Improved accumulation of poly- 3-hydroxybutyrate by a recombinant cyanobacterium Hideyuki Takahashi 1 , Masato Miyake 2 , Yutaka Tokiwa 2 , and Yasuo Asada 2 * 1 Research Institute of Innovative Technology for the Earth (RITE), NIBH-Branch, Higashi 1-1, Tsukuba, Ibaraki, 2 National Institute of Bioscience and Human-Technology, Higashi 1-1, Tsukuba, Ibaraki, 305. Japan. Synechococcus sp. PCC7942 was transformed with genes encoding poly-3-hydroxybutyrate(PHB)-synthesizing enzymes from Alcaligenes eutrophus by using a new plasmid vector. Under photoautotrophic and nitrogen-starved conditions, PHB content of the transformant was improved by CO 2 -enrichment in sparging. Supplement of acetate under nitrogen-starvation enhanced its PHB content by more than 25 %. The mol wt and distribution of PHB from the cyanobacterial transformant were similar to those by Escherichia coli transformant with the same genes as described above. Introduction Polyhydroxyalkanoates (PHAs) including poly- 3-hydroxybutyrate (PHB) are biodegradable polymers which are synthesized by many prokaryotes. Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) which is one of the PHA is now commercially produced by heterotrophic cultures of Alcaligenes eutrophus on glucose and propionate (Poirier et al., 1995a). Recently, higher plants into which PHB synthesis genes have been introduced have been used to produce PHB from CO 2 by photosynthesis. The trans- formant of Arabidopsis thaliana harboring PHB genes in a plastid, accumulated approximately 14% dry wt as PHB in its leaves (Nawrath et al., 1994). Oil crops such as soybean and rapeseed have been thought to be advantageous hosts for PHB accumulation because metabolic characteristics are partially common in oil and PHB synthesis (Poirier et al., 1995a). Cyanobacteria grow faster than higher plants and some can accumulate PHB under nitrogen or phosphorus lim- ited conditions (Stal, 1992). However, it is accumulated intracellularly only at a few percent of dry weight. Although we have isolated a potent PHB-accumulator (Miyake et al., 1996a), genetic engineering can be a useful tool to enhance the PHB-accumulating activity. We have previously reported that about 1% of PHB was accumu- lated by nitrogen-starved cultures of the transformant of Synechococcus sp. PCC7942 (Suzuki et al, 1996). Here, we report improved PHB accumulation under various culture conditions. Materials and methods Bacterial strains and cultures Synechococcus sp. PCC7942, which did not accumulate PHB, was used as a host since the transformants were easy to be analyzed for PHB accumulation. The cyanobacteria were grown at 30°C in BG-11 medium (Allen,1968) supple- mented with 30 mM HEPES (pH 7.8, adjusted with NaOH) under continuous illumination by fluorescent lamps (3 klux). The cultures were sparged with air or 5 % CO 2 (v/v) in air. In the case to examine effects of organic compounds, the transformant was cultivated in the medium containing 10 mM compounds. A transformant of Escherichia coli JM109 was cultivated at 37°C in Luria-Bertani medium (Sambrook et al., 1989) containing 1% glucose. DNA manipulations General DNA manipulations were according to a labor- atory manual (Sambrook et al., 1989). Transformation of Synechococcus sp. PCC7942 was done as reported pre- viously (Suzuki et al., 1996). Determination of PHB content PHB was extracted from the dry cells into chloroform, and cell materials were removed by filtration. PHB samples were purified by re-precipitation with hexane and dried according to Kawaguchi and Doi (1992). PHB content was determined by gas-chromatography as reported previously (Suzuki et al., 1996). Biotechnology Letters, Vol 20, No 2, February 1998, pp. 183–186 © 1998 Chapman & Hall Biotechnology Letters ⋅ Vol 20 ⋅ No 2 ⋅ 1998 183