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

Abstract

Background: Pseudomonas 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.

Results: The 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.

Conclusions: Sequential 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.

Figure 1

Growth of the P. putida KT2440 recombinants in shake flasks on E2 minimal medium containing 10 g L^-1glucose or xylose as the sole carbon source. The arrow represents the addition of IPTG. The experiments were performed in replicate flasks. Data points are the averages of the results of duplicate measurements.

Figure 2

Growth of P. putida KT2440 (pSLM1) in E2 minimal medium with 10 g L^-1 xylose in a 3.7 L bioreactor. Data points are the averages of the results of duplicate measurements.

Figure 3

Fed-batch experiment of P. putida KT2440 (pSLM1) grown on 0.2NE2 medium containing 1.8 g L^-1 xylose with linear feeding of 0.288 g L^-1h^-1 octanoic acid at OD₆₀₀ of 1 for 4 h. Nitrogen (∆), octanoic acid (□) and xylose (○) concentrations were measured. The logarithmic growth is represented by filled squares (■) and the PHA accumulation by filled triangles (▲). Data points are the averages of the results of duplicate measurements.

Figure 4

Hypothetical pathway for mcl-PHA accumulation from xylose in P. putida. Enhanced arrows: steps absent in wild-type P. putida strains; XylA: xylose isomerase; XylB: xylulokinase; PhaG: 3- hydroxyacyl-ACP:CoA transferase; PhaC: PHA polymerase.