Journal articles: 'Genetically engineered cyanobacteria'

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OSANAI, Takashi. "Hydrogen Production Using Genetically Engineered Cyanobacteria." Hyomen Kagaku 36, no. 2 (2015): 86–90. http://dx.doi.org/10.1380/jsssj.36.86.

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Govindasamy, Rajakumar, Ekambaram Gayathiri, Sathish Sankar, Baskar Venkidasamy, Palanisamy Prakash, Kaliaperumal Rekha, Varsha Savaner, Abirami Pari, Natesan Thirumalaivasan, and Muthu Thiruvengadam. "Emerging Trends of Nanotechnology and Genetic Engineering in Cyanobacteria to Optimize Production for Future Applications." Life 12, no. 12 (December 2, 2022): 2013. http://dx.doi.org/10.3390/life12122013.

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Nanotechnology has the potential to revolutionize various fields of research and development. Multiple nanoparticles employed in a nanotechnology process are the magic elixir that provides unique features that are not present in the component’s natural form. In the framework of contemporary research, it is inappropriate to synthesize microparticles employing procedures that include noxious elements. For this reason, scientists are investigating safer ways to produce genetically improved Cyanobacteria, which has many novel features and acts as a potential candidate for nanoparticle synthesis. In recent decades, cyanobacteria have garnered significant interest due to their prospective nanotechnological uses. This review will outline the applications of genetically engineered cyanobacteria in the field of nanotechnology and discuss its challenges and future potential. The evolution of cyanobacterial strains by genetic engineering is subsequently outlined. Furthermore, the recombination approaches that may be used to increase the industrial potential of cyanobacteria are discussed. This review provides an overview of the research undertaken to increase the commercial avenues of cyanobacteria and attempts to explain prospective topics for future research.

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Abalde-Cela, Sara, Anna Gould, Xin Liu, Elena Kazamia, Alison G. Smith, and Chris Abell. "High-throughput detection of ethanol-producing cyanobacteria in a microdroplet platform." Journal of The Royal Society Interface 12, no. 106 (May 2015): 20150216. http://dx.doi.org/10.1098/rsif.2015.0216.

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Ethanol production by microorganisms is an important renewable energy source. Most processes involve fermentation of sugars from plant feedstock, but there is increasing interest in direct ethanol production by photosynthetic organisms. To facilitate this, a high-throughput screening technique for the detection of ethanol is required. Here, a method for the quantitative detection of ethanol in a microdroplet-based platform is described that can be used for screening cyanobacterial strains to identify those with the highest ethanol productivity levels. The detection of ethanol by enzymatic assay was optimized both in bulk and in microdroplets. In parallel, the encapsulation of engineered ethanol-producing cyanobacteria in microdroplets and their growth dynamics in microdroplet reservoirs were demonstrated. The combination of modular microdroplet operations including droplet generation for cyanobacteria encapsulation, droplet re-injection and pico-injection, and laser-induced fluorescence, were used to create this new platform to screen genetically engineered strains of cyanobacteria with different levels of ethanol production.

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Sekar, Narendran, Rachit Jain, Yajun Yan, and Ramaraja P. Ramasamy. "Enhanced photo-bioelectrochemical energy conversion by genetically engineered cyanobacteria." Biotechnology and Bioengineering 113, no. 3 (September 18, 2015): 675–79. http://dx.doi.org/10.1002/bit.25829.

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Gao, Zhengxu, Hui Zhao, Zhimin Li, Xiaoming Tan, and Xuefeng Lu. "Correction: Photosynthetic production of ethanol from carbon dioxide in genetically engineered cyanobacteria." Energy & Environmental Science 9, no. 3 (2016): 1113. http://dx.doi.org/10.1039/c5ee90067k.

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Sekar, N., and R. P. Ramasamy. "Genetically Engineered Cyanobacteria Enhances Photocurrent Generation in Photo-bioelectrochemical Cell." ECS Transactions 69, no. 34 (December 28, 2015): 1–8. http://dx.doi.org/10.1149/06934.0001ecst.

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Wang, Yu, Fei Tao, Jun Ni, Chao Li, and Ping Xu. "Production of C3 platform chemicals from CO2 by genetically engineered cyanobacteria." Green Chemistry 17, no. 5 (2015): 3100–3110. http://dx.doi.org/10.1039/c5gc00129c.

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Gao, Zhengxu, Hui Zhao, Zhimin Li, Xiaoming Tan, and Xuefeng Lu. "Photosynthetic production of ethanol from carbon dioxide in genetically engineered cyanobacteria." Energy Environ. Sci. 5, no. 12 (2012): 9857–65. http://dx.doi.org/10.1039/c2ee22675h.

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Mohandass, ShylajaNaciyar, Mangalalakshmi Ragavan, Dineshbabu Gnanasekaran, Uma Lakshmanan, Prabaharan Dharmar, and Sushanta Kumar Saha. "Overexpression of Cu/Zn Superoxide Dismutase (Cu/Zn SOD) in Synechococcus elongatus PCC 7942 for Enhanced Azo Dye Removal through Hydrogen Peroxide Accumulation." Biology 10, no. 12 (December 10, 2021): 1313. http://dx.doi.org/10.3390/biology10121313.

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Discharge of recalcitrant azo dyes to the environment poses a serious threat to environmental health. However certain microorganisms in nature have developed their survival strategies by degrading these toxic dyes. Cyanobacteria are one such prokaryotic, photosynthetic group of microorganisms that degrade various xenobiotic compounds, due to their capability to produce various reactive oxygen species (ROS), and particularly the hydrogen peroxide (H2O2) when released in their milieu. The accumulation of H2O2 is the result of the dismutation of superoxide radicals by the enzyme superoxide dismutase (SOD). In this study, we have genetically modified the cyanobacterium Synechococcus elongatus PCC 7942 by integrating Cu/Zn SOD gene (sodC) from Synechococcus sp. PCC 9311 to its neutral site through homologous recombination. The overexpression of sodC in the derivative strain was driven using a strong constitutive promoter of the psbA gene. The derivative strain resulted in constitutive production of sodC, which was induced further during dye-treated growth. The genetically engineered Synechococcus elongatus PCC 7942 (MS-sodC+) over-accumulated H2O2 during azo dye treatment with a higher dye removal rate than the wild-type strain (WS-sodC−). Therefore, enhanced H2O2 accumulation through SODs overexpression in cyanobacteria may serve as a valuable bioremediation tool.

