Tailored Biosynthesis of Olefinic Medium-Chain-Length Poly[(R)-3-hydroxyalkanoates] in Pseudomonas putida GPo1 with Improved Thermal Properties Rene ´ Hartmann, †,‡,| Roland Hany, ‡ Thomas Geiger, ‡ Thomas Egli, § Bernard Witholt, | and Manfred Zinn* ,† Swiss Federal Laboratories for Materials Testing and Research (EMPA), Lerchenfeldstrasse 5, CH-9014 St. Gallen, Switzerland, Swiss Federal Laboratories for Materials Testing and Research (EMPA), Ueberlandstrasse 129, CH-8600 Duebendorf, Switzerland, Swiss Federal Institute for Environmental Science and Technology (EAWAG), Ueberlandstrasse 133, CH-8600 Duebendorf, Switzerland, and Swiss Federal Institute of Technology Zurich (ETHZ), Institute of Biotechnology, ETH-Hoenggerberg HPT, CH-8093 Zurich, Switzerland Received February 10, 2004; Revised Manuscript Received June 4, 2004 ABSTRACT: Mixtures of 5-phenylvalerate, octanoate, and 10-undecenoate were fed to a chemostat culture (dilution rate ) 0.1 h -1 ) of Pseudomonas putida GPo1 under well-defined dual-(C,N)-nutrient limited growth conditions. Five new, tailor-made copolymers were produced and consisted of poly(3-hydroxy-5- phenylvalerate-co-3-hydroxyalkanoates-co-3-hydroxy-ω-alkenoates), poly(HP-co-HA-co-HE), with increas- ing amounts of aromatic side chains (A, 0%; B, 3%; C, 19%; D, 42%; and E, 59%), approximately 10 mol % unsaturated side chains, and decreasing amounts of saturated side chains. On the basis of NMR analysis of polymer E, it was concluded that the incorporation of the substrates occurred randomly. The HP- content determined the glass transition temperature, which increased linearly from -38.7 °C for poly- (0%HP-co-90%HA-co-10%HE) to -6.0 °C for poly(59%HP-co-31%HA-co-10%HE). Introduction Poly-[(R)-3-hydroxyalkanoates] (PHAs) are biodegrad- able and biocompatible polymers of high molecular weight synthesized by a wide variety of microorgan- isms. 1,2 PHAs have received increased attention due to their potential applications in coatings, as medical implants, as controlled drug release systems or as a source of chiral monomers. 3-6 The mcl-PHAs (which contain medium-chain-length C 6 -C 12 alkanoate mono- mers) are mostly amorphous and soft-sticky with glass transition temperatures (T g ) between -44 and -30 °C and melting temperatures (T m ) between 39 and 61 °C. 7-9 Various mcl-PHAs with side chains containing func- tional groups, for example carbon-carbon double 10 and triple bonds, 11 acetoxy and ketone, 12 or aromatic groups 13-15 have been produced. Among the latter ones, mcl-PHAs containing phenyl groups, such as poly[(R)- 3-hydroxy-5-phenylvalerate] (T g ) 13 °C), 13 or (co-)- polymers from 6-phenylhexanoic acid, 7-phenylhepta- noic acid, and 8-phenyloctanoic acid (T g between -14.8 and -1.3 °C) 16 were completely amorphous but showed an increase in the glass transition temperature. The presence of functional groups in mcl-PHAs pro- vides sites for chemical modifications, which may be useful to modify physical properties or to create chemi- cal groups which cannot be directly introduced by biosynthesis. Polymer-analogous reactions on unsatur- ated mcl-PHAs have been studied in detail, and cross- linking, 17-19 epoxidation, 20 or conversion to carboxylic 21,22 and diol groups 23 have been reported so far. In this work, chemostatic culture conditions (also called continuous culture) were applied to produce tailored mcl-PHAs. A chemostat is essentially a perfu- sion reactor that is continuously supplied with sterile medium. The (culture) volume in the reactor is kept constant by the continuous removal of culture broth. Once such a system is in equilibrium (steady-state), cell number, nutrition, and productivity remain constant over time. 24 As we have shown previously, 25,26 Pseudomo- nas putida GPo1 can be grown under simultaneous limitation by carbon (C) and nitrogen (N) substrates in a chemostat. Under such growth conditions all fed C- and N-substrates are completely metabolized and there- fore no toxicity of C-substrates is detected. 27 In this study, different mixtures of octanoic, 10- undecenoic, and 5-phenylvaleric acid were used to produce mcl-PHAs (Scheme 1) in a chemostat under dual-(C,N)-limited growth conditions. This concept allowed the tailored synthesis of novel, olefinic PHAs * Corresponding author. Telephone: +41 71 274 7698. Fax: +41 71 274 7788. E-mail: manfred.zinn@empa.ch. † Swiss Federal Laboratories for Materials Testing and Research (EMPA), St. Gallen. ‡ Swiss Federal Laboratories for Materials Testing and Research (EMPA), Duebendorf. § Swiss Federal Institute for Environmental Science and Tech- nology (EAWAG). | Swiss Federal Institute of Technology Zurich (ETHZ). Scheme 1. Structural Formula of Poly(HP-co-HA-co-HE) a a Key: HP, 3-hydroxy-5-phenylvalerate; HA6, 3-hydroxy- hexanoate; HA8, 3-hydroxyoctanoate; HE7, 3-hydroxy-6-hep- tenoate; HE9, 3-hydroxy-8-nonenoate; HE11, 3-hydroxy-10- undecenoate. 6780 Macromolecules 2004, 37, 6780-6785 10.1021/ma040035+ CCC: $27.50 © 2004 American Chemical Society Published on Web 08/07/2004