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Effect of increased PHA synthase activity on polyhydroxyalkanoates biosynthesis in Synechocystis sp. PCC6803 - ScienceDirect

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International Journal of Biological Macromolecules Volume 30, Issue 2, 8 April 2002, Pages 97-104 Effect of increased PHA synthase activity on polyhydroxyalkanoates biosynthesis in Synechocystis sp. PCC6803 Author links open overlay panel Kumar Sudesh, Kazunori Taguchi , Yoshiharu Doi Show more Add to Mendeley Share Cite https://doi.org/10.1016/S0141-8130(02)00010-7 Get rights and content Abstract Polyhydroxyalkanoate (PHA) synthase activity in Synechocystis sp. PCC6803 was increased two-fold by introducing the PHA biosynthetic genes of Ralstonia eutropha. The resulting recombinant Synechocystis sp. PCC6803 strain was subjected to conditions that favor PHA accumulation and the effects of various carbon sources were studied. In addition, the fine structure of both wild-type and recombinant Synechocystis sp. PCC6803 was examined using freeze-fracture electron microscopy technique. The PHA granules in the recombinant Synechocystis sp. PCC6803 were localised near the thylakoid membranes. Maximum amount of PHA accumulation was obtained in the presence of acetate, where the number of granules in the recombinant cells ranged from 4 to 6 and their sizes were in the range of 70–240 nm. In comparison to wild-type Synechocystis sp. PCC6803, recombinant cells with increased PHA synthase activity showed only a marginal increase in PHA content suggesting that PHA synthase is not the rate limiting enzyme of PHA biosynthesis in Synechocystis sp. PCC6803. Introduction Intensive research on microbial polyhydroxyalkanoates (PHAs) has mainly been motivated by the fact that PHAs are potential biodegradable and biocompatible thermoplastic [1], [2], [3]. In order to mass-produce PHAs, various wild-type and recombinant bacteria have been employed in fermentation systems using various carbon sources [4] and several types of PHAs with useful physical properties have been identified and their biosynthesis mechanisms outlined [5]. Large amount of PHA accumulation (up to 80% of the bacterial dry cell weight) has been achieved in some recombinant bacteria [6]. Nevertheless, the cost of production is still a major obstacle for commercial purposes. In order to further reduce costs, the utilization of photosynthetic organisms as the production hosts are being explored. This had resulted in the development of transgenic plants carrying the PHA biosynthetic genes of bacteria [7]. Like higher plants, cyanobacteria are also oxygen-evolving photosynthetic bacteria [8] with the added advantage that some of them naturally possess the key enzyme in PHA biosynthesis, i.e. PHA synthase [9]. Recently, the complete PHA biosynthesis pathway in Synechocystis sp. PCC6803 was successfully determined [10] following hints provided by the available genome sequence [11]. Due to their minimal nutrient requirements, cyanobacteria are being investigated as potential production hosts for PHAs [12], [13], [14]. To date, only poly(3-hydroxybutyrate) [P(3HB)] homopolymer has been identified and relatively well characterized in most cyanobacteria. The contents of PHAs synthesized are in general low compared to the amounts synthesized by heterotrophic bacteria. Under nitrogen-limited conditions and in the presence of acetate, P(3HB) content can reach a maximum of about 10 wt% of the cellular dry weight [15], [16]. As in bacteria, the accumulated PHAs are stored in the form of granules and are slightly electron-dense [17]. Recently, it was noted that several strains from the genus Cyanothece seem to synthesize significant amounts of P(3HB) constitutively [18]. As for the incorporation of other hydroxyalkanoate units there seem to be some evidence for the incorporation of 3-hydroxyvalerate (3HV) units when cultures of Anabaena cylindrica were supplied with appropriate precursor substrates [19]. 