Squalene production under oxygen limitation by Schizochytrium sp. S31 in different cultivation systems.

The triterpene squalene is widely used in the food, cosmetics and pharmaceutical industries due to its antioxidant, antistatic and anti-carcinogenic properties. It is usually obtained from the liver of deep sea sharks, which are facing extinction. Alternative production organisms are marine protists from the family Thraustochytriaceae, which produce and store large quantities of various lipids. Squalene accumulation in thraustochytrids is complex, as it is an intermediate in sterol biosynthesis. Its conversion to squalene 2,3-epoxide is the first step in sterol synthesis and is heavily oxygen dependent. Hence, the oxygen supply during cultivation was investigated in our study. In shake flask cultivations, a reduced oxygen supply led to increased squalene and decreased sterol contents and yields. Oxygen-limited conditions were applied to bioreactor scale, where squalene accumulation and growth of Schizochytrium sp. S31 was determined in batch, fed-batch and continuous cultivation. The highest dry matter (32.03 g/L) was obtained during fed-batch cultivation, whereas batch cultivation yielded the highest biomass productivity (0.2 g/L*h-1). Squalene accumulation benefited from keeping the microorganisms in the growth phase. Therefore, the highest squalene content of 39.67 ± 1.34 mg/g was achieved by continuous cultivation (D = 0.025 h-1) and the highest squalene yield of 1131 mg/L during fed-batch cultivation. Volumetric and specific squalene productivity both reached maxima in the continuous cultivation at D = 0.025 h-1 (6.94 ± 0.27 mg/L*h-1 and 1.00 ± 0.03 mg/g*h-1, respectively). Thus, the choice of a suitable cultivation method under oxygen-limiting conditions depends heavily on the process requirements. KEY POINTS: • Measurements of respiratory activity and backscatter light of thraustochytrids • Oxygen limitation increased squalene accumulation in Schizochytrium sp. S31 • Comparison of different cultivation methods under oxygen-limiting conditions.

Co-Immobilization of RizA Variants with Acetate Kinase for the Production of Bioactive Arginyl Dipeptides.

The biocatalytic system comprised of RizA and acetate kinase (AckA) combines the specific synthesis of bioactive arginyl dipeptides with efficient ATP regeneration. Immobilization of this coupled enzyme system was performed and characterized in terms of activity, specificity and reusability of the immobilisates. Co-immobilization of RizA and AckA into a single immobilisate conferred no disadvantage in comparison to immobilization of only RizA, and a small addition of AckA (20:1) was sufficient for ATP regeneration. New variants of RizA were constructed by combining mutations to yield variants with increased biocatalytic activity and specificity. A selection of RizA variants were co-immobilized with AckA and used for the production of the salt-taste enhancers Arg-Ser and Arg-Ala and the antihypertensive Arg-Phe. The best variants yielded final dipeptide concentrations of 11.3 mM Arg-Ser (T81F_A158S) and 11.8 mM Arg-Phe (K83F_S156A), the latter of which represents a five-fold increase in comparison to the wild-type enzyme. T81F_A158S retained more than 50% activity for over 96 h and K83F_S156A for over 72 h. This study provides the first example of the successful co-immobilization of an l-amino acid ligase with an ATP-regenerating enzyme and paves the way towards a bioprocess for the production of bioactive dipeptides.

Co-Oxidative Transformation of Piperine to Piperonal and 3,4-Methylenedioxycinnamaldehyde by a Lipoxygenase from Pleurotus sapidus.

The valuable aroma compound piperonal with its vanilla-like olfactory properties is of high interest for the fragrance and flavor industry. A lipoxygenase (LOXPsa 1) of the basidiomycete Pleurotus sapidus was identified to convert piperine, the abundant pungent principle of black pepper (Piper nigrum), to piperonal and a second volatile product, 3,4-methylenedioxycinnamaldehyde, with a vanilla-like odor through an alkene cleavage. The reaction principle was co-oxidation, as proven by its dependence on the presence of linoleic or α-linolenic acid, common substrates of lipoxygenases. Optimization of the reaction conditions (substrate concentrations, reaction temperature and time) led to a 24-fold and 15-fold increase of the piperonal and 3,4-methylenedioxycinnamaldehyde concentration using the recombinant enzyme. Monokaryotic strains showed different concentrations of and ratios between the two reaction products.

Monokaryotic Pleurotus sapidus Strains with Intraspecific Variability of an Alkene Cleaving DyP-Type Peroxidase Activity as a Result of Gene Mutation and Differential Gene Expression.

