A novel, robust peptidyl-lys metalloendopeptidase from Trametes coccinea recombinantly expressed in Komagataella phaffii.

A novel peptidyl-lys metalloendopeptidase (Tc-LysN) from Tramates coccinea was recombinantly expressed in Komagataella phaffii using the native pro-protein sequence. The peptidase was secreted into the culture broth as zymogen (~38 kDa) and mature enzyme (~19.8 kDa) simultaneously. The mature Tc-LysN was purified to homogeneity with a single step anion-exchange chromatography at pH 7.2. N-terminal sequencing using TMTpro Zero and mass spectrometry of the mature Tc-LysN indicated that the pro-peptide was cleaved between the amino acid positions 184 and 185 at the Kex2 cleavage site present in the native pro-protein sequence. The pH optimum of Tc-LysN was determined to be 5.0 while it maintained ≥60% activity between pH values 4.5-7.5 and ≥30% activity between pH values 8.5-10.0, indicating its broad applicability. The temperature maximum of Tc-LysN was determined to be 60 °C. After 18 h of incubation at 80 °C, Tc-LysN still retained ~20% activity. Organic solvents such as methanol and acetonitrile, at concentrations as high as 40% (v/v), were found to enhance Tc-LysN's activity up to ~100% and ~50%, respectively. Tc-LysN's thermostability, ability to withstand up to 8 M urea, tolerance to high concentrations of organic solvents, and an acidic pH optimum make it a viable candidate to be employed in proteomics workflows in which alkaline conditions might pose a challenge. The nano-LC-MS/MS analysis revealed bovine serum albumin (BSA)'s sequence coverage of 84% using Tc-LysN which was comparable to the sequence coverage of 90% by trypsin peptides. KEY POINTS: •A novel LysN from Trametes coccinea (Tc-LysN) was expressed in Komagataella phaffii and purified to homogeneity •Tc-LysN is thermostable, applicable over a broad pH range, and tolerates high concentrations of denaturants •Tc-LysN was successfully applied for protein digestion and mass spectrometry fingerprinting.

Caproate production from Enset fiber in one-pot two-step fermentation using anaerobic fungi (Neocallimastix cameroonii strain G341) and Clostridium kluyveri DSM 555.

BACKGROUND: Lignocellulosic biomass plays a crucial role in creating a circular bioeconomy and minimizing environmental impact. Enset biomass is a byproduct of traditional Ethiopian Enset food processing that is thrown away in huge quantities. This study aimed to produce caproate from Enset fiber using Neocallimastix cameroonii strain G341 and Clostridium kluyveri DSM 555 in one-pot two-step fermentation. RESULTS: The process started by growing N. cameroonii on Enset fiber as a carbon source for 7 days. Subsequently, the fungal culture was inoculated with active C. kluyveri preculture and further incubated. The results showed that N. cameroonii grew on 0.25 g untreated Enset fiber as the sole carbon source and produced 1.16 mmol acetate, 0.51 mmol hydrogen, and 1.34 mmol formate. In addition, lactate, succinate, and ethanol were detected in small amounts, 0.17 mmol, 0.08 mmol, and 0.7 mmol, respectively. After inoculating with C. kluyveri, 0.3 mmol of caproate and 0.48 mmol of butyrate were produced, and hydrogen production also increased to 0.95 mmol compared to sole N. cameroonii fermentation. Moreover, after the culture was supplemented with 2.18 mmol of ethanol during C. kluyveri inoculation, caproate, and hydrogen production was further increased to 1.2 and 1.36 mmol, respectively, and the consumption of acetate also increased. CONCLUSION: A novel microbial cell factory was developed to convert untreated lignocellulosic Enset fiber into the medium chain carboxylic acid caproate and H2 by a co-culture of the anaerobic fungi N. cameroonii and C. kluyveri. This opens a new value chain for Enset farmers, as the process requires only locally available raw materials and low-price fermenters. As the caproate production was mainly limited by the available ethanol, the addition of locally produced ethanol-containing fermentation broth ("beer") would further increase the titer.

