Marine cyanobacteria as potential alternative source for GABA production

Journal Pre-proof Marine cyanobacteria as potential alternative source for GABA production

Katie Shiels, Patrick Murray, Sushanta Kumar Saha PII:

S2589-014X(19)30232-4

DOI:

https://doi.org/10.1016/j.biteb.2019.100342

Reference:

BITEB 100342

To appear in:

Bioresource Technology Reports

Received date:

28 August 2019

Revised date:

23 October 2019

Accepted date:

26 October 2019

Please cite this article as: K. Shiels, P. Murray and S.K. Saha, Marine cyanobacteria as potential alternative source for GABA production, Bioresource Technology Reports(2019), https://doi.org/10.1016/j.biteb.2019.100342

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© 2019 Published by Elsevier.

Journal Pre-proof

Marine cyanobacteria as potential alternative source for GABA production Katie Shiels, Patrick Murray and Sushanta Kumar Saha* Department of Applied Science and Shannon Applied Biotechnology Centre, Limerick Institute of Technology, Moylish Park, Limerick V94 E8YF, Ireland (ROI)

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*Correspondence: [email protected]; Tel.: +353 61 293536

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Journal Pre-proof Abstract GABA (-aminobutyric acid) is a non-proteinogenic amino acid that plays a significant role in various health functions and has been identified in cyanobacteria. This investigation screened seventeen fast-growing Irish marine cyanobacteria as potential sustainable producers of GABA. Twelve of the seventeen cyanobacteria tested positive for GAD (glutamate decarboxylase) activity in vitro and were tested for their GABA content using a spectrophotometric assay. Five of the twelve cyanobacterial extracts (Calothrix contarenii Chlorogloea

microcystoides

SABC022904,

Phormidium

africanum

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SABC022701,

SABC010301, P. angustissimum SABC022612 and P. laminosum SABC022613) showed

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the characteristic GABA peak with the amounts of GABA produced ranging from 0.99x104-

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72.84x104 nmol g-1 dry-weight biomass. These five cyanobacterial extracts were resolved by

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thin layer chromatography (TLC) to identify the GABA band, and the GABA bands from unstained TLC plate (same Rf values) were excised for verification of GABA

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spectrophotometrically. This is the first report on five potential Irish marine cyanobacteria as

Keywords:

Marine

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GABA producers.

cyanobacteria;

GAD

activity,

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spectrophotometric estimation.

Page 2 of 22

GABA

(-aminobutyric

acid),

Journal Pre-proof 1. Introduction: -Aminobutyric acid (GABA) is a non-proteinogenic, four carbon amino acid found both in prokaryotic and eukaryotic organisms including photosynthetic cyanobacteria. GABA performs essential functions in living organisms as an inhibitory neurotransmitter, hypotension inducer, diuretic and possess tranquilizing effects (Boonburapong et al., 2016; Jakobs et al., 1993). GABA administration was found to be positively associated with stimulation of immune cells (Abdou et al., 2006), treatments for sleeplessness, depression,

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autonomic disorders (Okada et al., 2000), and alcohol-related chronic symptoms (Oh et al., 2003). GABA was also reported to indirectly help regulate diabetic conditions in promoting

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insulin secretion from the pancreas.(Adeghate and Ponery, 2002; Hagiwara et al., 2004).

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Overall, due to the cited value-added benefits on human health, recently the use of GABA

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has received attention as a potential functional food ingredient. Currently, several GABAenriched food products are available in the market such as anaerobically treated tea leaves,

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water-soaked rice germ, red mold rice, tempeh-like fermented soybeans, yogurt, cheese and

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other fermented milk products (Thwe et al., 2011). In plants, GABA is an intermediate product of amino acid metabolism and is accumulated in

