Construction of a Shuttle Vector Using an Endogenous Plasmid From the Cyanobacterium Synechocystis sp. PCC6803

Jin, Haojie; Wang, Yan; Idoine, Adam; Bhaya, Devaki

doi:10.3389/fmicb.2018.01662

Cited by 42 publications

(62 citation statements)

References 58 publications

(74 reference statements)

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“…The chromosome of S. 6803 can be easily modified using the organism’s native homologous recombination mechanisms. In addition, several replicative plasmids have been used to modify S. 6803 without modifying the chromosome (Ferreira et al., 2018; Huang et al., 2010; Jin et al., 2018; Liu and Pakrasi, 2018). The genome of S. 6803 was sequenced in 1996 (Kaneko et al., 1996), and other genome projects listed on the CyanoBase website (http://genome.microbedb.jp/CyanoBase) have reached 376 cyanobacterial species.…”

Section: Introductionmentioning

confidence: 99%

Improving heterologous protein expression in Synechocystis sp. PCC 6803 for alpha-bisabolene production

Sebesta

Peebles

2020

Metabolic Engineering Communications

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Cyanobacterial biofuels have the potential to reduce the cost and climate impacts of biofuel production because primary carbon fixation and conversion to fuel are completed together in the cultivation of the cyanobacteria. Cyanobacterial biofuels, therefore, do not rely on costly organic carbon feedstocks that heterotrophs require, which reduces competition for agricultural resources such as arable land and freshwater. However, the published product titer achieved for most molecules of interest using cyanobacteria lag behind what has been achieved using yeast and Escherichia coli (E. coli) cultures. In Synechocystis sp. PCC 6803 (S. 6803), we attempted to increase the product titer of the sesquiterpene, bisabolene, which may be converted to bisabolane, a possible diesel replacement. We tested 19 strains of genetically modified S. 6803 with five different codon usage sequences of the bisabolene synthase from the grand fir tree (Abies grandis). At least three ribosome binding sites (most designed using the RBS Calculator) were tested for each codon usage sequence. We also tested strains with and without the farnesyl pyrophosphate synthase gene from E. coli. Bisabolene titers after five days of growth in continuous light ranged from un-detected to 7.8 mg/L. Bisabolene synthase abundance was measured and found to be well correlated with titer. Select strains were also tested in 12:12 light:dark cycles, where similar titers were reached after the same amount of light exposure time. One engineered strain was also tested in photobioreactors exposed to a simulated outdoor light pattern with maximum light intensity of 1600 μmol photons m−2 s−1. Here, the bisabolene titer reached 22.2 mg/L after 36 days of growth. Dramatic improvements in our ability to control gene expression in cyanobacteria such as S. 6803, and the co-utilization of additional metabolic engineering methods, are needed in order for these titers to improve to the levels reported for engineered E. coli.

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Section: Introductionmentioning

confidence: 99%

Improving heterologous protein expression in Synechocystis sp. PCC 6803 for alpha-bisabolene production

Sebesta

Peebles

2020

Metabolic Engineering Communications

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“…The BLASTP search of hypothetical protein RepC1 showed significant similarity with hypothetical replication protein of Synechocystis sp. PCC 6803 plasmid pCB2.4, which replicated by rolling circle mechanism (Yang and Mc Fadden, 1994, Berlaa and Pakrasi, 2012, Jin et al, 2018). The protein was shown to possess two partial conserved domains.…”

Section: Discussionmentioning

confidence: 99%

Isolation and characterization of two novel plasmids pCYM01 and pCYM02 of Cylindrospermum stagnale

