Production of medium-chain-length polyhydroxyalkanoates by sequential feeding of xylose and octanoic acid in engineered Pseudomonas putida KT2440

Abstract

BackgroundPseudomonas putida KT2440 is able to synthesize large amounts of medium-chain-length polyhydroxyalkanoates (mcl-PHAs). To reduce the substrate cost, which represents nearly 50% of the total PHA production cost, xylose, a hemicellulose derivate, was tested as the growth carbon source in an engineered P. putida KT2440 strain.ResultsThe genes encoding xylose isomerase (XylA) and xylulokinase (XylB) from Escherichia coli W3110 were introduced into P. putida KT2440. The recombinant KT2440 exhibited a XylA activity of 1.47 U and a XylB activity of 0.97 U when grown on a defined medium supplemented with xylose. The cells reached a maximum specific growth rate of 0.24 h-1 and a final cell dry weight (CDW) of 2.5 g L-1 with a maximal yield of 0.5 g CDW g-1 xylose. Since no mcl-PHA was accumulated from xylose, mcl-PHA production can be controlled by the addition of fatty acids leading to tailor-made PHA compositions. Sequential feeding strategy was applied using xylose as the growth substrate and octanoic acid as the precursor for mcl-PHA production. In this way, up to 20% w w-1 of mcl-PHA was obtained. A yield of 0.37 g mcl-PHA per g octanoic acid was achieved under the employed conditions.ConclusionsSequential feeding of relatively cheap carbohydrates and expensive fatty acids is a practical way to achieve more cost-effective mcl-PHA production. This study is the first reported attempt to produce mcl-PHA by using xylose as the growth substrate. Further process optimizations to achieve higher cell density and higher productivity of mcl-PHA should be investigated. These scientific exercises will undoubtedly contribute to the economic feasibility of mcl-PHA production from renewable feedstock.

Production of polyhydroxyalkanoates from dephenolised and fermented olive mill wastewaters by employing a pure culture of Cupriavidus necator

“…Two linear correlations were obtained when performing the Abs 600 vs. cell dry weight (CDW) calibration curve: one for the growth phase and another for the accumulation phase (data not shown). Therefore, absorbance measurements were also used to detect PHA production during the accumulation phase in agreement with [24][25][26]. PHAs were qualitatively and quantitatively determined by GC analyses, as described below.…”

Strategies for Large-scale Production of Polyhydroxyalkanoates

“…In a similar strategy, production of an mcl-PHA was attempted using xylose and octanoic acid. 68 It basically involved initiation of the cultivation as a batch fermentation on xylose, with linear feeding of octanoic acid at different point times during the exponential phase of the culture. Xylose was consumed by Pseudomonas putida KT2440 (pSML1) only for growth but not for mcl-PHA production, while octanoic acid was used as an mcl-PHA precursor.…”

Pilot-scale Production of Functionalized mcl-PHA from Grape Pomace Supplemented with Fatty Acids

“…Nevertheless, cheaper alternative carbon sources may be used for the growth phase in order to reduce the overall cost of the bioprocess. The strategy then consists of (1) producing large amounts of biomass from a non-costly substrate (= growth phase), and (2) adding the fatty acids that are necessary for the accumulation of tailor-made mcl-PHAstion phase 10 . Replacing fatty acids by carbohydrates for the growth phase also allows achieving higher cell densities, since the former can cause growth inhibition from concentrations starting at around 15-20 mmol L -1 , whereas sucrose is not problematic up to about 230 mmol L -1 for Pseudomonas 11 .…”