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response to different environmental stress conditions. GABA in plants help in the regulation of pH, carbon fluxes into the TCA cycle, nitrogen metabolism and protects from harmful effects of oxidative stress. Both in plants and animals, GABA is metabolized in the GABA shunt pathway that bypasses two steps of the tricarboxylic acid (TCA) cycle. The first step of GABA shunt is the irreversible decarboxylation of five carbon α-amino acid glutamate using an enzyme glutamate decarboxylase (GAD, EC 4.1.1.15). The intermediate step of GABA shunt is the reversible conversion of GABA to succinic semialdehyde by the enzyme GABA transaminase (GABA-T; EC 2.6.1.19). The final step of the GABA shunt is the irreversible oxidation of succinic semialdehyde to succinate, catalyzed by succinic semialdehyde dehydrogenase (SSADH; EC 1.2.1.16) (Bouché and Fromm, 2004). The important role of the GABA shunt pathway is to balance intracellular carbon and nitrogen (CN) metabolism,

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Journal Pre-proof which is also found in cyanobacteria. GABA is primarily formed in cyanobacteria from Lglutamate by the irreversible action of GAD enzyme and is called a GAD pathway. GABA accumulation in cyanobacteria was reported due to environmental conditions, such as increased salt concentration and acidic medium in marine Aphanothece halophytica (Boonburapong et al., 2016) and nitrogen status (nitrogen depletion as well as alternative nitrogen sources) in freshwater Synechocystis sp. PCC 6803 (Jantaro and Kanwal, 2017). Cyanobacteria are prokaryotic, photosynthetic organisms with a wide range of morphological

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diversity, comprising unicellular, filamentous and colonial forms. They are found in a wide

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variety of habitats including Arctic, Antarctic and hot springs, and can survive in extreme environmental conditions, such as drought and desiccation, a range of salinities, nitrogen

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starvation, anaerobiosis, heat and cold shock, photooxidation and UV or osmotic stress

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(Pham et al., 2017; Sinha and Häder, 1996; Whitton and Potts, 2002). However,

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photosynthetic organisms including cyanobacteria have several strategies to survive stressful environmental conditions by enhancing the production of various water-soluble and

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lipid-soluble compounds including GABA, and antioxidative enzymes (Jantaro and Kanwal, 2017; Saha et al., 2003; Saha et al., 2013) in the GABA shunt pathway is known to play an

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important role as a signaling molecule to respond to pH variation, osmotic or ionic and cold stress (Jantaro and Kanwal, 2017). Cyanobacteria are the oldest known fossils but have really made their mark only in recent years as a gold-mine of bio-active compounds and they are considered as bio-factories of structurally new and biologically active metabolites (Boonburapong et al., 2016). However, only some freshwater cyanobacteria and a few marine cyanobacteria have so far been studied in relation to GABA. Particularly, to best of our knowledge no Irish marine cyanobacteria were evaluated for the potential production of bioactive compounds such as GABA, which may have applications in health supplements. Therefore, the main objective of the present study was to screen Irish marine cyanobacteria from recently established biobank at Shannon Applied Biotechnology centre and to identify novel sustainable potential producers of GABA. Page 4 of 22

Journal Pre-proof 2. Materials and methods 2.1 Cyanobacteria and their cultivation conditions Seventeen

marine

cyanobacteria

(Calothrix

contarenii

SABC022701,

Chlorogloea

microcystoides SABC011701, C. microcystoides SABC022904, Leptolyngbya africana SABC011801, L. ectocarpi SABC012402, L. fragilis SABC012503, L. fragilis SABC031801, Phormidium africanum SABC010301, P. africanum SABC022001, P. angustissimum SABC020701, P. angustissimum SABC022612, P. angustissimum SABC022901, P. angustissimum SABC030403, P. laminosum SABC022613, Phormidium sp. SABC022903,

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Phormidium sp. SABC031703 and Spirulina subsalsa SABC051501) were obtained for this

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study from the biobank of Shannon Applied Biotechnology Centre, Limerick Institute of

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Technology. These cyanobacteria were grown in 250 mL Erlenmeyer flasks in triplicate, containing 100 mL of ASN-III medium (Rippka et al., 1979) by inoculating 2 mL of actively

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grown cultures. These flasks were incubated at 20 °C under the photosynthetically active

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radiation (PAR) illumination of 85 μmol photons m−2 s−1 for 16/8 h light/dark cycle. After 14 days of growth, biomass was harvested by centrifuging at 13,000 rpm at 4 °C for 5 min and

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were stored at -20 °C until extracted for spectrophotometric and TLC analysis for GABA.