Venkatesan

Raja

Hemaiswarya

et al. 2020

Saudi Journal of Biological Sciences

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Cyanobacteria play a vital role in supplying nitrogen into the soil and aquatic ecosystem. It has an extra chromosomal DNA, whose role is not yet defined well. Isolation and characterization of extra chromosomal DNA in cyanobacteria might help to understand its survival mechanism. Cylindrospermum stagnale isolated (and deposited in NRMCF 3001) from soil showed presence of four plasmids namely pCYLM01, pCYLM02, pCYLM03, and pCYLM04. The following plasmids pCYLM01 and pCYLM02 were subjected to restriction digestion using HindIII restriction enzyme and cloned into pBlueScriptSK(-) vector. The sequence of pCYLM01 contained 4 potential open reading frames (ORFs) that have amino acids in the range of 59–299. Among them, ORF1 shows high sequence homology to the bacterial replication initiator family protein as evident from BLASTP analysis. The analysis of 4359 bp plasmid pCYLM02 sequence revealed 7 ORFs which are longer than 50 amino acids in length. The ORF2 of pCYLM02 has 243 amino acids and is represented in the plasmid sequence from 3045 to 3776 bp. The ORF3 of pCYLM02 corresponds to the plasmid sequence from 2323 to 2976 and codes for a putative protein of 217 amino acids long. A number of small ORFs below 50 bp were also found in the sequence analysis.

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“…When determining the relative copy number of RSF1010 and pCC5.2 based shuttle vectors, results for the RSF1010 based plasmid indicate that it is maintained with a similar copy number to the genome of Synechocystis. In contrast, pCC5.2 is maintained in a 5 to 10 times higher copy number compared to the host genome, depending on the genomic reference gene [44]. These results suggest, that native origins are more favourable for shuttle vector applications with the aim of high expression rates.…”

Section: Dna Introduction and Modificationmentioning

confidence: 91%

“…On the other hand, self-replicating plasmids with different origins of replications as expression platforms were investigated. Here, plasmids harbouring either the heterologous broad-host range origin RSF1010 or the native origins of pCA2.4, pCB2.4 or pCC5.2 were compared with each other [44,45]. Comparing eYFP (enhanced yellow fluorescence protein) fluorescence intensities between RSF1010, pCA2.4 and pCB2.4 based shuttle vectors, 50% higher intensities were reached using the native origins [45].…”

Section: Dna Introduction and Modificationmentioning

confidence: 99%

Evaluation of New Genetic Toolkits and Their Role for Ethanol Production in Cyanobacteria

2019

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Since the public awareness for climate change has risen, increasing scientific effort has been made to find and develop alternative resources and production processes to reduce the dependency on petrol-based fuels and chemicals of our society. Among others, the biotechnological fuel production, as for example fermenting sugar-rich crops to ethanol, is one of the main strategies. For this purpose, various classical production systems like Escherichia coli or Saccharomyces cerevisiae are used and have been optimized via genetic modifications. Despite the progress made, this strategy competes for nutritional resources and agricultural land. To overcome this problem, various attempts were made for direct photosynthetic driven ethanol synthesis with different microalgal species including cyanobacteria. However, compared to existing platforms, the development of cyanobacteria as photoautotrophic cell factories has just started, and accordingly, the ethanol yield of established production systems is still unreached. This is mainly attributed to low ethanol tolerance levels of cyanobacteria and there is still potential for optimizing the cyanobacteria towards alternative gene expression systems. Meanwhile, several improvements were made by establishing new toolboxes for synthetic biology offering new possibilities for advanced genetic modifications of cyanobacteria. Here, current achievements and innovations of those new molecular tools are discussed.

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Construction of a Shuttle Vector Using an Endogenous Plasmid From the Cyanobacterium Synechocystis sp. PCC6803

Cited by 42 publications

References 58 publications

Improving heterologous protein expression in Synechocystis sp. PCC 6803 for alpha-bisabolene production

Improving heterologous protein expression in Synechocystis sp. PCC 6803 for alpha-bisabolene production

Isolation and characterization of two novel plasmids pCYM01 and pCYM02 of Cylindrospermum stagnale

Evaluation of New Genetic Toolkits and Their Role for Ethanol Production in Cyanobacteria

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