The basidiomycete Pleurotus sapidus produced a dye-decolorizing peroxidase (PsaPOX) with alkene cleavage activity, implying potential as a biocatalyst for the fragrance and flavor industry. To increase the activity, a daughter-generation of 101 basidiospore-derived monokaryons (MK) was used. After a pre-selection according to the growth rate, the activity analysis revealed a stable intraspecific variability of the strains regarding peroxidase and alkene cleavage activity of PsaPOX. Ten monokaryons reached activities up to 2.6-fold higher than the dikaryon, with MK16 showing the highest activity. Analysis of the PsaPOX gene identified three different enzyme variants. These were co-responsible for the observed differences in activities between strains as verified by heterologous expression in Komagataella phaffii. The mutation S371H in enzyme variant PsaPOX_high caused an activity increase alongside a higher protein stability, while the eleven mutations in variant PsaPOX_low resulted in an activity decrease, which was partially based on a shift of the pH optimum from 3.5 to 3.0. Transcriptional analysis revealed the increased expression of PsaPOX in MK16 as reason for the higher PsaPOX activity in comparison to other strains producing the same PsaPOX variant. Thus, different expression profiles, as well as enzyme variants, were identified as crucial factors for the intraspecific variability of the PsaPOX activity in the monokaryons.

A DyP-Type Peroxidase of Pleurotus sapidus with Alkene Cleaving Activity.

Alkene cleavage is a possibility to generate aldehydes with olfactory properties for the fragrance and flavor industry. A dye-decolorizing peroxidase (DyP) of the basidiomycete Pleurotus sapidus (PsaPOX) cleaved the aryl alkene trans-anethole. The PsaPOX was semi-purified from the mycelium via FPLC, and the corresponding gene was identified. The amino acid sequence as well as the predicted tertiary structure showed typical characteristics of DyPs as well as a non-canonical Mn2+-oxidation site on its surface. The gene was expressed in Komagataella pfaffii GS115 yielding activities up to 142 U/L using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) as substrate. PsaPOX exhibited optima at pH 3.5 and 40 °C and showed highest peroxidase activity in the presence of 100 µM H2O2 and 25 mM Mn2+. PsaPOX lacked the typical activity of DyPs towards anthraquinone dyes, but oxidized Mn2+ to Mn3+. In addition, bleaching of ß-carotene and annatto was observed. Biotransformation experiments verified the alkene cleavage activity towards the aryl alkenes (E)-methyl isoeugenol, α-methylstyrene, and trans-anethole, which was increased almost twofold in the presence of Mn2+. The resultant aldehydes are olfactants used in the fragrance and flavor industry. PsaPOX is the first described DyP with alkene cleavage activity towards aryl alkenes and showed potential as biocatalyst for flavor production.

Improvement in the Stability and Enzymatic Activity of Pleurotus sapidus Lipoxygenase Dissolved in Natural Deep Eutectic Solvents (NADESs).

Natural deep eutectic solvents (NADESs) can serve as solvents for enzymes, are biodegradable, and have low toxicities. Eight NADESs with different hydrogen bond acceptors and donors were tested to improve the stability and activity of a lipoxygenase from Basidiomycete Pleurotus sapidus (LOXPSA). Betaine:sorbitol:water (1:1:3, BSorbW) and betaine:ethylene glycol (1:3, BEtGly) had the best impact on the peroxidation of linoleic acid and the side reaction of piperine to the vanilla-like scented compound piperonal. The yield of piperonal in NADESs increased by 43% in BSorbW and 40% in BEtGly compared to the control. The addition of BSorbW also enhanced the enzyme's stability at various temperatures and increased its activity during incubation at 60 °C. The demonstrated improvement in lipoxygenase activity and stability indicates versatile applications in industry, expanding the potential uses of the enzyme.

The Nitrogen Content in the Fruiting Body and Mycelium of Pleurotus Ostreatus and Its Utilization as a Medium Component in Thraustochytrid Fermentation.

Following the idea of a circular bioeconomy, the use of side streams as substitutes for cultivation media (components) in bioprocesses would mean an enormous economic and ecological advantage. Costly compounds in conventional media for the production of the triterpene squalene in thraustochytrids are the main carbon source and complex nitrogen sources. Among other side streams examined, extracts from the spent mycelium of the basidiomycete Pleurotus ostreatus were best-suited to acting as alternative nitrogen sources in cultivation media for thraustochytrids. The total nitrogen (3.76 ± 0.01 and 4.24 ± 0.04%, respectively) and protein (16.47 ± 0.06 and 18.57 ± 0.18%, respectively) contents of the fruiting body and mycelium were determined. The fungal cells were hydrolyzed and extracted to generate accessible nitrogen sources. Under preferred conditions, the extracts from the fruiting body and mycelium contained 73.63 ± 1.19 and 89.93 ± 7.54 mM of free amino groups, respectively. Cultivations of Schizochytrium sp. S31 on a medium using a mycelium extract as a complex nitrogen source showed decelerated growth but a similar squalene yield (123.79 ± 14.11 mg/L after 216 h) compared to a conventional medium (111.29 ± 19.96 mg/L, although improvable by additional complex nitrogen source).