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However, for the GAD activity assay, 2 mL of the actively grown biomass (7-10 days old) were centrifuged at 3,000 rpm for 6 min at room temperature and used for the assay. Actively grown biomass used as inoculum for biomass production or for the GAD assay was incubated in the above-mentioned growth conditions. 2.2 GAD activity assay All cultures were tested for GAD activity by a rapid colorimetric assay modified from (Olier et al., 2004). For this in vitro assay, two pH indicator dyes were used such as bromophenol blue (BPB) and bromocresol purple (BCP) with an absorption peak at 427 nm and 425 nm respectively. The development of purple or blue color was considered as a positive result for GAD activity. Each culture was tested in duplicate for each indicator. Briefly, 2 mL of actively growing cyanobacterial cultures were pelleted by low-speed centrifugation at ambient

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Journal Pre-proof temperature and the resulting pellets were washed twice with deionized sterile water. The washed cells were then resuspended in 500 μL of test solution [1 g of L-glutamic acid, 300 µL of Triton X-100, 90 g of NaCl, and 0.05 g of indicator (either BCP or BPB) dissolved in 1 litre of deionized sterile water]. Each culture was tested in duplicate for each indicator and incubated at room temperature (~20 °C) for 4 hours. The change in color of the test solution to purple or blue was considered as positive for GAD activity. The test solution after reaction with active cyanobacterial biomass was centrifuged at 13,000 rpm for 2 min, and 100 μL of

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the clear supernatants were used for recording the absorption spectra in a 96-well plate

the appearance of a new peak at around 590 nm.

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2.3 GABA extraction

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reader, from 300-700 nm at 1 nm intervals. GAD activity positive absorption spectra showed

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GABA from selected cyanobacterial biomass was extracted by adopting a modified

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procedure of Kanwal and Incharoensakdi (Kanwal and Incharoensakdi, 2016) for the determination of intracellular GABA content. Briefly, 500 mg of fresh weight biomass of 14

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days old culture was resuspended in 10 mL of 75% aqueous ethanol by a gentle vortex. The

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tubes were then agitated in a rotary drum at 40 rpm and the content was extracted overnight in the dark at room temperature (approx. 20 °C). The tubes were centrifuged at 4600 rpm for 10 minutes at 4 °C and the clear supernatants were transferred to a new 35 mL tube. The ethanol was evaporated-off by placing the tubes in a water bath at 83 °C for 3-4 hours. The remaining content (~2.5 mL) was freeze-dried and stored at -20 °C until used. 2.4 Absorption spectra of assay mixture Presence of GABA in each extract and from the TLC bands was confirmed by absorption spectra analysis of the assay reaction mixture. Briefly, the freeze-dried extracts were reconstituted in 250 μL of Milli-Q water. Then, 50 µL was added to 100 µL of 0.2 M borate buffer pH 9, and 500 µL of 6% phenol was added. The assay solution was vortexed and kept on ice for 5 minutes for cooling. Then, 200 µL of 10-15% NaOCl was added and the assay Page 6 of 22

Journal Pre-proof solution was vortexed vigorously for one minute. The assay solution was then cooled again on ice before boiling the assay solution very carefully in a water bath at 99 °C for 10 minutes. The assay solution was allowed to cool to room temperature and centrifuged at 10,000 rpm for 30 sec. Finally, 100 µL of the clear supernatants were used to record the absorption spectra from 300-700 nm at 1 nm intervals in a 96-well plate reader (BioTek Synergy 4). To estimate the intracellular GABA content of selected cyanobacteria, a standard curve was prepared using a range of standard GABA (0-20 mM, Sigma) concentrations.