Optimization of L-malic acid production from acetate with Aspergillus oryzae DSM 1863 using a pH-coupled feeding strategy.

BACKGROUND: Malic acid, a dicarboxylic acid mainly used in the food industry, is currently produced from fossil resources. The utilization of low-cost substrates derived from biomass could render microbial processes economic. Such feedstocks, like lignocellulosic hydrolysates or condensates of fast pyrolysis, can contain high concentrations of acetic acid. Acetate is a suitable substrate for L-malic acid production with the filamentous fungus Aspergillus oryzae DSM 1863, but concentrations obtained so far are low. An advantage of this carbon source is that it can be used for pH control and simultaneous substrate supply in the form of acetic acid. In this study, we therefore aimed to enhance L-malate production from acetate with A. oryzae by applying a pH-coupled feeding strategy. RESULTS: In 2.5-L bioreactor fermentations, several feeding strategies were evaluated. Using a pH-coupled feed consisting of 10 M acetic acid, the malic acid concentration was increased about 5.3-fold compared to the batch process without pH control, resulting in a maximum titer of 29.53 ± 1.82 g/L after 264 h. However, it was not possible to keep both the pH and the substrate concentration constant during this fermentation. By using 10 M acetic acid set to a pH of 4.5, or with the repeated addition of NaOH, the substrate concentration could be maintained within a constant range, but these strategies did not prove beneficial as lower maximum titers and yields were obtained. Since cessation of malic acid production was observed in later fermentation stages despite carbon availability, a possible product inhibition was evaluated in shake flask cultivations. In these experiments, malate and succinate, which is a major by-product during malic acid production, were added at concentrations of up to 50 g/L, and it was found that A. oryzae is capable of organic acid production even at high product concentrations. CONCLUSIONS: This study demonstrates that a suitable feeding strategy is necessary for efficient malic acid production from acetate. It illustrates the potential of acetate as carbon source for microbial production of the organic acid and provides useful insights which can serve as basis for further optimization.

Enhanced Bioactivity of Tailor-Made Glycolipid Enriched Manuka Honey.

Glycolipids can be synthetized in deep eutectic solvents (DESs) as they possess low water content allowing a reversed lipase activity and thus enables ester formation. Based on this principle, honey can also serve as a media for glycolipid synthesis. Indeed, this supersaturated sugar solution is comparable in terms of physicochemical properties to the sugar-based DESs. Honey-based products being commercially available for therapeutic applications, it appears interesting to enhance its bioactivity. In the current work, we investigate if enriching medical grade honey with in situ enzymatically-synthetized glycolipids can improve the antimicrobial property of the mixture. The tested mixtures are composed of Manuka honey that is enriched with octanoate, decanoate, laurate, and myristate sugar esters, respectively dubbed GOH, GDH, GLH, and GMH. To characterize the bioactivity of those mixtures, first a qualitative screening using an agar well diffusion assay has been performed with methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Candida bombicola, Escherichia coli, and Pseudomonas putida which confirmed considerably enhanced susceptibility of these micro-organisms to the different glycolipid enriched honey mixtures. Then, a designed biosensor E. coli strain that displays a stress reporter system consisting of three stress-specific inducible, red, green, and blue fluorescent proteins which respectively translate to physiological stress, genotoxicity, and cytotoxicity was used. Bioactivity was, therefore, characterized, and a six-fold enhancement of the physiological stress that was caused by GOH compared to regular Manuka honey at a 1.6% (v/v) concentration was observed. An antibacterial agar well diffusion assay with E. coli was performed as well and demonstrated an improved inhibitory potential with GOH upon 20% (v/v) concentration.

Microwave-Assisted One-Pot Lipid Extraction and Glycolipid Production from Oleaginous Yeast Saitozyma podzolica in Sugar Alcohol-Based Media.