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Tan, Xiaoming, Wei Du, and Xuefeng Lu. "Photosynthetic and extracellular production of glucosylglycerol by genetically engineered and gel-encapsulated cyanobacteria." Applied Microbiology and Biotechnology 99, no. 5 (December 13, 2014): 2147–54. http://dx.doi.org/10.1007/s00253-014-6273-7.

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Lu, Xuefeng. "A perspective: Photosynthetic production of fatty acid-based biofuels in genetically engineered cyanobacteria." Biotechnology Advances 28, no. 6 (November 2010): 742–46. http://dx.doi.org/10.1016/j.biotechadv.2010.05.021.

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Verma, Samakshi, and Arindam Kuila. "Involvement of green technology in microalgal biodiesel production." Reviews on Environmental Health 35, no. 2 (June 25, 2020): 173–88. http://dx.doi.org/10.1515/reveh-2019-0061.

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AbstractAccording to the report of the renewable energy policy network for the 21st century published in 2014, biodiesel and bioethanol are the most used biofuels and are responsible for transportation worldwide. Biodiesel specially has shown an increase in production globally by 15 times by volume from 2002 to 2012. Promising feedstock of biodiesel are cyanobacteria and microalgae as they possess a shorter cultivation time (4 fold lesser) and high oil content (10 fold higher) than corn, jatropha and soybean (conventional oil-producing territorial plants). Various valuable natural chemicals are also produced from these organisms including food supplements such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), pigments, and vitamins. Additionally, cellular components of microalgae and cyanobacteria are connected with therapeutic characteristics such as anti-inflammatory, antioxidant, antiviral and immune stimulating. Commercialization of algal biodiesel (or other products) can be achieved by isolating and identifying the high-yielding strains that possess a faster growth rate. Indigenous strains can be genetically engineered into high-yielding transgenic strains. The present article discusses about the use of nanotechnology and genetic engineering approach for improved lipid accumulation in microalgae for biodiesel production.

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Nilsson, Astrid, Kiyan Shabestary, Miguel Brandão, and Elton P. Hudson. "Environmental impacts and limitations of third‐generation biobutanol: Life cycle assessment of n ‐butanol produced by genetically engineered cyanobacteria." Journal of Industrial Ecology 24, no. 1 (April 2019): 205–16. http://dx.doi.org/10.1111/jiec.12843.

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Guan, Wenna, Hui Zhao, Xuefeng Lu, Cong Wang, Menglong Yang, and Fali Bai. "Quantitative analysis of fatty-acid-based biofuels produced by wild-type and genetically engineered cyanobacteria by gas chromatography–mass spectrometry." Journal of Chromatography A 1218, no. 45 (November 2011): 8289–93. http://dx.doi.org/10.1016/j.chroma.2011.09.043.

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Wagner, Jonathan L., Daniel Lee-Lane, Mark Monaghan, Mahdi Sharifzadeh, and Klaus Hellgardt. "Recovery of excreted n-butanol from genetically engineered cyanobacteria cultures: Process modelling to quantify energy and economic costs of different separation technologies." Algal Research 37 (January 2019): 92–102. http://dx.doi.org/10.1016/j.algal.2018.11.008.

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Stevens, S. Edward, Randy C. Murphy, William J. Lamoreaux, and Lewis B. Coons. "A genetically engineered mosquitocidal cyanobacterium." Journal of Applied Phycology 6, no. 2 (April 1994): 187–97. http://dx.doi.org/10.1007/bf02186072.

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SHENG, Chun-Xia, and Ren-Qiu KONG. "WAX ESTER BIOSYNTHESIS IN A GENETICALLY ENGINEERED CYANOBACTERIUM." Acta Hydrobiologica Sinica 36, no. 3 (May 24, 2010): 652–55. http://dx.doi.org/10.3724/sp.j.1035.2010.00652.

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Omata, Tatsuo, Masayuki Ohmori, Nobuyuki Arai, and Teruo Ogawa. "Genetically engineered mutant of the cyanobacterium Synechococcus PCC 7942 defective in nitrate transport." Proceedings of the National Academy of Sciences 86, no. 17 (September 1989): 6612–16. http://dx.doi.org/10.1073/pnas.86.17.6612.

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Nitrate-grown cells of Synechococcus PCC 7942 (Anacystis nidulans R2) contain a 45-kDa protein as a major protein in the cytoplasmic membrane but ammonium-grown cells lack it. A mutant (M45) was constructed by inactivating the gene encoding the 45-kDa protein. M45 did not grow under low concentrations of nitrate but high concentrations of nitrate could support its growth, with the optimal concentration being 40-70 mM. The growth rate of M45 was as high as that of the wild-type cells when ammonium was the nitrogen source. The 45-kDa protein was absent in M45 irrespective of the growth conditions. The activities of nitrate and nitrite reductases were higher in M45 than in wild type. The rate of nitrate-dependent O2 evolution in wild type measured in the presence of L-methionine D,L-sulfoximine and D,L-glyceraldehyde showed saturation kinetics with respect to nitrate concentration in the external medium. The nitrate concentration required to produce half the maximal rate was 1 μM. In M45, the rate of nitrate-dependent O2 evolution was nearly zero at nitrate concentrations <1 mM and was linearly increased as the concentration increased. The presumed absence of nitrate transport in M45 demonstrated by these results suggested that the 45-kDa protein is a nitrate transporter.