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2.5 TLC analysis of GABA

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GABA from the cyanobacterial extracts was resolved through thin layer chromatography

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(TLC), and the GABA band was identified by comparing the Rf of a GABA standard ran simultaneously. Briefly, a freshly prepared solvent mixture of butanol, acetic acid and water

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(5:1:5, v/v) was poured into the TLC chamber. The lid of the chamber was placed tightly with

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silicone grease after placing thick filter papers to the sidewalls so that the inner atmosphere became saturated with the solvent. The TLC plate (20x20 cm size, silica gel 60 F254, Merck)

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was cut to the correct size or used as such. Using an HB pencil a straight line was drawn across the plate approximately one cm from the bottom. Freeze-dried extracts were

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reconstituted in 250 μL of Milli-Q water and filtered using 0.45 µm filter (Millipore UltrafreeMC centrifugal filter unit). 10 µL of filtered extracts along with 10 µL of amino acid GABA standards (20 ppm) were spotted gradually on the line approximately 1.5 cm apart. The spots were dried using hair dryer before placing the plate quickly and carefully inside the saturated chamber. The plate was placed as evenly as possible so that it leaned against the sidewall and the loading line remained just above the solvent upper-layer. The plate kept undisturbed for separation of compounds for about four hrs and removed once the solvent line was approximately five cm from the top. The solvent line was marked with a pencil for the calculation of Rf values, and the plate was allowed to dry. Finally, the dried plate was sprayed with ninhydrin reagent (0.3 g ninhydrin, 100 mL n-butanol and 3 mL acetic acid) and heated using a hairdryer until colored spots of amino acids appeared. The centre of the Page 7 of 22

Journal Pre-proof spots was marked and the unknown amino acids were identified comparing the relative front (Rf) values of standard amino acids. Once the extracts were confirmed to have GABA, a second run was undertaken in duplicate: one plate was developed to identify the GABA band and its Rf position, the second TLC plate containing undeveloped band was excised based on the appropriate Rf position. The excised bands were soaked in 100 µL Milli-Q water for 10 minutes at 50 °C in a water bath to elute the suspected GABA, and the content was centrifuged at 13,000 rpm for 5 minutes at 4

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°C. Then, the clear supernatants were transferred to a new tube. 50 µL of the supernatant

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was tested spectrophotometrically as described previously for the confirmation of GABA

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present in the TLC band. For simple understanding of the various work steps involved in this study is shown by a schematic flow-chart (Fig. 1).

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3. Results and discussion

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Marine cyanobacteria are emerging as biofactories for the production of structurally new and

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biologically active metabolites. With a view to screen Irish marine cyanobacteria as a potential producer of GABA (-aminobutyric acid), the present study was undertaken. GABA

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is a non-proteinogenic amino acid produced by both unicellular and filamentous cyanobacteria, however, its levels were reported highly dependent on cyanobacterial strains and their specific growth conditions. 3.1 GAD activity assay

In the present study, 7-10 days old actively growing cultures were screened for GAD enzyme activity, which converts glutamate to GABA. Twelve out of the seventeen cyanobacteria tested were positive for GAD enzyme activity as seen from the appearance of a characteristic absorption peak at 590 nm (Fig. 2). The peak values were categorized into low (A590 nm 0.1-0.3), moderate (A590 nm 0.3-0.6) and high (A590 nm 0.6-1.0) activities both in BPB and BCP indicators (Table 1). Of the twelve positive cultures for GAD activity, three (L. fragilis SABC031801, P. africanum SABC022001 and P. angustissimum SABC022901) Page 8 of 22

Journal Pre-proof showed low, six (C. microcystoides SABC022904, L. ectocarpi SABC012402, L. fragilis SABC012503, P. africanum SABC010301, P. angustissimum SABC022612 and Phormidium sp. SABC022903) moderate and remaining three (C. contarenii SABC022701, L. africana SABC011801 and P. laminosum SABC022613) showed high GAD activity in BPB indicator. In the BCP indicator, three (L. fragilis SABC031801, P. africanum SABC022001 and P. angustissimum

