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.

No abstract

No abstract

The Calvin-Benson-Bassham (CBB) cycle is the main pathway to fix atmospheric CO and store energy in carbon bonds, forming the precursors of most primary and secondary metabolites necessary for life. Speeding up the CBB cycle theoretically has positive effects on the subsequent growth and/or the end metabolite(s) production. Four CBB cycle enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase), transketolase (TK) and aldolase (FBA) were selected to be co-overexpressed with the ethanol synthesis enzymes pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) in the cyanobacterium Synechocystis PCC 6803. An inducible promoter, PnrsB, was used to drive PDC and ADH expression. When PnrsB was induced and cells were cultivated at 65 µmol photons m s, the RuBisCO-, FBP/SBPase-, TK-, and FBA-expressing strains produced 55%, 67%, 37% and 69% more ethanol and 7.7%, 15.1%, 8.8% and 10.1% more total biomass (the sum of dry cell weight and ethanol), respectively, compared to the strain only expressing the ethanol biosynthesis pathway. The ethanol to total biomass ratio was also increased in CBB cycle enzymes overexpressing strains. This study experimentally demonstrates that using the cells with enhanced carbon fixation, when the product synthesis pathway is not the main bottleneck, can significantly increase the generation of a product (exemplified with ethanol), which acts as a carbon sink.

The ribulose-1,5-bisphosphate (RuBP) oxygenation reaction catalyzed by Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is competing with carboxylation, being negative for both energy and carbon balances in photoautotrophic organisms. This makes RuBisCO one of the bottlenecks for oxygenic photosynthesis and carbon fixation. In this study, RuBisCO was overexpressed in the unicellular cyanobacterium Synechocystis PCC 6803. Relative RuBisCO levels in the engineered strains FL50 and FL52 increased 2.1 times and 1.4 times, respectively, and both strains showed increased growth, photosynthesis and in vitro RuBisCO activity. The oxygen evolution rate increased by 54% and 42% on per chlorophyll basis, while the in vitro RuBisCO activity increased by 52% and 8.6%, respectively. The overexpressed RuBisCO were tagged with a FLAG tag, in strain FL50 on the N terminus of the large subunit while in strain FL52 on the C terminus of the small subunit. The presence of a FLAG tag enhanced transcription of the genes encoding RuBisCO, and, with high possibility, also enhanced the initiation of translation or stability of the enzyme. However, when using a streptavidin-binding tag II (strep-tag II), we did not observe a similar effect. Tagged RuBisCO offers an opportunity for further studying RuBisCO expression and stability. Increased levels of RuBisCO can further improve photosynthesis and growth in the cyanobacterium Synechocystis PCC 6803 under certain growth conditions.

No abstract

This review discusses strategies to enhance oxygenic photoautotrophic carbon fixation with a focus on the Calvin–Benson–Bassham cycle.

Due to efficient photosynthetic capability, robust growth, and clear genetic background, cyanobacteria are recently used for production of different biofuel and biochemical molecules by genetic engineering and showed great potentials as the next-generation microbial cell factory. For improving the production of bio-products, a number of genetic modifications are important for cyanobacteria. However, the system-level genetic modification of cyanobacteria is limited by the lack of efficient method for marker recycling. In this investigation, we introduced the self-replicable shutter vectors harboring the flipase (FLP) gene from Saccharomyces cerevisiae into two mutants of Synechocystis sp. PCC6803 and Synechococcus elongatus PCC7942 whose genomes were inserted by a kanamycin resistance gene with flipase recombination target (FRT) flanking, respectively. Transcriptional analysis by reverse transcription polymerase chain reaction showed that FLP gene was transcripted in both the two cyanobacterial strains. Genotyping analysis indicated that FLP performed its function in vivo in both two cyanobacterial strains, and the following DNA sequencing analysis on the targeted loci further confirmed that FLP exactly excised and ligated the two FRT sites between which a kanamycin resistance gene is located. The homozygous mutants free of the kanamycin resistance gene cassette were obtained by conditional expression of FLP and further dilution plating. The shuttle vectors carrying the FLP gene were then lost in these mutants by growing in the absence of antibiotics and the further single colony separation. These results demonstrate that FLP/FRT recombination system is able to be applied to the construction of markerless mutant in both Synechocystis sp. PCC6803 and S. elongatus PCC7942.