Glycolipids are non-ionic surfactants occurring in numerous products of daily life. Due to their surface-activity, emulsifying properties, and foaming abilities, they can be applied in food, cosmetics, and pharmaceuticals. Enzymatic synthesis of glycolipids based on carbohydrates and free fatty acids or esters is often catalyzed using certain acyltransferases in reaction media of low water activity, e.g., organic solvents or notably Deep Eutectic Systems (DESs). Existing reports describing integrated processes for glycolipid production from renewables use many reaction steps, therefore this study aims at simplifying the procedure. By using microwave dielectric heating, DESs preparation was first accelerated considerably. A comparative study revealed a preparation time on average 16-fold faster than the conventional heating method in an incubator. Furthermore, lipids from robust oleaginous yeast biomass were successfully extracted up to 70% without using the pre-treatment method for cell disruption, limiting logically the energy input necessary for such process. Acidified DESs consisting of either xylitol or sorbitol and choline chloride mediated the one-pot process, allowing subsequent conversion of the lipids into mono-acylated palmitate, oleate, linoleate, and stearate sugar alcohol esters. Thus, we show strong evidence that addition of immobilized Candida antarctica Lipase B (Novozym 435®), in acidified DES mixture, enables a simplified and fast glycolipid synthesis using directly oleaginous yeast biomass.

Lipase-Catalyzed Production of Sorbitol Laurate in a "2-in-1" Deep Eutectic System: Factors Affecting the Synthesis and Scalability.

Surfactants, such as glycolipids, are specialty compounds that can be encountered daily in cleaning agents, pharmaceuticals or even in food. Due to their wide range of applications and, more notably, their presence in hygiene products, the demand is continuously increasing worldwide. The established chemical synthesis of glycolipids presents several disadvantages, such as lack of specificity and selectivity. Moreover, the solubility of polyols, such as sugars or sugar alcohols, in organic solvents is rather low. The enzymatic synthesis of these compounds is, however, possible in nearly water-free media using inexpensive and renewable building blocks. Using lipases, ester formation can be achieved under mild conditions. We propose, herein, a "2-in-1" system that overcomes solubility problems, as a Deep Eutectic System (DES) made of sorbitol and choline chloride replaces either a purely organic or aqueous medium. For the first time, 16 commercially available lipase formulations were compared, and the factors affecting the conversion were investigated to optimize this process, owing to a newly developed High-Performance Liquid Chromatography-Evaporative Light Scattering Detector (HPLC-ELSD) method for quantification. Thus, using 50 g/L of lipase formulation Novozym 435® at 50 °C, the optimized synthesis of sorbitol laurate (SL) allowed to achieve 28% molar conversion of 0.5 M of vinyl laurate to its sugar alcohol monoester when the DES contained 5 wt.% water. After 48h, the de novo synthesized glycolipid was separated from the media by liquid-liquid extraction, purified by flash-chromatography and characterized thoroughly by one- and two-dimensional Nuclear Magnetic Resonance (NMR) experiments combined to Mass Spectrometry (MS). In completion, we provide initial proof of scalability for this process. Using a 2.5 L stirred tank reactor (STR) allowed a batch production reaching 25 g/L in a highly viscous two-phase system.

Enzymatic Synthesis of Glucose Monodecanoate in a Hydrophobic Deep Eutectic Solvent.

Environmentally friendly and biodegradable reaction media are an important part of a sustainable glycolipid production in the transition to green chemistry. Deep eutectic solvents (DESs) are an ecofriendly alternative to organic solvents. So far, only hydrophilic DESs were considered for enzymatic glycolipid synthesis. In this study, a hydrophobic DES consisting of (-)-menthol and decanoic acid is presented for the first time as an alternative to hydrophilic DES. The yields in the newly introduced hydrophobic DES are significantly higher than in hydrophilic DESs. Different reaction parameters were investigated to optimize the synthesis further. Twenty milligrams per milliliter iCalB and 0.5 M glucose resulted in the highest initial reaction velocity for the esterification reaction, while the highest initial reaction velocity was achieved with 1.5 M glucose in the transesterification reaction. The enzyme was proven to be reusable for at least five cycles without significant loss of activity.