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Vermaas, W. F. J., J. G. K. Williams, A. W. Rutherford, P. Mathis, and C. J. Arntzen. "Genetically engineered mutant of the cyanobacterium Synechocystis 6803 lacks the photosystem II chlorophyll-binding protein CP-47." Proceedings of the National Academy of Sciences 83, no. 24 (December 1, 1986): 9474–77. http://dx.doi.org/10.1073/pnas.83.24.9474.

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Vermaas, W. F. J., M. Ikeuchi, and Y. Inoue. "Protein composition of the photosystem II core complex in genetically engineered mutants of the cyanobacterium Synechocystis sp. PCC 6803." Photosynthesis Research 17, no. 1-2 (1988): 97–113. http://dx.doi.org/10.1007/bf00047683.

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Patipong, Tanutcha, Takashi Hibino, Rungaroon Waditee-Sirisattha, and Hakuto Kageyama. "Efficient Bioproduction of Mycosporine-2-glycine, Which Functions as Potential Osmoprotectant, using Escherichia coli Cells." Natural Product Communications 12, no. 10 (October 2017): 1934578X1701201. http://dx.doi.org/10.1177/1934578x1701201017.

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Mycosporine-2-glycine (M2G) is known to be synthesized in halotolerant cyanobacterium Aphanothece halophytica. Escherichia coli cells in which the M2G synthetic genes of A. halophytica were introduced could synthesize M2G. Here, we report that M2G producing transformed E. coli cells showed salt tolerance compared to control cells. This result suggested that M2G could function as a potential osmoprotectant in E. coli. To our knowledge, this is the first report presenting the evidence that mycosporine-like amino acid confers salt tolerance on E. coli. Intracellular M2G content in the transformed E. coli cells were varied depending on NaCl concentration with maximum level at 0.75 M. Moreover, intracellular M2G level was affected by a supply of glycine with maximum level at 5 mM. In conclusion, we found that transformed E. coli cells could produce 205 μg of M2G/g fresh weight of cells under the best effective growth condition in this study. Thus, the results obtained here offer the potential for the bioproduction of mycosporine-like amino acids using the genetically engineered E. coli cells.

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Broddrick, Jared T., Benjamin E. Rubin, David G. Welkie, Niu Du, Nathan Mih, Spencer Diamond, Jenny J. Lee, Susan S. Golden, and Bernhard O. Palsson. "Unique attributes of cyanobacterial metabolism revealed by improved genome-scale metabolic modeling and essential gene analysis." Proceedings of the National Academy of Sciences 113, no. 51 (December 1, 2016): E8344—E8353. http://dx.doi.org/10.1073/pnas.1613446113.

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The model cyanobacterium,Synechococcus elongatusPCC 7942, is a genetically tractable obligate phototroph that is being developed for the bioproduction of high-value chemicals. Genome-scale models (GEMs) have been successfully used to assess and engineer cellular metabolism; however, GEMs of phototrophic metabolism have been limited by the lack of experimental datasets for model validation and the challenges of incorporating photon uptake. Here, we develop a GEM of metabolism inS. elongatususing random barcode transposon site sequencing (RB-TnSeq) essential gene and physiological data specific to photoautotrophic metabolism. The model explicitly describes photon absorption and accounts for shading, resulting in the characteristic linear growth curve of photoautotrophs. GEM predictions of gene essentiality were compared with data obtained from recent dense-transposon mutagenesis experiments. This dataset allowed major improvements to the accuracy of the model. Furthermore, discrepancies between GEM predictions and the in vivo dataset revealed biological characteristics, such as the importance of a truncated, linear TCA pathway, low flux toward amino acid synthesis from photorespiration, and knowledge gaps within nucleotide metabolism. Coupling of strong experimental support and photoautotrophic modeling methods thus resulted in a highly accurate model ofS. elongatusmetabolism that highlights previously unknown areas ofS. elongatusbiology.

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Diamond, Spencer, Darae Jun, Benjamin E. Rubin, and Susan S. Golden. "The circadian oscillator inSynechococcus elongatuscontrols metabolite partitioning during diurnal growth." Proceedings of the National Academy of Sciences 112, no. 15 (March 30, 2015): E1916—E1925. http://dx.doi.org/10.1073/pnas.1504576112.

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Synechococcus elongatusPCC 7942 is a genetically tractable model cyanobacterium that has been engineered to produce industrially relevant biomolecules and is the best-studied model for a prokaryotic circadian clock. However, the organism is commonly grown in continuous light in the laboratory, and data on metabolic processes under diurnal conditions are lacking. Moreover, the influence of the circadian clock on diurnal metabolism has been investigated only briefly. Here, we demonstrate that the circadian oscillator influences rhythms of metabolism during diurnal growth, even though light–dark cycles can drive metabolic rhythms independently. Moreover, the phenotype associated with loss of the core oscillator protein, KaiC, is distinct from that caused by absence of the circadian output transcriptional regulator, RpaA (regulator of phycobilisome-associated A). Although RpaA activity is important for carbon degradation at night, KaiC is dispensable for those processes. Untargeted metabolomics analysis and glycogen kinetics suggest that functional KaiC is important for metabolite partitioning in the morning. Additionally, output from the oscillator functions to inhibit RpaA activity in the morning, andkaiC-null strains expressing a mutant KaiC phosphomimetic, KaiC-pST, in which the oscillator is locked in the most active output state, phenocopies aΔrpaAstrain. Inhibition of RpaA by the oscillator in the morning suppresses metabolic processes that normally are active at night, andkaiC-null strains show indications of oxidative pentose phosphate pathway activation as well as increased abundance of primary metabolites. Inhibitory clock output may serve to allow secondary metabolite biosynthesis in the morning, and some metabolites resulting from these processes may feed back to reinforce clock timing.

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Cournoyer, Jason E., Sarah D. Altman, Yang-le Gao, Catherine L. Wallace, Dianwen Zhang, Guo-Hsuen Lo, Noah T. Haskin, and Angad P. Mehta. "Engineering artificial photosynthetic life-forms through endosymbiosis." Nature Communications 13, no. 1 (April 26, 2022). http://dx.doi.org/10.1038/s41467-022-29961-7.