SABC022901)

showed

low,

three

(L.

fragilis

SABC012503,

P.

angustissimum SABC022612 and P. laminosum SABC022613) moderate and the remaining

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six (C. contarenii SABC022701, C. microcystoides SABC022904, L. africana SABC011801, L. ectocarpi SABC012402, P. africanum SABC010301 and Phormidium sp. SABC022903)

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showed high activity. Of these, only two (C. contarenii SABC022701 and L. africana

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SABC011801) showed high and three (L. fragilis SABC031801, P. africanum SABC022001

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and P. angustissimum SABC022901) showed least GAD activities in both BPB and BCP indicators. Remaining cyanobacteria showed either moderate or high GAD activities in one

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or both indicators (Table 1). It's not surprising that certain cyanobacteria do not have gad

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gene encoding GAD enzyme (Jantaro and Kanwal, 2017) and those cyanobacterial strains still may produce GABA through degradation of other nitrogenous compounds as in plants

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(Evans, 1989) and also in cyanobacterium Synechocystis sp. PCC 6803 (Kanwal et al., 2014a). So, GAD assay results of this study were only indicative for selection of most likely strains for further characterization.

3.2 Spectrophotometric estimation of GABA Twelve cyanobacteria that showed positive GAD activity were considered for their biomass extraction and estimation of GABA spectrophotometrically. This GABA estimation assay resulted in blue color as positive for the presence of GABA in the specific extracts with an absorbance peak at around 630 nm (Fig. 3). Out of the twelve, only five extracts (C. contarenii SABC022701, C. microcystoides SABC022904, P. africanum SABC022001, P. angustissimum SABC022612 and P. laminosum SABC022613) showed detectable GABA

Page 9 of 22

Journal Pre-proof peak with the amounts of GABA ranging 0.99x104-72.84x104 nmol g-1 of dry weight biomass (Table 2). In Aphanothece halophytica accumulation of GABA was increased during the mid-log phase of growth under salt stress and also during acid stress upon glutamate supplementation (Boonburapong et al., 2016). However, in freshwater cyanobacterium Synechocystis sp. PCC 6803 accumulation of GABA was higher after the late-log phase of growth (Kanwal et al., 2014b). GABA is an intermediate amino acid of GABA shunt pathway, which may be

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available transiently, and may eventually be converted to succinate through the step-wise

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actions of GABA transaminase and succinic semialdehyde dehydrogenase enzymes respectively (Knoop et al., 2013; Shelp et al., 2012). Thus identification of a cyanobacterium

Synechocystis sp. PCC 6803 and genetically modified

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growth conditions specifically in

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as GABA producer is a challenging task. GABA accumulation was reportedly induced by

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Synechocystis sp. PCC 6803, by various exogenous carbon and nitrogenous sources and glucose supplementation (Kanwal and Incharoensakdi, 2019) and it was reported that GABA

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is transiently available intracellularly due to downstream activities of the GABA shunt pathway enzymes (Michaeli et al., 2011).

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3.3 Thin layer chromatography

The presence of GABA in cyanobacterial extracts was confirmed by TLC, and the partially purified GABA from TLC plate was further tested spectrophotometrically to validate. The five samples which were found positive for GABA spectrophotometrically were used for partial purification of GABA using thin layer chromatography (TLC). Each cyanobacterial extracts were run along with standard GABA and developed to determine the Rf position of the resolved GABA bands (Rf 0.84). Another, TLC plate was used to resolve each cyanobacterial GABA band that were excised based on the above Rf position. The eluted compounds were tested again spectrophotometrically as confirmation of GABA partial purification (Fig. 4) as well as to increase the confidence on GABA spectrophotometric method used for rapid screening of cyanobacterial extracts. It appears that Irish Page 10 of 22