Interfacial and Foaming Properties of Tailor-Made Glycolipids-Influence of the Hydrophilic Head Group and Functional Groups in the Hydrophobic Tail.

Glycolipids are a class of biodegradable surfactants less harmful to the environment than petrochemically derived surfactants. Here we discuss interfacial properties, foam stability, characterized in terms of transient foam height, gas volume fraction and bubble diameter as well as texture of seven enzymatically synthesized surfactants for the first time. Glycolipids consisting of different head groups, namely glucose, sorbitol, glucuronic acid and sorbose, combined with different C10 acyl chains, namely decanoate, dec-9-enoate and 4-methyl-nonanoate are compared. Equilibrium interfacial tension values vary between 24.3 and 29.6 mN/m, critical micelle concentration varies between 0.7 and 3.0 mM. In both cases highest values were found for the surfactants with unsaturated or branched tail groups. Interfacial elasticity and viscosity, however, were significantly reduced in these cases. Head and tail group both affect foam stability. Foams from glycolipids with sorbose and glucuronic acid derived head groups showed higher stability than those from surfactants with glucose head group, sorbitol provided lowest foam stability. We attribute this to different head group hydration also showing up in the time to reach equilibrium interfacial adsorption. Unsaturated tail groups reduced whereas branching enhanced foam stability compared to the systems with linear, saturated tail. Moreover, the tail group strongly influences foam texture. Glycolipids with unsaturated tail groups produced foams quickly collapsing even at smallest shear loads, whereas the branched tail group yielded a higher modulus than the linear tails. Normalized shear moduli for the systems with different head groups varied in a narrow range, with the highest value found for decylglucuronate.

Current status and perspectives of 2-phenylethanol production through biological processes.

2-Phenylethanol (2-PE), an important flavor and fragrance compound with a rose-like smell has been widely used in the cosmetics, perfume, and food industries. Traditional production of 2-PE was mainly through the extraction from plant materials or by chemical synthesis. However, the increasing demand of consumers for natural flavors cannot be met by these methods. Biological production of 2-PE has emerged to be an appealing solution due to an environmental friendly process and the definition of a "natural" product. In this review, we have comprehensively summarized the current status and perspectives for biological 2-PE production in terms of its advantages over classical chemical synthesis and extraction from natural plants. A comprehensive description of 2-PE synthetic pathways and global regulation mechanisms, strategies to increase 2-PE production, and the utilization of agro-industrial wastes as feedstocks has been systematically discussed. Furthermore, the application of in situ product removal techniques have also been highlighted.

Process characterization and influence of alternative carbon sources and carbon-to-nitrogen ratio on organic acid production by Aspergillus oryzae DSM1863.

L-Malic acid and fumaric acid are C4 dicarboxylic organic acids and considered as promising chemical building blocks. They can be applied as food preservatives and acidulants in rust removal and as polymerization starter units. Molds of the genus Aspergillus are able to produce malic acid in large quantities from glucose and other carbon sources. In order to enhance the production potential of Aspergillus oryzae DSM 1863, production and consumption rates in an established bioreactor batch-process based on glucose were determined. At 35 °C, up to 42 g/L malic acid was produced in a 168-h batch process with fumaric acid as a by-product. In prolonged shaking flask experiments (353 h), the suitability of the alternative carbon sources xylose and glycerol at a carbon-to-nitrogen (C/N) ratio of 200:1 and the influence of different C/N ratios in glucose cultivations were tested. When using glucose, 58.2 g/L malic acid and 4.2 g/L fumaric acid were produced. When applying xylose or glycerol, both organic acids are produced but the formation of malic acid decreased to 45.4 and 39.4 g/L, respectively. Whereas the fumaric acid concentration was not significantly altered when cultivating with xylose (4.5 g/L), it is clearly enhanced by using glycerol (9.3 g/L). When using glucose as a carbon source, an increase or decrease of the C/N ratio did not influence malic acid production but had an enormous influence on fumaric acid production. The highest fumaric acid concentrations were determined at the highest C/N ratio (300:1, 8.44 g/L) and lowest at the lowest C/N ratio (100:1, 0.7 g/L).

