Biotechnology Letters 23: 2057–2061, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. 2057 Increased poly-β -hydroxybutyrate (PHB) chain length by the modulation of PHA synthase activity in recombinant Escherichia coli Sang Jun Sim 1,∗ , Kristi D. Snell 2 , Byung Woo Kim 3 , Cho Kyun Rha 4 & Anthony J. Sinskey 5 1 Water Environment and Remediation Research Center, Korea Institute of Science and Technology, Seoul 136-791, Korea 2 Metabolix, Inc., MA 02142, USA 3 Department of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, Korea 4 Biomaterials Science and Engineering Laboratory, and 5 Department of Biology, Massachusetts Institute of Tech- nology, MA 02139, USA ∗ Author for correspondence (Fax: 82-2-958-5269; E-mail: simsj@kist.re.kr) Received 22 August 2001; Revisions requested 24 August 2001/26 September 2001; Revisions received 25 September 2001/18 October 2001; Accepted 18 October 2001 Key words: average molecular weight, PHA synthase, polyhydroxybutyrate Abstract A second promoter (P1) was inserted to the PHA (poly-β -hydroxyalkanoate) operon (pSP2) of Esherichia coli DH5α with an optimal E. coli ribosome binding site and a trc strong promoter (pSJS1) to obtain poly-β - hydroxybutyrate (PHB) with long chain length. When the inducer, IPTG was added to the culture at 0.4 mM, the average molecular weight was 1.1 × 10 6 Da. However, an even greater increase of the PHB average molecular weight to 2.5 × 10 7 Da was observed without IPTG being added. Introduction Poly-β -hydroxyalkanoates (PHAs) are intracellular carbon and energy storage materials that can be syn- thesized by many microorganisms under unfavorable growth conditions, such as limitation by nitrogen, phosphate, SO 2- 4 ,O 2 , or Mg 2+ (Anderson & Dawes 1990, Doi 1990, Haywood et al. 1990, Steinbüchel & Füchtenbusch 1998). PHAs also have been con- sidered as an attractive substitute for petroleum-based synthetic plastics because of their similar properties to synthetic polymers and complete biodegradabil- ity. However, the higher production cost of PHAs, in comparison with conventional petrochemical-based plastics, has been the main obstacle to their use in a wide range of applications (Byrom 1987, Poirier et al. 1995). In order to overcome such an obstacle, researches on poly-β -hydroxybutyrate (PHB) synthe- sis by agricultural production with transgenic plants have been conducted (Poirer et al. 1992, Pool 1989). PHA-copolymer [poly(3-hydroxybutyric-co-3-hy- droxyvaleric acid)], which possesses more desirable physical properties than PHB, is produced mainly by bacterial fermentation. However, its production cost is relatively high. On the other hand, genetically mod- ified (GM) plants are capable of producing only the PHB homopolymer, which is a serious limitation to its use in commodity products. There is a considerable in- terest in the synthesis of PHA-copolymer in GM plants (Poirier et al. 1995). As an alternative to copolymer production, production of homopolymer, PHB, with a wide range of average molecular weight has been investigated. It is then particularly important to control the average molecular weight and size distribution of biopolymer, which have been recognized as the most important physical properties of the polymer. This paper reports a biosynthesis of high molecular weight PHB through minimization of PHA synthase activity. We constructed a new plasmid by insert- ing a second non-inducible P1 promoter to the PHA operon (pSP2) with an optimal E. coli ribosome bind-