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# Enhancement of poly-3-hydroxybutyrate production in Synechocystis sp. PCC 6803 by overexpression of its native biosynthetic genes - ScienceDirect

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Bioresource Technology
Volume 214, August 2016, Pages 761-768
Enhancement of poly-3-hydroxybutyrate production in Synechocystis sp. PCC 6803 by overexpression of its native biosynthetic genes
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Wanthanee Khetkorn a b
, Aran Incharoensakdi a, Peter Lindblad c, 
Saowarath Jantaro a
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https://doi.org/10.1016/j.biortech.2016.05.014
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Highlights
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Native biosynthetic genes for PHB production overexpressed in Synechocystis.
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Overexpression of phaA and phaB resulted in the highest level of PHB.
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Metabolic engineering leads to increased production of the valuable polymer PHB.
Abstract
Synechocystis sp. PCC 6803 strains overexpressing pha genes were constructed and characterized for poly-3-hydroxybutyrate (PHB) production. These pha overexpressing strains showed slightly reduced growth rates. Under N-deprived condition, the strains overexpressing (OE) phaAB, phaEC and phaABEC showed significantly higher PHB contents than the wild type. The maximum PHB content, a 2.6-fold increase producing 26% PHB (dcw), was observed in OE phaAB cells grown for 9 days in N-deprived medium. Under this condition, these OE phaAB cells increased PHB production to 35% PHB (dcw) upon addition of 0.4% (w/v) acetate. Higher PHB granules in OE phaAB cells were clearly visualized by both Nile red staining and TEM imaging. All OE strains under N-deficient condition had increased glgX transcript levels. Overall results demonstrate an enhanced PHB production in Synechocystis cells overexpressing pha genes, particularly phaA and phaB, when grown in N-deprived medium containing 0.4% (w/v) acetate.
Introduction
In order to minimize the increasing problem of non-degradable plastic wastes, alternative sources of biodegradable plastics have received much attention, especially polyhydroxyalkanoates (PHAs). PHAs are biological origin-biodegradable polymers which gained wider applications in commercial market. Poly-β-hydroxybutyrate (PHB), a most common type of PHA, is found mainly as energy and carbon reserves inside unicellular cells that face stress conditions (Balaji et al., 2013). There are several naturally occurring microorganisms capable of PHB production under modified nutrient conditions. The fermentative or chemoautotrophic bacteria, such as Bacillus cereus SPV and Alcaligenes eutrophus can produce 38% and 85% PHB of dry cell weight (dcw), respectively (Valappil et al., 2007, Anderson and Dawes, 1990). The microalgae including cyanobacteria, such as Spirulina, Synechocystis sp. PCC 6803 and Nostoc muscorum can produce 73%, 28.8% and 60% PHB of dcw, respectively (Vincenzini et al., 1990, Panda et al., 2006, Abed et al., 2009). However, the wide uses of the PHB produced by bacteria for industrial applications are limited due to high cost of bacterial cultivation when compared to mainly employed petroleum derived plastics (Balaji et al., 2013). Cyanobacteria can be considered as sustainable and alternative sources of PHB production due to their photoautotrophic nature, i.e., able to fix CO2 with minimal nutrient consumption.
The metabolic pathways representing the conversion of acetyl-CoA to PHB biosynthesis and related pathways under light condition in cyanobacteria are shown in Fig. S1 (Supplementary material). The acetyl coenzyme A (acetyl-CoA), referred to as a hub of metabolism, is usually obtained from various routes including glycolysis pathway by oxidative conversion of pyruvate catalyzed by pyruvate dehydrogenase (EC 1.2.4.1), and glycogenolysis pathway by degrading glycogen into glucose-6-phosphate (G6P) which then enters glycolysis. The pathway from acetyl-CoA to PHB involved three catalytic steps. The first step is condensation of two molecules of acetyl-CoA into acetoacetyl-CoA by PHA-specific beta-ketothiolase (PhaA), followed by the reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA by acetoacetyl-CoA reductase (PhaB), and finally the (R)-3-hydroxybutyryl-CoA