Plant triterpenoids constitute a diverse class of organic compounds that play a major role in development, plant defence and environmental interaction. Several triterpenes have demonstrated potential as pharmaceuticals. One example is betulin, which has shown promise as a pharmaceutical precursor for the treatment of certain cancers and HIV. Major challenges for triterpenoid commercialization include their low production levels and their cost-effective purification from the complex mixtures present in their natural hosts. Therefore, attempts to produce these compounds in industrially relevant microbial systems such as bacteria and yeasts have attracted great interest. Here, we report the production of the triterpenes betulin and its precursor lupeol in the photosynthetic diatom Phaeodactylum tricornutum, a unicellular eukaryotic alga. This was achieved by introducing three plant enzymes in the microalga: a Lotus japonicus oxidosqualene cyclase and a Medicago truncatula cytochrome P450 along with its native reductase. The introduction of the L. japonicus oxidosqualene cyclase perturbed the mRNA expression levels of the native mevalonate and sterol biosynthesis pathway. The best performing strains were selected and grown in a 550-L pilot-scale photobioreactor facility. To our knowledge, this is the most extensive pathway engineering undertaken in a diatom and the first time that a sapogenin has been artificially produced in a microalga, demonstrating the feasibility of the photo-bio-production of more complex high-value, metabolites in microalgae.
15The genetic modification of microalgal strains for enhanced or modified metabolic activity 16 shows great promise for biotechnological exploitation. However, of key concern for many is the 17 safety of genetic modification technology and genetically modified organisms with regard to 18 both the environment and human health, and how these concerns are met will play a key role in 19 ensuring how successful commercialisation of genetically modified (GM) algae is achieved. 20Commercialisation opportunities for GM microalgae will inevitably require translation from
HighlightsEMS and UV mutagenesis of Nannochloropsis salina combined with FACS for mutant enrichment.Productivity of EMS mutants increased by 76% and showed range of FA profile changes.Dual EMS and UV mutants accumulated 3 fold more lipid than the wild type.Elevation in lipid content comes with a cost to growth rate impacting productivity.Mutants suitable for divergent industries generated (biofuel, high value PUFA production).
Highlights Hydrothermal liquefaction conditions were optimised for bio-crude production and nutrient recovery using the macroalga A. nodosum. Using the optimised conditions (345 °C; 30 °C min -1), liquefaction of 13 South West UK macroalgae species were carried out. Bio-crude yields of up to 29.9 % were obtained for HTL of U. lactuca. Phosphate levels of up to 236 ppm were detected in the aqueous phase products for HTL of S. chordalis. Biochemical compositions were not a clear predictor of product distribution. Varying particle size (between <125 μm and 1700 μm) did not have a strong effect on bio-crude recovery.
Microalgae are generating considerable interest for third generation biodiesel production. However, appropriate strain selection is proving challenging due to the significant variation in cellular physiology, metabolic potential and genetics observed even amongst strains deemed morphologically similar. Six strains of Nannochloropsis from the CCAP culture collection were assessed for their lipid productivity and cellular structure, as proxies for oil production and harvesting ease, to assess their suitability as biodiesel production platforms. Differences in growth rate and lipid accumulation across the strains were observed. N. oculata strain 849/7 showed significantly reduced doubling time compared to N. salina strain 849/3, whilst N. oceanica 849/10 produced the highest lipid content. In addition the six strains could be differentiated in to 3 distinct classes based on their cell wall thickness, which varied across the strains from 63-119 nm and which is independent of both species and geographical isolation location. The importance of these variations in ultrastructure and physiology for biodiesel production is discussed.
In this investigation, PHB producing cyanobacteria were converted through hydrothermal liquefaction (HTL) into propylene and a bio--oil suitable for advanced biofuel production. HTL of model compounds demonstrated that in contrast to proteins and carbohydrates, which react to produce a range of alternative intermediates, no synergistic effects were detected when converting PHB in the presence of algal biomass. Subsequently, Synechocystis cf. salina, which had accumulated 7.5 wt% PHB was converted via HTL (15 % dry weight loading at 340 °C). The reaction gave an overall propylene yield of 2.6 %, higher than that obtained from the analogous model compounds, in addition to a bio--oil with a low total nitrogen content of 4.6 %. No propylene was recovered from the alternative non--PHB producing cyanobacterial strains, Anabaena, Spirulina or Synechococcus, suggesting that PHB is the sole source of propylene. PHB producing microorganisms could therefore be used as a feedstock for a biorefinery to produce polypropylene and advanced biofuels, with the level of propylene being directly proportional to the accumulated amount of PHB.
The RSC chromatin remodeling complex has been implicated in contributing to DNA double-strand break (DSB) repair in a number of studies. Both survival and levels of H2A phosphorylation in response to damage are reduced in the absence of RSC. Importantly, there is evidence for two isoforms of this complex, defined by the presence of either Rsc1 or Rsc2. Here, we investigated whether the two isoforms of RSC provide distinct contributions to DNA damage responses. First, we established that the two isoforms of RSC differ in the presence of Rsc1 or Rsc2 but otherwise have the same subunit composition. We found that both rsc1 and rsc2 mutant strains have intact DNA damage-induced checkpoint activity and transcriptional induction. In addition, both strains show reduced non-homologous end joining activity and have a similar spectrum of DSB repair junctions, suggesting perhaps that the two complexes provide the same functions. However, the hypersensitivity of a rsc1 strain cannot be complemented with an extra copy of RSC2, and likewise, the hypersensitivity of the rsc2 strain remains unchanged when an additional copy of RSC1 is present, indicating that the two proteins are unable to functionally compensate for one another in DNA damage responses. Rsc1, but not Rsc2, is required for nucleosome sliding flanking a DNA DSB. Interestingly, while swapping the domains from Rsc1 into the Rsc2 protein does not compromise hypersensitivity to DNA damage suggesting they are functionally interchangeable, the BAH domain from Rsc1 confers upon Rsc2 the ability to remodel chromatin at a DNA break. These data demonstrate that, despite the similarity between Rsc1 and Rsc2, the two different isoforms of RSC provide distinct functions in DNA damage responses, and that at least part of the functional specificity is dictated by the BAH domains.
Here we report recombinant expression and activity of the Saccharomyces cerevisiae type 2 diacylglycerol acyltransferase DGA1 functioning in parallel with the native Nannochloropsis salina genes. Expression of DGA1 shifted the chain length distribution of fatty acids produced and reflected an oleoyl-CoA substrate preference. Effect on the total FAME content was moderate and elevated by a maximum of 38%. Expression of the DGA1 transgene varied throughout the culture life cycle and evidence of growth dependent environmental silencing of the transgene was observed. This is to our knowledge the first example of silencing and subsequent resetting in a transgenic microalga. Results from this study add valuable insights into the efficacy of algal genetic engineering and use of these microorganisms as bio-platforms for chemical manufacture.
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