Journal Pre-proof cyanobacteria possess high GABA content compared to previously reported cyanobacteria (Table 2). Interestingly, of these five potential GABA producers, only C. contarenii SABC022701 showed high GAD activities in both BPB and BCP indicators with highest GABA content, while the least GAD activities in both indicators were recorded for P. africanum SABC022001 with least GABA content. Remaining three cyanobacteria showed either moderate or high GAD activities in one or both indicators with variable GABA content (Tables 1 and 2). It was reported earlier that GAD is the most likely enzyme responsible for

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GABA shunt in cyanobacteria (Xiong et al. 2014). The results of the present study however possibly suggest that GABA production in cyanobacteria may not only be GAD pathway-

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dependent but also due to polyamine degradation in certain cyanobacteria (Jantaro and

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Kanwal, 2017). It was hypothesised that spermidine (polyamine) is converted to -

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aminobutanal, which is subsequently converted to GABA. This hypothesis was supported by the finding that intracellular GABA content of both wild-type Synechocystis and gad

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Synechocystis was increased upon exogenous supply of spermidine (Kanwal et al., 2014a).

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It was also found through bioinformatics analysis that cyanobacteria possess the putative genes encoding enzymes involved in GABA production from spermidine degradation (Knoop

4. Conclusions

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et al., 2013).

Overall, this preliminary screening identified five marine cyanobacteria from Irish habitats for the first time as potential producers of GABA. This study paves the way for further growth and characterization study for enhanced production of GABA in marine cyanobacteria. Competing interests The authors declare that they have no competing interests. Authors’ contributions

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Journal Pre-proof Katie Shiels carried out research work, collected and analysed the data, and also wrote the initial draft of the manuscript. SKS supervised the research work and drafting as well as editing of the manuscript. PM co-supervised the above postgraduate student (KS) research work and assisted in editing the final draft of the manuscript. Acknowledgements This research work was supported by GRO Bursary awards from Limerick Institute of

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Technology to Katie Shiels (KS) and she is thankful for the award.

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Journal Pre-proof References: Abdou, A.M., Higashiguchi, S., Horie, K., Kim, M., Hatta, H., Yokogoshi, H., 2006. Relaxation and immunity enhancement effects of γ-Aminobutyric acid (GABA) administration in humans. BioFactors 26, 201–208. https://doi.org/10.1002/biof.5520260305 Adeghate, E., Ponery, A.S., 2002. GABA in the endocrine pancreas: Cellular localization and function in normal and diabetic rats. Tissue Cell 34, 1–6. https://doi.org/10.1054/tice.2002.0217 Boonburapong, B., Laloknam, S., Incharoensakdi, A., 2016. Accumulation of gammaaminobutyric acid in the halotolerant cyanobacterium Aphanothece halophytica under salt and acid stress. J. Appl. Phycol. 28, 141–148. https://doi.org/10.1007/s10811-0150523-7

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Shelp, B.J., Bozzo, G.G., Trobacher, C.P., Zarei, A., Deyman, K.L., Brikis, C.J., 2012. Hypothesis/review: Contribution of putrescine to 4-aminobutyrate (GABA) production in response to abiotic stress. Plant Sci. 193–194, 130–135. https://doi.org/10.1016/j.plantsci.2012.06.001 Sinha, R.P., Häder, D.P., 1996. Photobiology and ecophysiology of rice field cyanobacteria. Photochem. Photobiol. 64, 887–896. https://doi.org/10.1111/j.17511097.1996.tb01852.x Thwe, S.M., Kobayashi, T., Luan, T., Shirai, T., Onodera, M., Hamada-Sato, N., Imada, C., 2011. Isolation, characterization, and utilization of γ-aminobutyric acid (GABA)producing lactic acid bacteria from Myanmar fishery products fermented with boiled rice. Fish. Sci. 77, 279–288. https://doi.org/10.1007/s12562-011-0328-9 Whitton, B.A., Potts, M., 2002. Introduction to the Cyanobacteria, 1st ed, The Ecology of Cyanobacteria, Their Diversity in Time and Space. Kluwer Academic Publishers, Dordrecht. https://doi.org/10.1007/0-306-46855-7 Xiong, W., Brune, D., Vermaas, W.F.J., 2014. The γ-aminobutyric acid shunt contributes to closing the tricarboxylic acid cycle in Synechocystis sp. PCC 6803. Mol. Microbiol. 93, 786–796. https://doi.org/10.1111/mmi.12699