condensation into PHB by PHA synthase (PhaEC). To date, enhancement strategy of PHB production from biosources encompasses not only nutrient modification but also genetically engineered changes that enable cells to produce PHB at higher levels than its natural capacity. Nitrogen and phosphorus starvation could stimulate PHB accumulation by effectively diverting energy and carbon reserves towards PHB synthesis and its content may be increased further by supplementing a precursor acetate into the growth medium leading to an increased flux of acetyl-CoA needed for PHB synthesis (Panda et al., 2006, Abed et al., 2009, Wu et al., 2001, Asada et al., 1999). Synechocystis sp. PCC 6803 contains two genes, phaC and phaE, of PHA synthase, which are located in the same operon (Hein et al., 1998). The other two genes in PHB biosynthesis pathway, phaA and phaB, were identified and the sequence analysis showed that both genes are also located in the same orientation in the chromosome (Takahashi et al., 1998). Previously, several heterologous expression studies have been conducted to increase PHB production by cyanobacteria (Sudesh et al., 2002, Takahashi et al., 1998, Wang et al., 2013, Osanai et al., 2013, Hondo et al., 2015). However, the engineered cyanobacteria capable of high PHB production have not been reported.
In this study, we have overexpressed the native Synechocystis sp. PCC 6803 PHB biosynthetic genes and optimized the conditions for PHB production with respect to nutrient modification. The co-overexpression of phaA and phaB resulted in the highest PHB level in the overexpressing strain grown in N-deprived medium supplemented with acetate.
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Section snippets
Strains and normal growth conditions
Wild type Synechocystis sp. PCC 6803 was grown in BG11 medium containing 17.6 mM NaNO3 as a source of nitrogen and buffered with 20 mM HEPES-NaOH (pH = 7.5) as described previously (Baebprasert et al., 2010). All overexpressing (OE) strains (phaAB, phaEC and phaABEC) were grown in BG11 medium containing 30 μg/mL chloramphenicol. The cultures with the initial cell density of an OD730 ∼ 0.1 were incubated aerobically under continuous illumination of 40 μE/m2/s on a rotatory shaker at 160 rpm and 30 °C. The 
Overexpression of native PHB biosynthesis genes in Synechocystis sp. PCC 6803 cells
In the present study, the native phaAB, phaEC and phaABEC operons, each cloned separately in a plasmid construct where the respective genes were linked with a chloramphenicol resistance cassette, were placed between the flanking regions of the psbA2 gene of Synechocystis sp. PCC 6803. Each obtained plasmid containing phaAB, phaEC or phaABEC was used for double homologous recombination into the Synechocystis chromosome, in which the psbA2 gene was replaced by phaAB, phaEC and phaABEC constructs, 
Conclusion
All pha genes were overexpressed in Synechocystis sp. PCC 6803 using the psbA2 promoter to drive the gene expression. The overexpressing strains had the highest ability to produce PHB. The strain overexpressing phaAB gene highly accumulated PHB up to 35% of dcw after growth for 9 days in nitrogen-deprived medium containing 0.4% (w/v) acetate. This study demonstrates a higher PHB productivity than those previously reported in other engineered cyanobacterial strains (Table 3). Therefore, a
Acknowledgements
This work was supported by the research grant (fiscal year 2012) provided by the National Research Council of Thailand – Thailand, the Knut and Alice Wallenberg Foundation – Sweden, and the Swedish Energy Agency – Sweden. W. Khetkorn wishes to thank the Graduate School of Chulalongkorn University for post-doctoral fellowship. A. Incharoensakdi also thanks Chulalongkorn University for the grant on Frontier Research Energy Cluster, and Thailand Research Fund (IRG 5780008).
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Citation Excerpt :

Thus, genetic engineering or mutation techniques have been applied in cyanobacteria to improve PHAs synthesis that will further help to enhance the economic feasibility. Synechocystis sp. strain was genetically modified with overexpressing pha genes that resulted into 2.6 folds increase in PHB production under N-limited medium (Khetkorn et al., 2016). Carpine et al. reported highest PHB productivity of 7.3 mg/L·d after optimal genetic modifications (Carpine et al., 2017).

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