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


RESULTS
Here, 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.


CONCLUSIONS
Our 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.
Plant Physiology Department, Institute of Biological Science, University of Rostock, Albert-Einstein-Str. 3, 18059 Rostock, Germany.
Nadin
N
Pade
Analytic Chemistry Department, University of Rostock, Dr.-Lorenz-Weg 1, 18059 Rostock, Germany.
Sabrina
S
Erdmann
Algenol Biofuels Germany GmbH, Magnusstr. 11, 12489 Berlin, Germany.
Heike
H
Enke
Department of Molecular Physiology, Applied Metabolome Analysis, Max-Planck-Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany.
Frederik
F
Dethloff
Algenol Biofuels Germany GmbH, Magnusstr. 11, 12489 Berlin, Germany.
Ulf
U
Dühring
Genetics & Experimental Bioinformatics, Institute of Biology III, University of Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany.
Jens
J
Georg
Algenol Biofuels Germany GmbH, Magnusstr. 11, 12489 Berlin, Germany.
Juliane
J
Wambutt
Department of Molecular Physiology, Applied Metabolome Analysis, Max-Planck-Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany.
Joachim
J
Kopka
Genetics & Experimental Bioinformatics, Institute of Biology III, University of Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany.
Wolfgang R
WR
Hess
Analytic Chemistry Department, University of Rostock, Dr.-Lorenz-Weg 1, 18059 Rostock, Germany.
Ralf
R
Zimmermann
Algenol Biofuels Germany GmbH, Magnusstr. 11, 12489 Berlin, Germany.
Dan
D
Kramer
Plant Physiology Department, Institute of Biological Science, University of Rostock, Albert-Einstein-Str. 3, 18059 Rostock, Germany.
Martin
M
Hagemann
Cyanobacteria; Glucosylglycerol; Isoprene; Metabolome; Promoter; Salinity; Transcriptome