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# Prospect of Synechocystis sp. PCC 6803 for synthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) - ScienceDirect

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Algal Research
Volume 50, September 2020, 101994
Prospect of Synechocystis sp. PCC 6803 for synthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate)
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Sadaf Tanweer
, 
Bhabatarini Panda
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https://doi.org/10.1016/j.algal.2020.101994
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Highlights
•
First report for poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer synthesis in cyanobacteria without genetic modification.
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1H-NMR analysis confirmed the synthesis of copolymer in the test organism.
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Increasing concentration of γ-butyrolactone decreased the polymer content.
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Material properties found comparable with polymers from other sources.
Abstract
The rapidly growing use of petroleum-based plastics is contributing to severe environmental pollution, thereby putting the environment to a hard test. Research for alternative plastics is essential to substitute conventional plastics with a certain grade of biodegradability. One such potential material to replace petrochemical-based plastics is microbially originated polyhydroxyalkanoates. Among them, poly-β-hydroxybutyrate (PHB) is the most common and well-characterized member. However, studies demonstrate that the properties of PHB such as brittleness, low extension-to-break, and lack of flexibility limit its possible application whereas; copolymers have the properties which can overcome the limitations of PHB. In this study, Synechocystis sp. PCC 6803, a unicellular, non-diazotrophic cyanobacterium has been found to accumulate poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer under nitrogen and phosphorus-deficient conditions either using γ-butyrolactone alone or along with acetate as carbon source. The maximum copolymer accumulation i.e. 37.64% dry cell weight was recorded when the cultures were supplemented with 0.4% acetate and 0.01% γ-butyrolactone. The purity and mole fraction of the polymer was confirmed by proton nuclear magnetic resonance and fourier transform infrared spectroscopy. The material properties were assessed and found comparable with other polymers from bacteria and cyanobacteria. Thus, the test organism has the potential to be utilized for large scale biopolymer production and applications in various fields.
Introduction
Sustainable bioplastic production is one of the effective methods to overcome the growing problem of plastic pollution. Polyhydroxyalkanoates (PHA) are a class of bioplastics, synthesized by many prokaryotes as carbon and energy reserve materials under the conditions of unfavorable growth. Production of these biodegradable plastics by heterotrophic microorganisms has been restricted due to its high carbon and energy demands. To overcome this difficulty, autotrophic organisms are being explored as an alternative source for the production of biodegradable plastics. Attempts have also been made for the accumulation of PHA in higher plants by introducing PHA synthase genes from bacteria but its yield is found to be commercially and economically less sustainable [1]. Photosynthetic microorganisms such as cyanobacteria and algae are also tried as alternative sources for PHA production [2].
Out of the various classes of microbially synthesized PHAs, poly-β-hydroxybutyrate (PHB) is the most extensively studied one. The biodegradability and biocompatibility properties of PHB distinguish itself from the non-biodegradable plastics of the present day. Even though the environment-friendly property of PHB has created interest among the researchers and industries, the constrained physico-mechanical properties such as melting temperature and brittleness of the polymer have made it less suitable for wider industrial applications. Therefore, attempts have been made to improve the mechanical properties of PHB by synthesizing various copolymers, and thus have made it more suitable for various specific industrial applications. Depending upon the microbial strains and carbon precursors used for the synthesis of PHA, the types and compositions of polymers, thus synthesized, vary with the changing material properties [3]. PHB is the most commonly occurring short-chain length polymer accumulated by many bacteria and cyanobacteria under photoautotrophic conditions or with supplementation of an even number of carbon sources. Besides PHB, more than 130 different types of PHA have been reported so far in bacteria [4]. Subsequently, monomers such as hydroxybutyrate (HB), hydroxyvalerate (HV), hydroxyhexanoate (HHx), etc. polymerize with 3HB monomer to form various copolymers. Like many bacterial strains, few cyanobacteria can also synthesize such copolymers under the supplementation of appropriate carbon precursors. The first copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate), P (3HB-co-3HV) accumulation (2% w/dcw) was reported in Anabaena cylindrica 10C under the propionate-supplemented condition [5]. Thereafter, Synechocystis sp. PCC 6803 (45%), Aulosira fertilissima CCC 444 (77%) and Nostoc muscurom (69%) have also been reported to accumulate a higher amount of P (3HB-co-3HV) copolymer under propionate and valerate supplemented conditions [[6], [7], [8]]. Other monomer compositions, except 3HB and 3HV, are rarely reported from cyanobacteria.
Among various types of PHAs synthesized yet, the PHA containing 4-hydroxybutyrate (4HB) monomers are promising candidates for therapeutic applications [9]. Poly (3-hydroxybutyrate-co-4-hydroxybutyrate) [shortly known as P(3HB-co-4HB)] copolymer is one such short-chain length PHAs (scl-PHAs) that have the potential to be used as a biomaterial, as both 3-hydroxybutyrate and 4-hydroxybutyrate are common metabolites present in human body [9]. This copolymer has also improved material properties than the other scl-PHAs that can be tailor-made for wider medical applications. Structurally, the 4HB monomer differs from 3HB, the former lacking an alkyl side group attached to the polymer backbone. Further, an extended distance between the ester groups in the polymer backbone differentiates 4HB from 3HB [10]. Due to this difference in structure between P(3HB) and P(4HB), the physico-mechanical properties of the polymers are considerably different, the former being highly brittle and crystalline whereas, the latter being highly ductile and flexible. Therefore, incorporation of 4HB in the homopolymer of 3HB to form a copolymer of P(3HB-co-4HB) is the desired process to modify the physico-mechanical properties of the polymer. The synthesis of P(3HB-co-4HB) copolymer is carried out in bacteria when the addition of carbon sources such as γ-butyrolactone, butyric acid, 1,4-butanediol, 1,6-hexanediol or 1,8-octanediol are present in the PHA production media, which incorporates 4HB monomer to 3HB. In the case of a few bacteria, the 4HB monomer can also be catabolized to 3HB thus leading to the production of P(3HB-co-4HB) copolymer [11]. The key enzyme involved in the synthesis of this copolymer is PHA synthase.