Intensification of Enzymatic Sorbityl Laurate Production in Dissolved and Neat Systems under Conventional and Microwave Heating.

Glycolipids such as sugar alcohol esters have been demonstrated to be relevant for numerous applications across various domains of specialty. The use of organic solvents and, more recently, deep eutectic solvents (DESs) to mediate lipase-supported bioconversions is gaining potential for industrial application. However, many challenges and limitations remain such as extensive time of production and relatively low productivities among others, which must be solved to strengthen such a biocatalytic process in industry. In this context, this study focuses on the intensification of sorbityl laurate production, as a model biocatalyzed reaction using Novozym 435, investigating the relevance of temperature, heating method, and solvent system. By increasing the reaction temperature from 50 to 90 °C, the space-time yield and product yield were considerably enhanced for reactions in DES and the organic solvent 2M2B, irrespective of the heating method (conventional or microwave heating). However, positive effects in 2M2B were more pronounced with conventional heating as 98% conversion yield was reached within 90 min at 90 °C, equating thus to a nearly 4-fold increase in performance yielding 118.0 ± 3.6 g/(L·h) productivity. With DES, the overall yield and space-time yield were lower with both heating methods. However, microwave heating enabled a 2-fold increase in both performance parameters when the reaction temperature was increased from 50 to 90 °C. Compared to conventional heating, a 7-fold increase in space-time yield at 50 °C and a 16-fold increase at 90 °C were achieved in DES by microwave heating. Furthermore, microwave irradiation enabled the usage of a neat, solvent-free system, representing an initial proof of concept with productivities of up to 13.3 ± 2.3 g/(L·h).

Engineering Scheffersomyces segobiensis for palmitoleic acid-rich lipid production.

Palmitoleic acid (POA; C16:1) is an essential high-value ω-7-conjugated fatty acid with beneficial bioactivities and potential applications in the nutraceutical and pharmaceutical industries. Previously, the oleaginous yeast Scheffersomyces segobiensis DSM27193 has been identified as a promising production host as an alternative for POA extraction from plant or animal sources. Here, the POA-producing capacity of this host was further expanded by optimizing the fermentation process and molecular strain engineering. Specifically, a dual fermentation strategy (O-S dynamic regulation strategy) focused on the substrate and dissolved oxygen concentration was designed to eliminate ethanol and pyruvate accumulation during fermentation. Key genes influencing POA production, such as jen, dgat, ole were identified on the transcriptional level and were subsequently over-expressed. Furthermore, the phosphoketolase (Xpk)/phosphotransacetylase (Pta) pathway was introduced to improve the yield of the precursor acetyl-CoA from glucose. The resulting cell factory SS-12 produced 7.3 g/L of POA, corresponding to an 11-fold increase compared to the wild type, presenting the highest POA titre reported using oleaginous yeast to date. An economic evaluation based on the raw materials, utilities and facility-dependent costs showed that microbial POA production using S. segobiensis can supersede the current extraction method from plant oil and marine fish. This study reports the construction of a promising cell factory and an effective microbial fermentation strategy for commercial POA production.

Detoxification of a pyrolytic aqueous condensate from wheat straw for utilization as substrate in Aspergillus oryzae DSM 1863 cultivations.

