value-added chemicals is to design and develop new strains that can be translated into well-controlled fermentation processes using bioreactors. The aim of this study was to assess the influence of various fed-batch strategies in the performance of metabolically engineered Pseudomonas putida strains, Δgcd and Δgcd-pgl, for improving production of medium-chain-length polyhydroxyalkanoates (mcl-PHAs) using glucose as the only carbon source. First we developed a fed-batch process that comprised an initial phase of biomass accumulation based on an exponential feeding carbon-limited strategy. For the mcl-PHA accumulation stage, three induction techniques were tested under nitrogen limitation. The substrate-pulse feeding was more efficient than the constant-feeding approach to promote the accumulation of the desirable product. Nonetheless, the most efficient approach for maximum PHA synthesis was the application of a dissolved-oxygen-stat feeding strategy (DO-stat), where P. putida Δgcd mutant strain showed a final PHA content and specific PHA productivity of 67% and 0.83 g·l(-1)·h(-1), respectively. To our knowledge, this mcl-PHA titer is the highest value that has been ever reported using glucose as the sole carbon and energy source. Our results also highlighted the effect of different fed-batch strategies upon the extent of realization of the intended metabolic modification of the mutant strains.