Insights into isoprene production using the cyanobacterium Synechocystis sp. PCC 6803

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0503-4) contains supplementary material, which is available to authorized users.

Bio-production of gaseous alkenes: ethylene, isoprene, isobutene

“…For example, researchers have codon-optimized ispS for expression in Synechocystis sp. PCC6803 [ 53 ], removed the chloroplast targeting peptide [ 48 , 54 ], site mutagenesis [ 55 ], and performed directed evolution [ 56 ]. Additionally, to overcome the low substrate affinity of ISPS for DMAPP, a fusion protein of overexpression of S. cerevisiae IDI and P. alba ISPS was generated.…”

Cyanobacteria as Chassis for Industrial Biotechnology: Progress and Prospects

“…Metabolomics, transcriptomics and proteomics have been used to improve biofuel tolerances in Synechocystis 6803, which provided valuable correlations between genes, protein expression and production titers of different biotechnological products [63,64]. In a study on isoprene production from Synechocystis 6803, the metabolomic and transcriptomic profiles were analysed under metabolic stress [64].…”

“…In a study on isoprene production from Synechocystis 6803, the metabolomic and transcriptomic profiles were analysed under metabolic stress [64]. The metabolomic profile suggested that the limiting factor of isoprene production under stress might be due to an insufficient precursor level, while the transcriptome revealed an upregulation of RNA characteristic of acclimation to stress [64].…”

“…In a study on isoprene production from Synechocystis 6803, the metabolomic and transcriptomic profiles were analysed under metabolic stress [64]. The metabolomic profile suggested that the limiting factor of isoprene production under stress might be due to an insufficient precursor level, while the transcriptome revealed an upregulation of RNA characteristic of acclimation to stress [64]. Tolerance of Synechocystis 6803 towards other products, like butanol, was investigated using transcriptomics and it was revealed that the gene encoding HspA, a small heat shock protein, was induced when Synechocystis 6803 was grown in the presence of butanol.…”

Optimizing cyanobacterial product synthesis: Meeting the challenges

“…As shown in studies of intracellular changes of Synechocystis sp. PCC 6803 during ethylene 20 or isoprene production, 21 recent approaches increasingly incorporate quantitative analysis of cellular parameters.…”