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Fig. 1. Schematic showing the flow of work undertaken in this study.

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Fig. 2. Absorption spectra of the GAD assay reagent containing either BPB (A) or BCP (B). The change in absorption peak to 590 nm indicated positive GAD activity in twelve cyanobacteria.

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Fig. 3. Absorption spectra of the reaction mixture of GABA spectrophotometric assay showing a characteristic peak for GABA at 630 nm in five cyanobacterial extracts (2-6) and control GABA standard (1).

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Fig. 4. Representative thin layer chromatography (TLC) plates showing identification and partial purification of GABA bands. L1 of unstained plate slice showing marked position used for excision of GABA band. L2 and L3 of stained plate respectively showing standard GABA band and the GABA band of crude extracts of Calothrix contarenii SABC022701.

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Journal Pre-proof Table 1. Level of GAD activity in terms of absorbance values at 590 nm for specific pH indicators after in vitro reaction with fresh cyanobacterial cells. Cyanobacteria

Level of GAD activity in bromophenol blue +++ ++

Level of GAD activity in bromocresol purple +++ +++

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Calothrix contarenii SABC022701 Chlorogloea microcystoides SABC022904 Leptolyngbya africana SABC011801 +++ +++ L. ectocarpi SABC012402 ++ +++ L. fragilis SABC012503 ++ ++ L. fragilis SABC031801 + + Phormidium africanum SABC010301 ++ +++ P. africanum SABC022001 + + P. angustissimum SABC022612 ++ ++ P. angustissimum SABC022901 + + P. laminosum SABC022613 +++ ++ Phormidium sp. SABC022903 ++ +++ Note: +, low GAD activity (A590 nm 0.1-0.3); ++, moderate GAD activity (A590 nm 0.3-0.6); +++, high GAD activity (A590 nm 0.6-1).

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Journal Pre-proof Table 2. Relative GABA content of Irish marine cyanobacteria and the cyanobacteria studied earlier. GABA content nmol g-1 biomass (DW)

Cyanobacteria

Reference

2.06 ± 0.10

(Boonburapong et al., 2016)

Arthrospira platensis

2.27 ± 0.11

(Boonburapong et al., 2016)

Anabaena siamensis TISTR 8012

0.83 ± 0.03

(Boonburapong et al., 2016)

Anabaena sp. PCC 7120

0.50 ± 0.01

(Boonburapong et al., 2016)

72.84x104 ± 3875

This study

Chlorogloea microcystoides SABC022904

0.99x104 ± 254.8

This study

Phormidium africanum SABC022001

1.04x104 ± 322.78

This study

1.62x104 ± 164.36

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4.99x104 ± 770.51

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Synechococcus sp. PCC 7942 Synechocystis sp. PCC 6803

This study

1.38 ± 0.09

(Boonburapong et al., 2016)

0.93 ± 0.06

(Boonburapong et al., 2016)

Note: DW, dry weight biomass; ±, standard deviations (n=3).

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Graphical Abstract

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Journal Pre-proof Highlights for review  Identified five marine cyanobacteria as potential producers of GABA.  Irish marine cyanobacteria screened for potential producers of GABA.  Twelve out of seventeen cyanobacteria proved positive for GAD activity.  GABA was detected both spectrophotometrically and by thin layer chromatography.

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 GABA concentrations were ranging from 4x104-406x104 nmol g-1 fresh weight

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biomass.

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