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AbstractThe evolutionary origin of the photosynthetic eukaryotes drastically altered the evolution of complex lifeforms and impacted global ecology. The endosymbiotic theory suggests that photosynthetic eukaryotes evolved due to endosymbiosis between non-photosynthetic eukaryotic host cells and photosynthetic cyanobacterial or algal endosymbionts. The photosynthetic endosymbionts, propagating within the cytoplasm of the host cells, evolved, and eventually transformed into chloroplasts. Despite the fundamental importance of this evolutionary event, we have minimal understanding of this remarkable evolutionary transformation. Here, we design and engineer artificial, genetically tractable, photosynthetic endosymbiosis between photosynthetic cyanobacteria and budding yeasts. We engineer various mutants of model photosynthetic cyanobacteria as endosymbionts within yeast cells where, the engineered cyanobacteria perform bioenergetic functions to support the growth of yeast cells under defined photosynthetic conditions. We anticipate that these genetically tractable endosymbiotic platforms can be used for evolutionary studies, particularly related to organelle evolution, and also for synthetic biology applications.

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Cournoyer, Jason E., Sarah D. Altman, Yang-le Gao, Catherine L. Wallace, Dianwen Zhang, Guo-Hsuen Lo, Noah T. Haskin, and Angad P. Mehta. "Engineering artificial photosynthetic life-forms through endosymbiosis." Nature Communications 13, no. 1 (April 26, 2022). http://dx.doi.org/10.1038/s41467-022-29961-7.

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AbstractThe evolutionary origin of the photosynthetic eukaryotes drastically altered the evolution of complex lifeforms and impacted global ecology. The endosymbiotic theory suggests that photosynthetic eukaryotes evolved due to endosymbiosis between non-photosynthetic eukaryotic host cells and photosynthetic cyanobacterial or algal endosymbionts. The photosynthetic endosymbionts, propagating within the cytoplasm of the host cells, evolved, and eventually transformed into chloroplasts. Despite the fundamental importance of this evolutionary event, we have minimal understanding of this remarkable evolutionary transformation. Here, we design and engineer artificial, genetically tractable, photosynthetic endosymbiosis between photosynthetic cyanobacteria and budding yeasts. We engineer various mutants of model photosynthetic cyanobacteria as endosymbionts within yeast cells where, the engineered cyanobacteria perform bioenergetic functions to support the growth of yeast cells under defined photosynthetic conditions. We anticipate that these genetically tractable endosymbiotic platforms can be used for evolutionary studies, particularly related to organelle evolution, and also for synthetic biology applications.

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Cournoyer, Jason E., Sarah D. Altman, Yang-le Gao, Catherine L. Wallace, Dianwen Zhang, Guo-Hsuen Lo, Noah T. Haskin, and Angad P. Mehta. "Engineering artificial photosynthetic life-forms through endosymbiosis." Nature Communications 13, no. 1 (April 26, 2022). http://dx.doi.org/10.1038/s41467-022-29961-7.

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AbstractThe evolutionary origin of the photosynthetic eukaryotes drastically altered the evolution of complex lifeforms and impacted global ecology. The endosymbiotic theory suggests that photosynthetic eukaryotes evolved due to endosymbiosis between non-photosynthetic eukaryotic host cells and photosynthetic cyanobacterial or algal endosymbionts. The photosynthetic endosymbionts, propagating within the cytoplasm of the host cells, evolved, and eventually transformed into chloroplasts. Despite the fundamental importance of this evolutionary event, we have minimal understanding of this remarkable evolutionary transformation. Here, we design and engineer artificial, genetically tractable, photosynthetic endosymbiosis between photosynthetic cyanobacteria and budding yeasts. We engineer various mutants of model photosynthetic cyanobacteria as endosymbionts within yeast cells where, the engineered cyanobacteria perform bioenergetic functions to support the growth of yeast cells under defined photosynthetic conditions. We anticipate that these genetically tractable endosymbiotic platforms can be used for evolutionary studies, particularly related to organelle evolution, and also for synthetic biology applications.

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"Enhanced Photo-Bioelectrochemical Energy Conversion By Genetically Engineered Cyanobacteria." ECS Meeting Abstracts, 2015. http://dx.doi.org/10.1149/ma2015-02/39/1619.

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Sebesta, Jacob, Wei Xiong, Michael T. Guarnieri, and Jianping Yu. "Biocontainment of Genetically Engineered Algae." Frontiers in Plant Science 13 (March 2, 2022). http://dx.doi.org/10.3389/fpls.2022.839446.

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Algae (including eukaryotic microalgae and cyanobacteria) have been genetically engineered to convert light and carbon dioxide to many industrially and commercially relevant chemicals including biofuels, materials, and nutritional products. At industrial scale, genetically engineered algae may be cultivated outdoors in open ponds or in closed photobioreactors. In either case, industry would need to address a potential risk of the release of the engineered algae into the natural environment, resulting in potential negative impacts to the environment. Genetic biocontainment strategies are therefore under development to reduce the probability that these engineered bacteria can survive outside of the laboratory or industrial setting. These include active strategies that aim to kill the escaped cells by expression of toxic proteins, and passive strategies that use knockouts of native genes to reduce fitness outside of the controlled environment of labs and industrial cultivation systems. Several biocontainment strategies have demonstrated escape frequencies below detection limits. However, they have typically done so in carefully controlled experiments which may fail to capture mechanisms of escape that may arise in the more complex natural environment. The selection of biocontainment strategies that can effectively kill cells outside the lab, while maintaining maximum productivity inside the lab and without the need for relatively expensive chemicals will benefit from further attention.

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Yang, Ruigang, Lingyun Zhu, Tao Li, Lv-yun Zhu, Zi Ye, and Dongyi Zhang. "Photosynthetic Conversion of CO2 Into Pinene Using Engineered Synechococcus sp. PCC 7002." Frontiers in Bioengineering and Biotechnology 9 (December 17, 2021). http://dx.doi.org/10.3389/fbioe.2021.779437.