BACKGROUND: The pyrolytic aqueous condensate (PAC) formed during the fast pyrolysis of wheat straw contains a variety of organic carbons and might therefore potentially serve as an inexpensive substrate for microbial growth. One of its main components is acetic acid, which was recently shown to be a suitable carbon source for the filamentous fungus Aspergillus oryzae. However, the condensate also contains numerous toxic compounds that inhibit fungal growth and result in a tolerance of only about 1%. Therefore, to enable the use of the PAC as sole substrate for A. oryzae cultivations, a pretreatment seems to be necessary. RESULTS: Various conditions for treatments with activated carbon, overliming, rotary evaporation and laccase were evaluated regarding fungal growth and the content of inhibitory model substances. Whereas the first three methods considerably increased the fungal tolerance to up to 1.625%, 12.5% and 30%, respectively, the enzymatic treatment did not result in any improvement. The optimum carbon load for the treatment with activated carbon was identified to be 10% (w/v) and overliming should ideally be performed at 100 °C and an initial pH of 12. The best detoxification results were achieved with rotary evaporation at 200 mbar as a complete removal of guaiacol and a strong reduction in the concentration of acetol, furfural, 2-cyclopenten-1-one and phenol by 84.9%, 95.4%, 97.7% and 86.2%, respectively, were observed. Subsequently, all possible combinations of the effective single methods were performed and rotary evaporation followed by overliming and activated carbon treatment proved to be most efficient as it enabled growth in 100% PAC shake-flask cultures and resulted in a maximum cell dry weight of 5.21 ± 0.46 g/L. CONCLUSION: This study provides a comprehensive insight into the detoxification efficiency of a variety of treatment methods at multiple conditions. It was revealed that with a suitable combination of these methods, PAC toxicity can be reduced to such an extent that growth on pure condensate is possible. This can be considered as a first important step towards a microbial valorization of the pyrolytic side-stream with A. oryzae.

RNA-Seq Based Transcriptome Analysis of Aspergillus oryzae DSM 1863 Grown on Glucose, Acetate and an Aqueous Condensate from the Fast Pyrolysis of Wheat Straw.

Due to its acetate content, the pyrolytic aqueous condensate (PAC) formed during the fast pyrolysis of wheat straw could provide an inexpensive substrate for microbial fermentation. However, PAC also contains several inhibitors that make its detoxification inevitable. In our study, we examined the transcriptional response of Aspergillus oryzae to cultivation on 20% detoxified PAC, pure acetate and glucose using RNA-seq analysis. Functional enrichment analysis of 3463 significantly differentially expressed (log2FC >2 & FDR < 0.05) genes revealed similar metabolic tendencies for both acetate and PAC, as upregulated genes in these cultures were mainly associated with ribosomes and RNA processing, whereas transmembrane transport was downregulated. Unsurprisingly, metabolic pathway analysis revealed that glycolysis/gluconeogenesis and starch and sucrose metabolism were upregulated for glucose, whereas glyoxylate and the tricarboxylic acid (TCA) cycle were important carbon utilization pathways for acetate and PAC, respectively. Moreover, genes involved in the biosynthesis of various amino acids such as arginine, serine, cysteine and tryptophan showed higher expression in the acetate-containing cultures. Direct comparison of the transcriptome profiles of acetate and PAC revealed that pyruvate metabolism was the only significantly different metabolic pathway and was overexpressed in the PAC cultures. Upregulated genes included those for methylglyoxal degradation and alcohol dehydrogenases, which thus represent potential targets for the further improvement of fungal PAC tolerance.

Parameters Influencing Lipase-Catalyzed Glycolipid Synthesis by (Trans-)Esterification Reaction.

Glycolipids are biodegradable, non-toxic surfactants with a wide range of applications. Enzymatic esterification or transesterification facilitated in reaction media of low water activity is a reaction strategy for the production of tailor-made glycolipids as a high structural diversity can be achieved. Organic solvents, ionic liquids, and deep eutectic solvents have been applied as reaction media. However, several challenges need to be addressed for efficient (trans-)esterification reactions, especially for the lipophilization of polar substrates. Therefore, crucial parameters in (trans-)esterification reactions in conventional and non-conventional media are discussed and compared in this review with a special focus on glycolipid synthesis.

Effect of process mode, nitrogen source and temperature on L-malic acid production with Aspergillus oryzae DSM 1863 using acetate as carbon source.

