Bioengineering of poly(β-hydroxyalkanoates) for advanced material applications: incorporation of cyano and nitrophenoxy side chain substituents

Novel functional poly(β-hydroxyalkanoates), PHAs, with hyperpolarizable side groups were prepared. Pseudomonas oleovorans ATCC 29347 and Pseudomonas putida KT 2442 were selected as biocatalysts. These organisms were first grown on citrate (40 mM) before polymer formation was studied in a second stage. The carbon sources in second-stage cultivations were mixtures (total of 15 mM) of octanoate (OA) with either 7.5 or 10 mM para-cyanophenoxyhexanoate (CPH), para-cyanophenoxyvalerate (CPV), para-cyanophenoxybutyrate (CPB), or para-nitrophenoxyhexanoate (NPH). For both organisms, only small deviations in the number of colony-forming units per millilitre were observed when second-stage cultivations containing only 15 mM octanoate were compared with those using mixtures of OA with either CPB, CPV, CPH, or NPH. PHA volumetric yields as a function of organism, carbon source mixture, and culture time are reported. The percent incorporations of substituted phenoxy side groups in the polymer products were determined by1H NMR spectroscopy. Pseudomonas putida formed PHAs with up to 24.2% 3-hydroxy-6-(para-cyanophenoxy)hexanoate side groups when 5 mM OA and 10 mM CPH were used. A dramatic decrease in the percent incorporation of para-cyanophenoxy (CP) by P. putida was observed when the relatively shorter CP-substituted n-alkanoate chain CPV and CPB cosubstrates were used. Use of NPH in place of CPH had deleterious effects on both polymer formation and percent incorporation of substituted phenoxy side groups in P. putida. Pseudomonas oleovorans formed PHAs with only up to ~2% CP side chains when a combination of OA and CPH was used and little to no CP side groups when a combination of OA and CPV or CPB was used. Substitution of NPH in place of CPH led to a modest increase (up to ~5%) in substituted phenoxy side groups. Thus, a new route to first-generation chiral polymer structures for nonlinear optical applications was demonstrated.Key words: Pseudomonas oleovorans, Pseudomonas putida, poly(β-hydroxyalkanoate), poly(3-hydroxy-6-para-cyanophenoxyhexanoate), poly(3-hydroxy-6-para-nitrophenoxyhexanoate), poly(β-hydroxy-para-cyanophenoxyvalerate).