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Metabolic engineering of cyanobacteria has received much attention as a sustainable strategy to convert CO2 to various longer carbon chain fuels. Pinene has become increasingly attractive since pinene dimers contain high volumetric energy and have been proposed to act as potential aircraft fuels. However, cyanobacteria cannot directly convert geranyl pyrophosphate into pinene due to the lack of endogenous pinene synthase. Herein, we integrated the gene encoding Abies grandis pinene synthase into the model cyanobacterium Synechococcus sp. PCC 7002 through homologous recombination. The genetically modified cyanobacteria achieved a pinene titer of 1.525 ± 0.l45 mg L−1 in the lab-scale tube photobioreactor with CO2 aeration. Specifically, the results showed a mixture of α- and β-pinene (∼33:67 ratio). The ratio of β-pinene in the product was significantly increased compared with that previously reported in the engineered Escherichia coli. Furthermore, we investigated the photoautotrophic growth performances of Synechococcus overlaid with different concentrations of dodecane. The work demonstrates that the engineered Synechococcus is a suitable potential platform for β-pinene production.

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Liu, X., D. Brune, W. Vermaas, and R. Curtiss. "Production and secretion of fatty acids in genetically engineered cyanobacteria." Proceedings of the National Academy of Sciences, March 29, 2010. http://dx.doi.org/10.1073/pnas.1001946107.

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Nguyen, Thu, Cherrelle Barnes, Sana Sherazi, Jason Agola, Lesley Greene, and James W. Lee. "Bio‐risk assessment research on genetically engineered cyanobacteria for sustainable biofuels." FASEB Journal 33, S1 (April 2019). http://dx.doi.org/10.1096/fasebj.2019.33.1_supplement.lb301.

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Liberton, Michelle, Anindita Bandyopadhyay, and Himadri B. Pakrasi. "Enhanced Nitrogen Fixation in a glgX-Deficient Strain of Cyanothece sp. Strain ATCC 51142, a Unicellular Nitrogen-Fixing Cyanobacterium." Applied and Environmental Microbiology 85, no. 7 (February 1, 2019). http://dx.doi.org/10.1128/aem.02887-18.

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ABSTRACT Cyanobacteria are oxygenic photosynthetic prokaryotes with important roles in the global carbon and nitrogen cycles. Unicellular nitrogen-fixing cyanobacteria are known to be ubiquitous, contributing to the nitrogen budget in diverse ecosystems. In the unicellular cyanobacterium Cyanothece sp. strain ATCC 51142, carbon assimilation and carbohydrate storage are crucial processes that occur as part of a robust diurnal cycle of photosynthesis and nitrogen fixation. During the light period, cells accumulate fixed carbon in glycogen granules to use as stored energy to power nitrogen fixation in the dark. These processes have not been thoroughly investigated, due to the lack of a genetic modification system in this organism. In bacterial glycogen metabolism, the glgX gene encodes a debranching enzyme that functions in storage polysaccharide catabolism. To probe the consequences of modifying the cycle of glycogen accumulation and subsequent mobilization, we engineered a strain of Cyanothece 51142 in which the glgX gene was genetically disrupted. We found that the ΔglgX strain exhibited a higher growth rate than the wild-type strain and displayed a higher rate of nitrogen fixation. Glycogen accumulated to higher levels at the end of the light period in the ΔglgX strain, compared to the wild-type strain. These data suggest that the larger glycogen pool maintained by the ΔglgX mutant is able to fuel greater growth and nitrogen fixation ability. IMPORTANCE Cyanobacteria are oxygenic photosynthetic bacteria that are found in a wide variety of ecological environments, where they are important contributors to global carbon and nitrogen cycles. Genetic manipulation systems have been developed in a number of cyanobacterial strains, allowing both the interruption of endogenous genes and the introduction of new genes and entire pathways. However, unicellular diazotrophic cyanobacteria have been generally recalcitrant to genetic transformation. These cyanobacteria are becoming important model systems to study diurnally regulated processes. Strains of the Cyanothece genus have been characterized as displaying robust growth and high rates of nitrogen fixation. The significance of our study is in the establishment of a genetic modification system in a unicellular diazotrophic cyanobacterium, the demonstration of the interruption of the glgX gene in Cyanothece sp. strain ATCC 51142, and the characterization of the increased nitrogen-fixing ability of this strain.

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Wu, Xiao-Xi, Jian-Wei Li, Su-Fang Xing, Hui-Ting Chen, Chao Song, Shu-Guang Wang, and Zhen Yan. "Establishment of a resource recycling strategy by optimizing isobutanol production in engineered cyanobacteria using high salinity stress." Biotechnology for Biofuels 14, no. 1 (August 30, 2021). http://dx.doi.org/10.1186/s13068-021-02023-8.

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Abstract Background Isobutanol is an attractive biofuel with many advantages. Third-generation biorefineries that convert CO2 into bio-based fuels have drawn considerable attention due to their lower feedstock cost and more ecofriendly refining process. Although autotrophic cyanobacteria have been genetically modified for isobutanol biosynthesis, there is a lack of stable and convenient strategies to improve their production. Results In this study, we first engineered Synechococcus elongatus for isobutanol biosynthesis by introducing five exogenous enzymes, reaching a production titer of 0.126 g/L at day 20. It was then discovered that high salinity stress could result in a whopping fivefold increase in isobutanol production, with a maximal in-flask titer of 0.637 g/L at day 20. Metabolomics analysis revealed that high salinity stress substantially altered the metabolic profiles of the engineered S. elongatus. A major reason for the enhanced isobutanol production is the acceleration of lipid degradation under high salinity stress, which increases NADH. The NADH then participates in the engineered isobutanol-producing pathway. In addition, increased membrane permeability also contributed to the isobutanol production titer. A cultivation system was subsequently developed by mixing synthetic wastewater with seawater to grow the engineered cyanobacteria, reaching a similar isobutanol production titer as cultivation in the medium. Conclusions High salinity stress on engineered cyanobacteria is a practical and feasible biotechnology to optimize isobutanol production. This biotechnology provides a cost-effective approach to biofuel production, and simultaneously recycles chemical nutrients from wastewater and seawater.

