ABSTRACT
BACKGROUND: Cyanobacteria have emerged as highly efficient organisms for the production of chemicals and biofuels. Yet, the productivity of the cell has been low for commercial application. Cyanobacterial photobiotransformations utilize photosynthetic electrons to form reducing equivalents, such as NADPH-to-fuel biocatalytic reactions. These photobiotransformations are a measure to which extent photosynthetic electrons can be deviated toward heterologous biotechnological processes, such as the production of biofuels. By expressing oxidoreductases, such as YqjM from Bacillus subtilis in Synechocystis sp. PCC 6803, a high specific activity was obtained in the reduction of maleimides. Here, we investigated the possibility to accelerate the NAD(P)H-consuming redox reactions by addition of carbohydrates as exogenous carbon sources such as D-Glucose under light and darkness. RESULTS: A 1.7-fold increase of activity (150 µmol min-1 gDCW-1) was observed upon addition of D-Glucose at an OD750 = 2.5 (DCW = 0.6 g L-1) in the biotransformation of 2-methylmaleimide. The stimulating effect of D-Glucose was also observed at higher cell densities in light and dark conditions as well as in the reduction of other substrates. No increase in both effective photosynthetic yields of Photosystem II and Photosystem I was found upon D-Glucose addition. However, we observed higher NAD(P)H fluorescence when D-Glucose was supplemented, suggesting increased glycolytic activity. Moreover, the system was scaled-up (working volume of 200 mL) in an internally illuminated Bubble Column Reactor exhibiting a 2.4-fold increase of specific activity under light-limited conditions. CONCLUSIONS: Results show that under photoautotrophic conditions at a specific activity of 90 µmol min-1 gDCW-1, the ene-reductase YqjM in Synechocystis sp. PCC 6803 is not NAD(P)H saturated, which is an indicator that an increase of the rates of heterologous electron consuming processes for catalysis and biofuel production will require funnelling further reducing power from the photosynthetic chain toward heterologous processes.
ABSTRACT
The microalga Nannochloropsis oceanica is considered a promising platform for the sustainable production of high-value lipids and biofuel feedstocks. However, current lipid yields of N. oceanica are too low for economic feasibility. Gaining fundamental insights into the lipid metabolism of N. oceanica could open up various possibilities for the optimization of this species through genetic engineering. Therefore, the aim of this study was to discover novel genes associated with an elevated neutral lipid content. We constructed an insertional mutagenesis library of N. oceanica, selected high lipid mutants by five rounds of fluorescence-activated cell sorting, and identified disrupted genes using a novel implementation of a rapid genotyping procedure. One particularly promising mutant (HLM23) was disrupted in a putative APETALA2-like transcription factor gene. HLM23 showed a 40%-increased neutral lipid content, increased photosynthetic performance, and no growth impairment. Furthermore, transcriptome analysis revealed an upregulation of genes related to plastidial fatty acid biosynthesis, glycolysis and the Calvin-Benson-Bassham cycle in HLM23. Insights gained in this work can be used in future genetic engineering strategies for increased lipid productivity of Nannochloropsis.
