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Evaluation of promoters and ribosome binding sites for biotechnological applications in the unicellular cyanobacterium Synechocystis sp. PCC 6803.

ABSTRACT: For effective metabolic engineering, a toolbox of genetic components that enables predictable control of gene expression is needed. Here we present a systematic study of promoters and ribosome binding sites in the unicellular cyanobacterium Synechocystis sp. PCC 6803. A set of metal ion inducible promoters from Synechocystis were compared to commonly used constitutive promoters, by measuring fluorescence of a reporter protein in a standardized setting to allow for accurate comparisons of promoter activity. The most versatile and useful promoter was found to be PnrsB, which from a relatively silent expression could be induced almost 40-fold, nearly up to the activity of the strong psbA2 promoter. By varying the concentrations of the two metal ion inducers Ni2+ and Co2+, expression from the promoter was highly tunable, results that were reproduced with PnrsB driving ethanol production. The activities of several ribosomal binding sites were also measured, and tested in parallel in Synechocystis and Escherichia coli. The results of the study add useful information to the Synechocystis genetic toolbox for biotechnological applications.

SUBMITTER: Englund E

PROVIDER: S-EPMC5114575 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Evaluation of promoters and ribosome binding sites for biotechnological applications in the unicellular cyanobacterium Synechocystis sp. PCC 6803.

Englund Elias E Liang Feiyan F Lindberg Pia P

Scientific reports 20161118

For effective metabolic engineering, a toolbox of genetic components that enables predictable control of gene expression is needed. Here we present a systematic study of promoters and ribosome binding sites in the unicellular cyanobacterium Synechocystis sp. PCC 6803. A set of metal ion inducible promoters from Synechocystis were compared to commonly used constitutive promoters, by measuring fluorescence of a reporter protein in a standardized setting to allow for accurate comparisons of promote ...[more]

PMID: 27857166

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Project description:Cyanophycin is a carbon/nitrogen storage polymer widely distributed in most cyanobacterial strains and in a few heterotrophic bacteria. It is a nonribosomal polypeptide consisting of equimolar amounts of aspartate and arginine. Here, we focused on the physiological function and cell biology of cyanophycin in the unicellular nondiazotrophic cyanobacterium Synechocystis sp. strain PCC 6803. To study the cellular localization of the cyanophycin-synthesizing enzyme CphA during cyanophycin synthesis and degradation, we fused it to green fluorescent protein. When CphA was inactive, it localized diffusely in the cytoplasm. When cyanophycin synthesis was triggered, CphA first aggregated into foci and later localized on the surface of cyanophycin granules. In the corresponding cell extracts, localization of CphA on the cyanophycin granule surface required Mg2+ During cyanophycin degradation, CphA dissociated from the granule surface and returned to its inactive form in the cytoplasm. To investigate the physiological role of cyanophycin, we compared wild-type cells with a CphA-deficient mutant. Under standard laboratory conditions, the ability to synthesize cyanophycin did not confer a growth advantage. To mimic the situation in natural habitats, cells were cultured with a fluctuating and limiting nitrogen supplementation and/or day/night cycles. Under all of these conditions, cyanophycin provided a fitness advantage to the wild type over the mutant lacking cyanophycin. During resuscitation from nitrogen starvation, wild-type cells accumulated cyanophycin during the night and used it as an internal nitrogen source during the day. This demonstrates that cyanophycin can be used as a temporary nitrogen storage to uncouple nitrogen assimilation from photosynthesis.IMPORTANCE We clarified the elusive biological function of cyanophycin in the nondiazotrophic cyanobacterium Synechocystis sp. PCC 6803. Cyanophycin is a dynamic carbon/nitrogen storage polymer (multi-arginyl-l-polyaspartate) that is conditionally present in most cyanobacteria and a few heterotrophic bacteria as cellular inclusion granules. Here, we show that the cyanophycin-synthesizing enzyme CphA in the nonactive state localizes diffusely in the cytoplasm. When cyanophycin synthesis is triggered, active CphA first aggregates into foci and then covers the surface of mature cyanophycin granules, which in vitro requires Mg2+ as a cofactor. Cyanophycin accumulation enables Synechocystis sp. to optimize nitrogen assimilation under nitrogen-poor conditions, in particular when the nitrogen supply fluctuates and during day/night cycles, by allowing continuous nitrogen assimilation and storage. Therefore, cyanophycin provides the wild-type cyanobacterium with a clear fitness advantage over non-cyanophycin-producing cells in natural environments with fluctuating nitrogen supply.

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

Evaluation of promoters and ribosome binding sites for biotechnological applications in the unicellular cyanobacterium Synechocystis sp. PCC 6803.

Publications

Evaluation of promoters and ribosome binding sites for biotechnological applications in the unicellular cyanobacterium Synechocystis sp. PCC 6803.

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