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Lehmann, Martin, Evgenia Vamvaka, Alejandro Torrado, Peter Jahns, Marcel Dann, Lea Rosenhammer, Amel Aziba, Dario Leister, and Thilo Rühle. "Introduction of the Carotenoid Biosynthesis α-Branch Into Synechocystis sp. PCC 6803 for Lutein Production." Frontiers in Plant Science 12 (July 6, 2021). http://dx.doi.org/10.3389/fpls.2021.699424.

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Lutein, made by the α-branch of the methyl-erythritol phosphate (MEP) pathway, is one of the most abundant xanthophylls in plants. It is involved in the structural stabilization of light-harvesting complexes, transfer of excitation energy to chlorophylls and photoprotection. In contrast, lutein and the α-branch of the MEP pathway are not present in cyanobacteria. In this study, we genetically engineered the cyanobacterium Synechocystis for the missing MEP α-branch resulting in lutein accumulation. A cassette comprising four Arabidopsis thaliana genes coding for two lycopene cyclases (AtLCYe and AtLCYb) and two hydroxylases (AtCYP97A and AtCYP97C) was introduced into a Synechocystis strain that lacks the endogenous, cyanobacterial lycopene cyclase cruA. The resulting synlut strain showed wild-type growth and only moderate changes in total pigment composition under mixotrophic conditions, indicating that the cruA deficiency can be complemented by Arabidopsis lycopene cyclases leaving the endogenous β-branch intact. A combination of liquid chromatography, UV-Vis detection and mass spectrometry confirmed a low but distinct synthesis of lutein at rates of 4.8 ± 1.5 nmol per liter culture at OD730 (1.03 ± 0.47 mmol mol–1 chlorophyll). In conclusion, synlut provides a suitable platform to study the α-branch of the plastidic MEP pathway and other functions related to lutein in a cyanobacterial host system.

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Hayashi, Yuuki, and Munehito Arai. "Recent advances in the improvement of cyanobacterial enzymes for bioalkane production." Microbial Cell Factories 21, no. 1 (December 12, 2022). http://dx.doi.org/10.1186/s12934-022-01981-4.

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AbstractThe use of biologically produced alkanes has attracted considerable attention as an alternative energy source to petroleum. In 2010, the alkane synthesis pathway in cyanobacteria was found to include two small globular proteins, acyl-(acyl carrier protein [ACP]) reductase (AAR) and aldehyde deformylating oxygenase (ADO). AAR produces fatty aldehydes from acyl-ACPs/CoAs, which are then converted by ADO to alkanes/alkenes equivalent to diesel oil. This discovery has paved the way for alkane production by genetically modified organisms. Since then, many studies have investigated the reactions catalyzed by AAR and ADO. In this review, we first summarize recent findings on structures and catalytic mechanisms of AAR and ADO. We then outline the mechanism by which AAR and ADO form a complex and efficiently transfer the insoluble aldehyde produced by AAR to ADO. Furthermore, we describe recent advances in protein engineering studies on AAR and ADO to improve the efficiency of alkane production in genetically engineered microorganisms such as Escherichia coli and cyanobacteria. Finally, the role of alkanes in cyanobacteria and future perspectives for bioalkane production using AAR and ADO are discussed. This review provides strategies for improving the production of bioalkanes using AAR and ADO in cyanobacteria for enabling the production of carbon–neutral fuels.

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Tóth, Gábor Szilveszter, Vilja Siitonen, Lauri Nikkanen, Lucija Sovic, Pauli Kallio, Robert Kourist, Sergey Kosourov, and Yagut Allahverdiyeva. "Photosynthetically produced sucrose by immobilized Synechocystis sp. PCC 6803 drives biotransformation in E. coli." Biotechnology for Biofuels and Bioproducts 15, no. 1 (December 27, 2022). http://dx.doi.org/10.1186/s13068-022-02248-1.

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Abstract Background Whole-cell biotransformation is a promising emerging technology for the production of chemicals. When using heterotrophic organisms such as E. coli and yeast as biocatalysts, the dependence on organic carbon source impairs the sustainability and economic viability of the process. As a promising alternative, photosynthetic cyanobacteria with low nutrient requirements and versatile metabolism, could offer a sustainable platform for the heterologous production of organic compounds directly from sunlight and CO2. This strategy has been applied for the photoautotrophic production of sucrose by a genetically engineered cyanobacterium, Synechocystis sp. PCC 6803 strain S02. As the key concept in the current work, this can be further used to generate organic carbon compounds for different heterotrophic applications, including for the whole-cell biotransformation by yeast and bacteria. Results Entrapment of Synechocystis S02 cells in Ca2+-cross-linked alginate hydrogel beads improves the specific sucrose productivity by 86% compared to suspension cultures during 7 days of cultivation under salt stress. The process was further prolonged by periodically changing the medium in the vials for up to 17 days of efficient production, giving the final sucrose yield slightly above 3000 mg l−1. We successfully demonstrated that the medium enriched with photosynthetically produced sucrose by immobilized Synechocystis S02 cells supports the biotransformation of cyclohexanone to ε-caprolactone by the E. coli WΔcscR Inv:Parvi strain engineered to (i) utilize low concentrations of sucrose and (ii) perform biotransformation of cyclohexanone to ε-caprolactone. Conclusion We conclude that cell entrapment in Ca2+-alginate beads is an effective method to prolong sucrose production by the engineered cyanobacteria, while allowing efficient separation of the cells from the medium. This advantage opens up novel possibilities to create advanced autotroph–heterotroph coupled cultivation systems for solar-driven production of chemicals via biotransformation, as demonstrated in this work by utilizing the photosynthetically produced sucrose to drive the conversion of cyclohexanone to ε-caprolactone by engineered E. coli.