Impact of Agro-Industrial Side-Streams on Sesquiterpene Production by Submerged Cultured Cerrena unicolor.

The quality and harvest of essential oils depend on a large number of factors, most of which are hard to control in an open-field environment. Therefore, Basidiomycota have gained attention as a source for biotechnologically produced terpenoids. The basidiomycete Cerrena unicolor (Cun) was cultivated in submerged culture, and the production of sesquiterpenoids was analyzed via stir bar sorptive extraction (SBSE), followed by thermo-desorption gas chromatography coupled with mass spectrometry (TDS-GC-MS). Identification of aroma-active sesquiterpenoids was supported by GC, coupled with an olfactory detection port (ODP). Following the ideal of a circular bioeconomy, Cun was submerged (up-scalable) cultivated, and supplemented with a variety of food industrial side-streams. The effects of the different supplementations and of pure fatty acids were evaluated by liquid extraction and analysis of the terpenoids via GC-MS. As sesquiterpenoid production was enhanced by the most by lipid-rich side-streams, a cultivation with 13C-labeled acetate was conducted. Data confirmed that lipid-rich side-streams enhanced the sesquiterpene production through an increased acetyl-CoA pool.

Mycelium vs. Fruiting Bodies of Edible Fungi-A Comparison of Metabolites.

Edible mushrooms are widely appreciated for their appealing flavours, low caloric values and high content of presumably health-protecting metabolites. Their long history of safe use together with the looming worldwide food crisis have revived the idea of generating meat analogues and protein isolates by the controlled fermentation of mycelia of these edible fungi as a dietary option. The occurrence of proteins, polysaccharides, smaller metabolites, metal ions and toxins in mycelia and fruiting bodies is compared among the three most popular species, Agaricus bisporus (button mushroom), Pleurotus ostreatus (oyster mushroom), Lentinus edodes (shiitake) and some closely related species. Large effects of substrate chemistry, strain, developmental stage and ecological interactions result in a wide variation of the concentrations of some metabolites in both mycelial cells and fruiting bodies. This is obviously a result of the high adaptation abilities required to survive in natural habitats. Fungal bioprocesses are decoupled from agricultural production and can be operated anytime, anywhere, and on any scale according to demand. It is concluded that fungal biomass, if produced under food-grade conditions and on an industrial scale, could provide a safe and nutritious meat substitute and protein isolates with a high biological value for future vegan foods.

Production of natural colorants by liquid fermentation with Chlorociboria aeruginascens and Laetiporus sulphureus and prospective applications.

The replacement of potentially hazardous synthetic dyes with natural dyes and pigments are of great interest for a sustainable economy. In order to obtain cost-efficient, environmentally friendly and competitive products, improvements in the cultivation and extraction of pigment-producing organisms and in dyeing processes are necessary. In our study, we were able to scale up the production of xylindein by Chlorociboria aeruginascens from 3 to 70 L bioreactor cultivations. We have identified important bioprocess parameters like low shear stress (150 rpm, tip speed <0.5 m/s) for optimal pigment yield (4.8 mg/L/d). Additionally, we have demonstrated the potential of laetiporic acid production by Laetiporus sulphureus in various cultivation systems and media, achieving dried biomass concentrations of almost 10 g/L with a 7 L bioreactor cultivation after 17 days. Extractions performed at 70°C and 15 min incubation time showed optimal results. To the best of our knowledge, we have described for the first time the use of this pigment in silk dyeing, which results in a brilliant hue that cannot easily be produced by other natural pigments.

A synthetic RNA-based biosensor for fructose-1,6-bisphosphate that reports glycolytic flux.

RNA-based sensors for intracellular metabolites are a promising solution to the emerging issue of metabolic heterogeneity. However, their development, i.e., the conversion of an aptamer into an in vivo-functional intracellular metabolite sensor, still harbors challenges. Here, we accomplished this for the glycolytic flux-signaling metabolite, fructose-1,6-bisphosphate (FBP). Starting from in vitro selection of an aptamer, we constructed device libraries with a hammerhead ribozyme as actuator. Using high-throughput screening in yeast with fluorescence-activated cell sorting (FACS), next-generation sequencing, and genetic-environmental perturbations to modulate the intracellular FBP levels, we identified a sensor that generates ratiometric fluorescent readout. An abrogated response in sensor mutants and occurrence of two sensor conformations-revealed by RNA structural probing-indicated in vivo riboswitching activity. Microscopy showed that the sensor can differentiate cells with different glycolytic fluxes within yeast populations, opening research avenues into metabolic heterogeneity. We demonstrate the possibility to generate RNA-based sensors for intracellular metabolites for which no natural metabolite-binding RNA element exits.