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From Science & Research

From Science & Research PHA from sunlight New route for bioplastics production in cyanobacteria via photosynthesis By: Minami Matsui RIKEN Center for Sustainable Resource Science Yokohama, Japan Photosynthesis PhaA Cupriavidus necator PhaB Cupriavidus necator PhaC Chromobacterium sp. Cyano bacteria Acetyl-CoA Acetoacetyl-CoA (R)-3-Hydroxybutyryl-CoA Polyhydroxyalkanoate (PHA) Heterotrophic bacteria Carbon source (sugars) Malonyl-CoA NphT7 Streptomyces sp. Figure 1: Metabolic pathways for PHA production. For the production of PHA in cyanobacteria, the genes, phaA, phaB and phaC were introduced. The condensation reaction of two acetyl-CoA compounds to form acetoacetyl-CoA by PhaA was hypothesized to be thermodynamically unfavorable in cyanobacteria under photosynthetic conditions. Therefore, PhaA was replaced by NphT7 that catalyzes the irreversible condensation of acetyl-CoA and malonyl-CoA to give acetoacetyl-CoA. In the past few decades, among all of the bio-based polymers, polyhydroxyalkanoates (PHA) have gained significantly in interest since they were shown to be completely biodegradable in appropriate environments. Another attractive feature of PHAs, apart from their biodegradability, is that they can be synthesized from renewable resources, allowing a sustainable production on a large scale. PHA is a type of storage inclusion that is naturally synthesized by numerous micro-organisms under unfavorable growth conditions. However, the commercialization of PHA has been ongoing, but with limited success due to its high production cost. The use of heterotrophic bacteria for PHA production calls for culture requirements and the supply of carbon sources that contribute significantly to the cost of production. Cyanobacteria, endowed with a photosynthetic system to fix carbon dioxide in a reduced form, are an ideal biosynthetic machine for sustainable production of various industrially important products, such as PHA. The conversion of atmospheric carbon dioxide into a biopolymer by cyanobacteria eliminates the use of costly external carbon sources and helps to achieve a carbon neutral bioplastic production process. The current bottleneck for photosynthetic PHA production using plant and other photosynthetic micro-organisms is to achieve production at an economically viable level. Minami Matsui, Nyok Sean Lau and colleagues from the RIKEN Synthetic Genomics Research Team in collaboration with Sudesh Kumar at Universiti Sains Malaysia have genetically engineered a cyanobacterium to address the challenges in terms of cost and productivity. The genetically modified variant of the cyanobacterium, Synechocystis sp. strain 6803, synthesized an encouraging level of PHA as high as 14% of the dried cellular biomass. So far, this is the highest level achieved in completely photoautotrophic PHA production without the provision of any carbon source. The addition of a carbon source in a small amount (0.4% acetate) had improved PHA production to 41% of the dry weight. Although cyanobacteria have relatively simple nutrient requirements, the provision of exogenous carbon source was found to boost PHA production approximately three-fold. Nonetheless, the amount of carbon source provided was very much lower compared to that required by heterotrophic bacteria to achieve the same PHA production level. In this modified strain, the carbon flux to PHA biosynthetic pathway was enhanced by the introduction of acetoacetyl-CoA synthase from Streptomyces sp. CL190, an enzyme that catalyzes the irreversible condensation of acetyl-CoA and malonyl-CoA to give acetoacetyl-CoA. In addition, a highly active PHA polymerizing enzyme, PHA synthase from Chromobacterium sp., was also introduced to improve the strain’s production efficiency. To better understand the mechanism that leads to the enhanced photoautotrophic PHA production, gene expression in the PHA overproducer was compared with its unmodified counterpart. It is surprising to find that the activities of enzymes directly involved in PHA synthesis are not the critical factors responsible for the overproduction of PHA in 20 bioplastics MAGAZINE [03/14] Vol. 9

From Science & Research Figure 2: Microscope image shows the likely accumulation of PHA in genetically modified cyanobacteria. Upper left: Image of cells after staining with nile-red pigment that shows lipid and polymer inclusions. Upper right: Image of cyanobacterial cells. Bottom: Merged image of upper two images. It shows that PHA is accumulating in cyanobacterias cells. the modified strain. On the other hand, genes encoding proteins involved in several aspects of photosynthetic activities were significantly upregulated in the PHA overproducer compared to the control strain. Results from this study suggest that cyanobacterial cells may utilize enhanced photosynthesis capability to drive the product formation. During PHA formation, the pool of carbon in cyanobacterial cells was constantly being used for the synthesis. In order to cope with the higher production demand, the cyanobacterial cells may increase the carbon fixing capacity to replenish the pool of carbon that was lost to PHA synthesis. At the same time, the flow of newly fixed carbon into cellular processes other than PHA (e.g. amino acids biosynthesis) was limited. Based on the findings of this study, future work can be done to engineer cyanobacteria for the production of various chemicals or biofuels and a similar approach can likely be extended to higher plants. It is hope that the development of a new route for the production of biopolymer only by solar energy will provide a platform for the shift of production process from petroleum-based to bio-based. Reference Lau, N.S., Foong, C.P., Kurihara, Y., Sudesh, K. & Matsui, M. RNA-Seq analysis provides insights for understanding photoautotrophic polyhydroxyalkanoate production in recombinant Synechocystis sp. PLoS One. 2014 Jan 22; 9(1): e86368. doi: 10.1371/journal.pone.0086368 (2014). in Raw Materials, Machinery & Products Free of Charge from the Industrial Sector and the Plastics Markets for Plastics. for Plastics & Additives, Machinery & Equipment, Subcontractors and Services. for Specialists and Executive Staff in the Plastics Industry bioplastics MAGAZINE [03/14] Vol. 9 21

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