Transcriptional profiling and flux measurements of polyhydroxybutyrate production in Synechocystis

Abstract

(cont.) to determine the CO₂ uptake rates and PHB production rates of strains engineered for enhanced CO₂ fixation and PHB production respectively.

The metabolism of Synechocystis PCC6803 cells has been investigated using full-genome DNA micro-arrays and C14 tracer techniques. Full-genome (3169 genes) DNA micro-arrays were used to probe transcript levels of Synechocystis cells grown under a variety of medium conditions. Canonical discriminant analysis was used to identify transcript levels that allowed discrimination between growth media conditions, and allowed predictions of polyhydroxybutyrate (PHB) levels. Phosphate-related genes were found to alter in response to phosphate limitation and were found to include differentially regulated multi-gene families. Nitrogen-related genes were not found to be substantially reflective of nitrogen limitation under the conditions studied. Finally, transcription of PHA biosynthetic pathway genes were found to reflect the media conditions of greatest PHB accumulation, suggesting that constitutive over-expression of the PHA biosynthetic genes may lead to greater PHB accumulation levels. A methodology using C14 tracers was developed for the accurate measurement of CO₂ uptake rates and the partitioning of the fixed carbon into different biosynthetic fractions. These techniques were applied to the characterization of WT Synechocystis cells in late exponential phase. A stoichiometric model of Synechocystis metabolism was used to determine constraints between the measurements. A balance on C14 counts was obtained and significant levels of secreted compounds were not detected. The measured carbon fixation rates were found to be consistent with the observed growth rates, but inconsistent with measurements of oxygen evolution in the light and uptake in the dark made using a Clarke Electrode apparatus. These techniques may be used in future studies