The use of ascomycetous fungi as pigment producers opens the way to an alternative to synthetic dyes, especially in the red-dye industries, which have very few natural pigment alternatives. The present paper aimed to bio-prospect and screen out 15 selected ascomycetous fungal strains, originating from terrestrial and marine habitats belonging to seven different genera (Penicillium, Talaromyces, Fusarium, Aspergillus, Trichoderma, Dreschlera, and Paecilomyces). We identified four strains, Penicillium purpurogenum rubisclerotium, Fusarium oxysporum, marine strains identified as Talaromyces spp., and Trichoderma atroviride, as potential red pigment producers. The extraction of the pigments is a crucial step, whereby the qualitative and quantitative compositions of each fungal extract need to be respected for reliable identification, as well as preserving bioactivity. Furthermore, there is a growing demand for more sustainable and cost-effective extraction methods. Therefore, a pressurized liquid extraction technique was carried out in this study, allowing a greener and faster extraction step of the pigments, while preserving their chemical structures and bioactivities in comparison to conventional extraction processes. The protocol was illustrated with the production of pigment extracts from P. purpurogenum rubisclerotium and Talaromyces spp. Extracts were analyzed by high-performance liquid-chromatography combined with photodiode array-detection (HPLC-DAD) and high-resolution mass spectrometry (UHPLC-HRMS). The more promising strain was the isolate Talaromyces spp. of marine origin. The main polyketide pigment produced by this strain has been characterized as N-threoninerubropunctamine, a non-toxic red Monascus-like azaphilone pigment.

Demand for microbial colorants is now becoming a competitive research topic for food, cosmetics and pharmaceutics industries. In most applications, the pigments of interest such as polyketide-based red pigments from fungal submerged cultures are extracted by conventional liquid–liquid extraction methods requiring large volumes of various organic solvents and time. To address this question from a different angle, we proposed, here, to investigate the use of three different aqueous two-phase extraction systems using either ammonium- or imidazolium-based ionic liquids. We applied these to four fermentation broths of Talaromyces albobiverticillius (deep red pigment producer), Emericella purpurea (red pigment producer), Paecilomyces marquandii (yellow pigment producer) and Trichoderma harzianum (yellow-brown pigment producer) to investigate their selective extraction abilities towards the detection of polyketide-based pigments. Our findings led us to conclude that (i) these alternative extraction systems using ionic liquids as greener extractant means worked well for this extraction of colored molecules from the fermentation broths of the filamentous fungi investigated here; (ii) tetrabutylammonium bromide, [N4444]Br-, showed the best pigment extraction ability, with a higher putative affinity for azaphilone red pigments; (iii) the back extraction and recovery of the fungal pigments from ionic liquid phases remained the limiting point of the method under our selected conditions for potential industrial applications. Nevertheless, these alternative extraction procedures appeared to be promising ways for the detection of polyketide-based colorants in the submerged cultures of filamentous fungi.

We demonstrate the use of two-stage dynamic metabolic control to manipulate feedback regulation in central metabolism and improve stationary phase biosynthesis in engineered E. coli . Specifically, we report the impact of dynamic control over two enzymes: citrate synthase, and glucose-6-phosphate dehydrogenase, on stationary phase fluxes. Firstly, reduced citrate synthase levels lead to a reduction in α -ketoglutarate, which is an inhibitor of sugar transport, resulting in increased stationary phase glucose uptake and glycolytic fluxes. Reduced glucose-6-phosphate dehydrogenase activity activates the SoxRS regulon and expression of pyruvate-ferredoxin oxidoreductase, which is in turn responsible for large increases in acetyl-CoA production. The combined reduction in citrate synthase and glucose-6-phosphate dehydrogenase, leads to greatly enhanced stationary phase metabolism and the improved production of citramalic acid enabling titers of 126±7g/L. These results identify pyruvate oxidation via the pyruvate-ferredoxin oxidoreductase as a “central” metabolic pathway in stationary phase E. coli , which coupled with ferredoxin reductase comprise a pathway whose physiologic role is maintaining NADPH levels. Highlights: Dynamic reduction in α -keto-glutarate pools alleviate inhibition of PTS dependent transport improving stationary phase sugar uptake. Dynamic reduction in glucose-6-phosphate dehydrogenase activates pyruvate flavodoxin/ferredoxin oxidoreductase and improves stationary acetyl-CoA flux. Pyruvate flavodoxin/ferredoxin oxidoreductase is responsible for large stationary phase acetyl-CoA fluxes under aerobic conditions. Production of citramalate to titers 126 ± 7g/L at > 90 % of theoretical yield.

We report improved strain and bioprocess robustness as a result of the dynamic deregulation of central metabolism using two-stage dynamic control. Dynamic control is implemented using combinations of CRISPR interference and controlled proteolysis to reduce levels of central metabolic enzymes in the context of a standardized two-stage bioprocesses. Reducing the levels of key enzymes alters metabolite pools resulting in deregulation of the metabolic network. The deregulated network is more robust to environmental conditions improving process robustness, which in turn leads to predictable scalability from high throughput small scale screens to fully instrumented bioreactors as well as to pilot scale production. Additionally, as these two-stage bioprocesses are standardized, a need for traditional process optimization is minimized. Predictive high throughput approaches that translate to larger scales are critical for metabolic engineering programs to truly take advantage of the rapidly increasing throughput and decreasing costs of synthetic biology. In this work we demonstrate that the improved robustness of E. coli strains engineered for the improved scalability of the important industrial chemicals alanine, citramalate and xylitol, from microtiter plates to pilot reactors.