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"Retraction for Liu et al., Production and secretion of fatty acids in genetically engineered cyanobacteria." Proceedings of the National Academy of Sciences 107, no. 29 (July 2, 2010): 13189. http://dx.doi.org/10.1073/pnas.1008568107.

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Villacreses-Freire, Daniela, Franziska Ketzer, and Christine Rösch. "Advanced Metabolic Engineering Approaches and Renewable Energy to Improve Environmental Benefits of Algal Biofuels: LCA of Large-scale Biobutanol Production with Cyanobacteria Synechocystis PCC6803." BioEnergy Research, October 13, 2021. http://dx.doi.org/10.1007/s12155-021-10323-y.

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AbstractWith modern genetic engineering tools, microorganisms can become resilient green cell factories to produce sustainable biofuels directly. Compared to non-engineered algae and cyanobacteria, the photon conversion efficiency can be significantly increased. Furthermore, simplified harvesting processes are feasible since the novel microorganisms are excreting the biofuels or their precursors continuously and directly into the cultivation media. Along with higher productivity and direct product harvesting, it is expected that environmental benefits can be achieved, especially for climate protection. A life cycle assessment (LCA) for biobutanol production with the genetically engineered cyanobacteria Synechocystis PCC6803 is performed to test this hypothesis. A prospective and upscaled approach was applied to assess the environmental impacts at large-scale production (20 ha plant) for better comparability with conventional butanol production. The LCA results show that the engineering of microorganisms can improve the environmental impact, mainly due to the higher productivity compared to non-engineered cyanobacteria. However, the nevertheless high electricity demand required for the cultivation and harvesting process overcompensates this benefit. According to the scenario calculations, a more favourable climate gas balance can be achieved if renewable electricity is used. Then, greenhouse gas emissions are reduced to 3.1 kg CO2 eq/kg biobutanol, corresponding to 20% more than the fossil reference: (2.45 kg CO2 eq./kg 1-butanol). The results indicate the importance of genetic engineering and the energy transition towards renewable electricity supply to take full advantage of the environmental potential of microorganisms as future green cell factories for sustainable biofuel production. Besides, the necessity of developing different scenarios for perspective and upscaled LCA for a fairer comparison with mature reference technologies is demonstrated.

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Deshpande, Arnav, Jeremiah Vue, and John Morgan. "Combining Random Mutagenesis and Metabolic Engineering for Enhanced Tryptophan Production in Synechocystis sp. Strain PCC 6803." Applied and Environmental Microbiology 86, no. 9 (March 6, 2020). http://dx.doi.org/10.1128/aem.02816-19.

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ABSTRACT Tryptophan (Trp) is an essential aromatic amino acid that has value as an animal feed supplement, as the amount found in plant-based sources is insufficient. An alternative to production by engineered microbial fermentation is to have tryptophan biosynthesized by a photosynthetic microorganism that could replace or supplement both the plant and industrially used microbes. We selected Synechocystis sp. strain PCC 6803, a model cyanobacterium, as the host and studied metabolic engineering and random mutagenesis approaches. Previous work on engineering heterotrophic microbes for improved Trp titers has targeted allosteric feedback regulation in enzymes 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase (DAHPS) and anthranilate synthase (AS) as major bottlenecks in the shikimate pathway. In this work, the genes encoding feedback-resistant enzymes from Escherichia coli, aroGfbr and trpEfbr, were overexpressed in the host wild-type (WT) strain. Separately, the WT strain was subjected to random mutagenesis and selection using an amino acid analog to isolate tryptophan-overproducing strains. The randomly mutagenized strains were sequenced in order to identify the mutations that resulted in the desirable phenotypes. Interestingly, the tryptophan overproducers had mutations in the gene encoding chorismate mutase (CM), which catalyzes the conversion of chorismate to prephenate. The best tryptophan overproducer from random mutagenesis was selected as a host for metabolic engineering where aroGfbr and trpEfbr were overexpressed. The best strain developed produced 212 ± 23 mg/liter of tryptophan after 10 days of photoautotrophic growth under 3% (vol/vol) CO2. We demonstrated that a combination of random mutagenesis and metabolic engineering was superior to either individual approach. IMPORTANCE Aromatic amino acids such as tryptophan are primarily used as additives in the animal feed industry and are typically produced using genetically engineered heterotrophic organisms such as Escherichia coli. This involves a two-step process, where the substrate such as molasses is first obtained from plants followed by fermentation by heterotrophic organisms. We have engineered photoautotrophic cyanobacterial strains by a combination of random mutagenesis and metabolic engineering. These strains grow on CO2 as the sole carbon source and utilize light as the sole energy source to produce tryptophan, thus converting the two-step process into a single step. Our results show that combining random mutagenesis and metabolic engineering was superior to either approach alone. This study also builds a foundation for further engineering of cyanobacteria for industrial tryptophan production.

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Kukil, Kateryna, and Pia Lindberg. "Expression of phenylalanine ammonia lyases in Synechocystis sp. PCC 6803 and subsequent improvements of sustainable production of phenylpropanoids." Microbial Cell Factories 21, no. 1 (January 10, 2022). http://dx.doi.org/10.1186/s12934-021-01735-8.

