Microbial conversion of biomass to fatty acids (FA) and products derived thereof is an attractive alternative to the traditional oleochemical production route from animal and plant lipids. This study examined if NADPH-costly FA biosynthesis could be enhanced by overexpressing the transcription factor Stb5 in Saccharomyces cerevisiae. Stb5 activates expression of multiple genes encoding enzymes within the pentose phosphate pathway (PPP) and other NADPH-producing reactions. Overexpression of STB5 led to a decreased growth rate and an increased free fatty acid (FFA) production during growth on glucose. The improved FFA synthetic ability in the glucose phase was shown to be independent of flux through the oxidative PPP. RNAseq analysis revealed that STB5 overexpression had wide-ranging effects on the transcriptome in the batch phase, and appeared to cause a counterintuitive phenotype with reduced flux through the oxidative PPP. During glucose limitation, when an increased NADPH supply is likely less harmful, an overall induction of the proposed target genes of Stb5 (eg. GND1/2, TAL1, ALD6, YEF1) was observed. Taken together, the strategy of utilizing STB5 overexpression to increase NADPH supply for reductive biosynthesis is suggested to have potential in strains engineered to have strong ability to consume excess NADPH, alleviating a potential redox imbalance.

Metabolically engineered cyanobacteria have the potential to mitigate anthropogenic CO2 emissions by converting CO2 into renewable fuels and chemicals. Yet, better understanding of metabolic regulation in cyanobacteria is required to develop more productive strains that can make industrial scale-up economically feasible. The aim of this study was to find the cause for the previously reported inconsistency between oscillating transcription and constant protein levels under day-night growth conditions. To determine whether translational regulation counteracts transcriptional changes, Synechocystis sp. PCC 6803 was cultivated in an artificial day-night setting and the level of transcription, translation and protein was measured across the genome at different time points using mRNA sequencing, ribosome profiling and quantitative proteomics. Furthermore, the effect of protein turnover on the amplitude of protein oscillations was investigated through in silico simulations using a protein mass balance model. Our experimental analysis revealed that protein oscillations were not dampened by translational regulation, as evidenced by high correlation between translational and transcriptional oscillations (r = 0.88) and unchanged protein levels. Instead, model simulations showed that these observations can be attributed to a slow protein turnover, which reduces the effect of protein synthesis oscillations on the protein level. In conclusion, these results suggest that cyanobacteria have evolved to govern diurnal metabolic shifts through allosteric regulatory mechanisms in order to avoid the energy burden of replacing the proteome on a daily basis. Identification and manipulation of such mechanisms could be part of a metabolic engineering strategy for overproduction of chemicals.

The importance of biofuels is growing for both environmental and economic point of view. The usage of wastes in bio-ethanol production is investigated in our paper. Cellulose contained wastes i.e. byproduct of tobacco is used for experiments. The paper is focused mainly on the investigation of the enzyme recovery that followed the saccharification process. The membrane filtration combined with sonication was used for enzyme recovery. Our results show that recovered enzyme did not lose its activity. The patterns were originated from fermented solutions made from tobacco byproducts. Based on our results, the ultrasound accompanied by a membrane separation would be a possible and effective operation of bio-ethanol production technology.