3HV units have also been detected in photosynthetic microbial mats where the mole percent ratio of repeating units were generally 1:1 (3HB:3HV) [20]. This indicates that at least some cyanobacteria have the biochemical machinery to produce and incorporate hydroxyalkanoate monomers other than 3HB. In this study, we have investigated various aspects of PHA biosynthesis by the model cyanobacterium Synechocystis sp. PCC6803. PHA biosynthetic genes of Ralstonia eutropha were introduced and their effect on PHA biosynthesis by Synechocystis sp. PCC6803 was studied. To detect the presence of small amounts of PHA (<5 wt%) from as little as 0.5 mg freeze-dried cells, Fourier transform infrared (FT-IR) spectroscopy was found to be applicable. Freeze-fracture electron microscopy was used to identify and locate precisely PHA granules in vivo. Various carbon sources were tested for their abilities to promote PHA biosynthesis and to incorporate hydroxyalkanoate monomers other than 3HB. Access through your organization

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Access through your organization Section snippets Strains and growth conditions Synechocystis sp. PCC6803 (ATCC 27184) [21] was cultured in 250 ml tripple-baffled Erlenmeyer flasks containing 50 ml of BG11 medium at 30 °C under continuous illumination with cool white fluorescent light (∼150 μEinstein m-2 s-1). The cultures were shaken at 100 rpm on a rotary shaker placed inside an incubator saturated with 1% CO2. Growth was monitored by measuring the OD730 of the cultures that were grown in triplicates. For nitrogen-limited conditions BG11 medium without sodium nitrate was Phenotype of the recombinant Synechocystis sp. PCC6803 Fig. 1 shows the growth of wild-type and recombinant Synechocystis strains used in this study. As can be seen, both strains showed good growth characteristics under the conditions used in this study (∼150 μEinstein m-2 s-1, 1% CO2 saturation). The PHA synthase activity was determined at the end of the eighth day when the cultures were still in the growth phase. In the wild-type, the specific activity of PHA synthase was 0.2 U mg-1 while that of the recombinant was 0.5 U mg-1. However, no PHA Discussion In order to rapidly identify the presence of PHAs from small amounts of cells (∼0.5 mg), especially from streaks on agar plates, we found that Nile blue A staining together with FT-IR analysis to be very efficient. PHA contents as low as 3–5 wt% of the dry cell weight can be detected by these methods. These techniques are expected to be useful in screening large numbers of recombinant cyanobacteria or even cyanobacteria from nature for enhanced PHA producing ability. Recently it has been shown Conclusions Small amounts of PHAs in Synechocystis sp. PCC6803 can be effectively identified by using Nile blue A staining and FT-IR analysis of the dry cells. In addition, the precise location of the PHA granules can be determined by using freeze-fracture electron microscopy. Based on these analyses it was found that the number and size of PHA granules accumulated by Synechocystis sp. PCC6803 are comparable to those accumulated by many natural PHA-producing bacteria. Increased PHA synthase activity Acknowledgments This work was supported by the grant for Ecomolecular Science Research at RIKEN Institute. Part of this work was presented at the 10th International Symposium on Phototrophic Prokaryotes, Barcelona. References (33) K. Sudesh et al. Prog. Polym. Sci. (2000) A. Steinbüchel et al. Trends. Biotechnol. (1998) Y. Asada et al. Int. J. Biol. Macromol. (1999) N.G. Carr Biochim. Biophys. Acta (1966) L.J. Stal FEMS Microbiol. Rev. (1992) L. Lama et al. Phytochemistry (1996) J. Davison et al. Gene (1987) K. Sudesh et al. Int. J. Biol. Macromol. (1999) A.J. Anderson et al. Microbiol. Rev. (1990) Y. Doi Microbial polyesters (1990) View more references Cited by (91) Microalgae as source of polyhydroxyalkanoates (PHAs) — A review 2019, International Journal of Biological Macromolecules Show abstract Engineering cyanobacteria to generate high-value products 2011, Trends in Biotechnology Show abstract Optimization of cultural and nutritional conditions for accumulation of poly-β-hydroxybutyrate in Synechocystis sp. PCC 6803 2006, Bioresource Technology Show abstract Process design for microbial plastic factories: Metabolic engineering of polyhydroxyalkanoates 2003, Current Opinion in Biotechnology Show abstract Mini-review: Biosynthesis of poly(hydroxyalkanoates) 2009, Polymer Reviews Applications of cyanobacteria in biotechnology 2009, Journal of Applied Microbiology View all citing articles on Scopus View full text Copyright © 2002 Elsevier Science B.V. All rights reserved.