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Exploring native genetic elements as plug-in tools for synthetic biology in the cyanobacterium Synechocystis sp. PCC 6803.

ABSTRACT: The unicellular cyanobacterium Synechocystis sp. PCC 6803 has been widely used as a photoautotrophic host for synthetic biology studies. However, as a green chassis to capture CO2 for biotechnological applications, the genetic toolbox for Synechocystis 6803 is still a limited factor.We systematically characterized endogenous genetic elements of Synechocystis 6803, including promoters, ribosome binding sites, transcription terminators, and plasmids. Expression from twelve native promoters was compared by measuring fluorescence from the reporter protein EYFP in an identical setup, exhibiting an 8000-fold range of promoter activities. Moreover, we measured the strength of twenty native ribosome binding sites and eight native terminators, indicating their influence on the expression of the reporter genes. In addition, two shuttle vectors, pCA-UC118 and pCB-SC101, capable of replication in both Synechocystis 6803 and E. coli were constructed. Expression of reporter proteins were significantly enhanced in cells containing these new plasmids, thus providing superior gene expression platforms in this cyanobacterium.The results of this study provide useful and well characterized native tools for bioengineering work in the model cyanobacterium Synechocystis 6803.

SUBMITTER: Liu D

PROVIDER: S-EPMC5868059 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Exploring native genetic elements as plug-in tools for synthetic biology in the cyanobacterium Synechocystis sp. PCC 6803.

Liu Deng D Pakrasi Himadri B HB

Microbial cell factories 20180326 1

<h4>Background</h4>The unicellular cyanobacterium Synechocystis sp. PCC 6803 has been widely used as a photoautotrophic host for synthetic biology studies. However, as a green chassis to capture CO<sub>2</sub> for biotechnological applications, the genetic toolbox for Synechocystis 6803 is still a limited factor.<h4>Results</h4>We systematically characterized endogenous genetic elements of Synechocystis 6803, including promoters, ribosome binding sites, transcription terminators, and plasmids. E ...[more]

PMID: 29580240

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Project description:BackgroundCyanobacteria are phototrophic prokaryotes that convert inorganic carbon as CO2 into organic compounds at the expense of light energy. They need only inorganic nutrients and can be cultivated to high densities using non-arable land and seawater. This has made cyanobacteria attractive organisms for the production of biofuels and chemical feedstock. Synechocystis sp. PCC 6803 is one of the most widely used cyanobacterial model strains. Based on its available genome sequence and genetic tools, Synechocystis has been genetically modified to produce different biotechnological products. Efficient isoprene production is an attractive goal because this compound is widely used as chemical feedstock.ResultsHere, we report on our attempts to generate isoprene-producing strains of Synechocystis using a plasmid-based strategy. As previously reported, a codon-optimized plant isoprene synthase (IspS) was expressed under the control of different Synechocystis promoters that ensure strong constitutive or light-regulated ispS expression. The expression of the ispS gene was quantified by qPCR and Western blotting, while the amount of isoprene was quantified using GC-MS. In addition to isoprene measurements in the headspace of closed culture vessels, single photon ionization time-of-flight mass spectrometry (SPI-MS) was applied, which allowed online measurements of isoprene production in open-cultivation systems under various conditions. Under standard conditions, a good correlation existed between ispS expression and isoprene production rate. The cultivation of isoprene production strains under NaCl-supplemented conditions decreased isoprene production despite enhanced ispS mRNA levels. The characterization of the metabolome of isoprene-producing strains indicated that isoprene production might be limited by insufficient precursor levels. Transcriptomic analysis revealed the upregulation of mRNA and regulatory RNAs characteristic of acclimation to metabolic stress.ConclusionsOur best production strains produced twofold higher isoprene amounts in the presence of low NaCl concentrations than previously reported strains. These results will guide future attempts to establish isoprene production in cyanobacterial hosts.

Dataset Information

Exploring native genetic elements as plug-in tools for synthetic biology in the cyanobacterium Synechocystis sp. PCC 6803.

Publications

Exploring native genetic elements as plug-in tools for synthetic biology in the cyanobacterium Synechocystis sp. PCC 6803.

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