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Abstract Background Phenylpropanoids represent a diverse class of industrially important secondary metabolites, synthesized in plants from phenylalanine and tyrosine. Cyanobacteria have a great potential for sustainable production of phenylpropanoids directly from CO2, due to their photosynthetic lifestyle with a fast growth compared to plants and the ease of generating genetically engineered strains. This study focuses on photosynthetic production of the starting compounds of the phenylpropanoid pathway, trans-cinnamic acid and p-coumaric acid, in the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). Results A selected set of phenylalanine ammonia lyase (PAL) enzymes from different organisms was overexpressed in Synechocystis, and the productivities of the resulting strains compared. To further improve the titer of target compounds, we evaluated the use of stronger expression cassettes for increasing PAL protein levels, as well as knock-out of the laccase gene slr1573, as this was previously reported to prevent degradation of the target compounds in the cell. Finally, to investigate the effect of growth conditions on the production of trans-cinnamic and p-coumaric acids from Synechocystis, cultivation conditions promoting rapid, high density growth were tested. Comparing the different PALs, the highest specific titer was achieved for the strain AtC, expressing PAL from Arabidopsis thaliana. A subsequent increase of protein level did not improve the productivity. Production of target compounds in strains where the slr1573 laccase had been knocked out was found to be lower compared to strains with wild type background, and the Δslr1573 strains exhibited a strong phenotype of slower growth rate and lower pigment content. Application of a high-density cultivation system for the growth of production strains allowed reaching the highest total titers of trans-cinnamic and p-coumaric acids reported so far, at around 0.8 and 0.4 g L−1, respectively, after 4 days. Conclusions Production of trans-cinnamic acid, unlike that of p-coumaric acid, is not limited by the protein level of heterologously expressed PAL in Synechocystis. High density cultivation led to higher titres of both products, while knocking out slr1573 did not have a positive effect on production. This work contributes to capability of exploiting the primary metabolism of cyanobacteria for sustainable production of plant phenylpropanoids.

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Kopka, Joachim, Stefanie Schmidt, Frederik Dethloff, Nadin Pade, Susanne Berendt, Marco Schottkowski, Nico Martin, et al. "Systems analysis of ethanol production in the genetically engineered cyanobacterium Synechococcus sp. PCC 7002." Biotechnology for Biofuels 10, no. 1 (March 6, 2017). http://dx.doi.org/10.1186/s13068-017-0741-0.

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Ketseoglou, Irene, and Gustav Bouwer. "The persistence and ecological impacts of a cyanobacterium genetically engineered to express mosquitocidal Bacillus thuringiensis toxins." Parasites & Vectors 9, no. 1 (May 10, 2016). http://dx.doi.org/10.1186/s13071-016-1544-z.

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Talley, Ethan, and Whitney Holden. "Combating Algae Blooms." Journal of Student Research 10, no. 2 (July 1, 2021). http://dx.doi.org/10.47611/jsrhs.v10i2.1464.

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Microcystis aeruginosa is a common freshwater cyanobacterium that can form toxic algal blooms that harm other species and the environment. This project studied the effects of the antimicrobial peptide Cecropin A on the growth of M. aeruginosa to assess Cecropin A’s effectiveness as a tool to combat algal blooms and limit their environmental impacts. In this study, different concentrations of Cecropin A were tested on M. aeruginosa, the growth of which was then measured using a plate count. Each concentration of Cecropin A tested resulted in a significant decrease in M. aeruginosa growth compared to the control group, indicating the effectiveness of this peptide at inhibiting M. aeruginosa. Because Cecropin A is a peptide, bacteria can be genetically engineered to produce it for anti-algal applications. This study also analyzed the effects of Cecropin A on the non-pathogenic E. coli K12 in order to study development of antibiotic resistance in this bacterium and determine its feasibility for anti-algal applications such as producing or distributing Cecropin A. The effects of Cecropin A were tested on successive generations to determine if this strain of bacterium can build up a resistance to Cecropin A that would make it a suitable candidate to produce large quantities of this peptide. The results over three 24-hour periods of exposure to Cecropin A seem to indicate a development of resistance to Cecropin A by E. coli K12, suggesting that this bacterium may be suitable for production and/or distribution of Cecropin A for anti-bloom control efforts.

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Satagopan, Sriram, Katherine A. Huening, and F. Robert Tabita. "Selection of Cyanobacterial (Synechococcus sp. Strain PCC 6301) RubisCO Variants with Improved Functional Properties That Confer Enhanced CO2-Dependent Growth of Rhodobacter capsulatus, a Photosynthetic Bacterium." mBio 10, no. 4 (July 23, 2019). http://dx.doi.org/10.1128/mbio.01537-19.

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ABSTRACT Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a ubiquitous enzyme that catalyzes the conversion of atmospheric CO2 into organic carbon in primary producers. All naturally occurring RubisCOs have low catalytic turnover rates and are inhibited by oxygen. Evolutionary adaptations of the enzyme and its host organisms to changing atmospheric oxygen concentrations provide an impetus to artificially evolve RubisCO variants under unnatural selective conditions. A RubisCO deletion strain of the nonsulfur purple photosynthetic bacterium Rhodobacter capsulatus was previously used as a heterologous host for directed evolution and suppressor selection studies that led to the identification of a conserved hydrophobic region near the active site where amino acid substitutions selectively impacted the enzyme’s sensitivity to O2. In this study, structural alignments, mutagenesis, suppressor selection, and growth complementation with R. capsulatus under anoxic or oxygenic conditions were used to analyze the importance of semiconserved residues in this region of Synechococcus RubisCO. RubisCO mutant substitutions were identified that provided superior CO2-dependent growth capabilities relative to the wild-type enzyme. Kinetic analyses of the mutant enzymes indicated that enhanced growth performance was traceable to differential interactions of the enzymes with CO2 and O2. Effective residue substitutions also appeared to be localized to two other conserved hydrophobic regions of the holoenzyme. Structural comparisons and similarities indicated that regions identified in this study may be targeted for improvement in RubisCOs from other sources, including crop plants. IMPORTANCE RubisCO catalysis has a significant impact on mitigating greenhouse gas accumulation and CO2 conversion to food, fuel, and other organic compounds required to sustain life. Because RubisCO-dependent CO2 fixation is severely compromised by oxygen inhibition and other physiological constraints, improving RubisCO’s kinetic properties to enhance growth in the presence of atmospheric O2 levels has been a longstanding goal. In this study, RubisCO variants with superior structure-functional properties were selected which resulted in enhanced growth of an autotrophic host organism (R. capsulatus), indicating that RubisCO function was indeed growth limiting. It is evident from these results that genetically engineered RubisCO with kinetically enhanced properties can positively impact growth rates in primary producers.

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Journal articles on the topic 'Genetically engineered cyanobacteria'

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