To date, few strains of bacteria i.e. Alcaligenes latus [12], Comamonas testosteronii [13], Hydrogenphaga pseudoflava [14], Comamonas acidovorans [15], Ralstonia eutropha [16], Cupriavidus sp. USMAA1020 [17], Cupriavidus necator DSM 545 [18] and Burkholderia sacchari [19] have been reported to accumulate P(3HB-co-4HB) naturally by using various carbon sources. However, non-engineered cyanobacteria are not reported so far to be accumulating P(3HB-co-4HB) copolymer by using any carbon sources. A metabolically engineered Synechococcus sp. PCC 7002 has been reported to produce the P(3HB-co-4HB) copolymer, amounting up to 4.5% w/dcw under photoautotrophic condition, by introducing a biosynthetic pathway for the synthesis of 4HB monomers [20].
Attempts were made in this study to synthesize P(3HB-co-4HB) copolymer by a non-engineered cyanobacterium i.e., Synechocystis sp. PCC 6803, and further characterization of the copolymers thus synthesized was carried out to compare the material properties with the commercially available biopolymers. The test cyanobacterium Synechocystis sp. PCC 6803 was reported to accumulate a homopolymer of PHB under photoautotrophic conditions and the polymer accumulation was boosted up to 32% w/dcw by acetate supplementation under chemoheterotrophy and nutrient deficiency (N-deficiency, P-deficiency and oxygen limitations) [21]. Therefore, in this study, experiments have been designed to study their combined effect on polymer accumulation in the test cyanobacterium by modifying the medium in a two-stage cultivation process. Further, γ-butyrolactone was added as a sole carbon source or in combination with acetate to study its effect on copolymer production by the test cyanobacterium.
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Section snippets
Test organism and experimental conditions
Axenic cultures of Synechocystis sp. PCC 6803 (source: Pasteur Culture Collection of Cyanobacteria, Pasteur Institute, Paris, France) were grown in 250 ml Erlenmeyer flasks containing 100 ml of BG-11 medium (Himedia) [22] at 27 ± 2 °C under illumination with 18 W fluorescent lamps at the light intensity of 75 μmol photon m−2 s−1 PAR and photoperiod of 14:10 h. The pH of the media was maintained at 8.5 with Tris-HCl (4 mM) buffer. Cultures were allowed to grow up to 14 days (log phase) under
Polymer accumulation under microscopic observation
Polymer accumulation was obtained in a two-step process. In the first step, a complete BG-11 medium was used for increasing the biomass of cyanobacteria. Then, the biomass was harvested and transferred to the modified media (MM) supplemented with various carbon sources alone and in combination for polymer yield. The Nile red staining of the cells from the second phase depicted the PHA inclusions in it (Fig. 1). PHA granules were confirmed as gold-yellow color inclusions under the fluorescent
Discussion
When cyanobacterial cultures from the second phase were observed under a fluorescent microscope after staining with Nile red, they exhibited gold-yellow inclusions of PHA granules (Fig. 1) as reported earlier by Gopi [26]. Four elements mainly C, H, O, and N play a vital role in the normal functioning of cells. Microorganisms obtain H and O from water, whereas C and N are made available from the surroundings depending on their mode of nutrition and media composition. The modified media used for 
Conclusion
Synechocystis sp. PCC 6803 is capable of synthesizing P(3HB-co-4HB) copolymer by using γ-butyrolactone as a carbon source. The advantage of using this substrate is its low cost and easy availability as compared to other carbon sources such as 1, 4 butanediol and 4-hydroxybutyric acid. To the knowledge of the authors, this is the first-ever report of non-engineered cyanobacteria capable of accumulating P(3HB-co-4HB) copolymer by using a carbon source. A maximum copolymer content (37.64%) was
Statement of informed consent, human/animal rights
No conflicts, informed consent, human or animal rights applicable.
Author contributions
B. Panda and S. Tanweer designed the experiments. S. Tanweer performed the experiments. Both the authors analyzed the results and prepared the manuscript.
Declaration of author agreement
All the authors listed have approved the manuscript and agreed to authorship and submission of the manuscript to Algal Research.
Declaration of competing interest
The authors declare that there is no conflict of interest.
Acknowledgement
Miss Sadaf Tanweer is thankful to DST, Govt. of India for providing financial assistance through INSPIRE fellowship (IF170733). Financial support provided by Seed Grant (Dev.I/536/3488/2017), Utkal University and Department of S & T (3365/ST (Bio.)-02/2017), Odisha, India is thankfully acknowledged. Instrumentation facilities provided by DST-FIST to Utkal University, OUAT, CSIR-IMMT and IIT-Kharagpur are also highly acknowledged. Thanks are also due to Dr. D. Roy, Faculty in English, CAEPHT,
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