Malic acid, mainly used as acidulant and taste enhancer in the food industry, is currently produced from fossil resources. In this study, microbial L-malate production with the filamentous fungus A. oryzae using the carbon source acetate was evaluated. Acetate is for example contained in biomass-derived substrates such as lignocellulosic hydrolysates and condensates of fast pyrolysis, thus avoiding competition with food production. Since research on malic acid synthesis from acetate is limited and reported productivities and yields are low, this work aimed to improve the process. First, different cultivation temperatures were tested. This parameter was found to affect the ratio between malic and succinic acid, which is the major by-product of organic acid production with A. oryzae. At 32°C, the malate share was highest (53.7 ± 1.6%), while it was lowest at 38°C (43.3 ± 1.1%) whereas succinate represented the main product (51.5 ± 1.0%). Besides the temperature, the type of nitrogen source was also found to affect malate synthesis as well as biomass production. In the pre-culture, the biomass concentration was increased by a factor of 3.4-3.9, and germination started earlier with the complex nitrogen sources yeast extract, casein hydrolysate and peptone compared to the defined nitrogen source (NH4)2SO4. Especially with yeast extract, malate synthesis in the main culture was accelerated and the titer obtained after 48 h was about 2.6 times higher than that quantified with (NH4)2SO4. To reduce substrate inhibition in acetate medium, fed-batch and repeated-batch processes were evaluated using (NH4)2SO4 or yeast extract as nitrogen source. In the fed-batch process, the period of malate production was extended, and the maximum product concentration was increased to 11.49 ± 1.84 g/L with (NH4)2SO4 and 12.08 ± 1.25 g/L with yeast extract. In the repeated-batch process, the total acid production was highest within the first 240 h of fermentation, but optimization is required to maintain high production rates in later cycles. The lessons learned in this study will help in the development of further process strategies to maximize malate production using acetate as alternative substrate to the commonly used glucose.

Lipase-Mediated Mechanoenzymatic Synthesis of Sugar Esters in Dissolved Unconventional and Neat Reaction Systems.

Mechanochemical and biocatalytic approaches in modern research are two major assets to develop greener processes. In the present study, these modular tools of sustainability are pointed toward the production of versatile and daily employed compounds such as surfactants. Toward this aim, glycolipids, a class of nonionic surfactants composed of ubiquitous and primary metabolites such as sugar and fatty acid moieties, represent a promising alternative to petroleum-derived surface-active agents. Therefore, the combination of biocatalysis with mechanochemistry aiming at glycolipid synthesis seemed a logical step that was taken in this study for the first time. The monoacylated model compound glucose-6-O-decanoate was synthesized with the help of a bead mill apparatus using two different unconventional dissolved reaction systems, namely, menthol-based hydrophobic deep eutectic solvents and 2-methyl-2-butanol, thus reaching up to 12% yield in the latter based on the conversion of vinyl decanoate, after only 90 min of reaction. In addition, a neat reaction system using an excess of vinylated fatty ester as an adjuvant allowed a 27 mM/h space-time yield. The overall significant increase in productivities, up to 6 times, compared to standard heating and shaking methods, shows the tremendous potential of mechanoenzymatic synthesis.

Metabolic shift towards increased biohydrogen production during dark fermentation in the anaerobic fungus Neocallimastix cameroonii G341.

BACKGROUND: Anaerobic fungi of the phylum Neocallimastigomycota have a high biotechnological potential due to their robust lignocellulose degrading capabilities and the production of several valuable metabolites like hydrogen, acetate, formate, lactate, and ethanol. The metabolism of these fungi, however, remains poorly understood due to limitations of the current cultivation strategies in still-standing bottles, thereby restricting the comprehensive evaluation of cultivation conditions. RESULTS: We describe the analysis of growth conditions and their influence on the metabolism of the previously isolated fungus Neocallimastix cameroonii G341. We established a bioreactor process in a stirred tank, enabling cultivation under defined conditions. The optimal growth temperature for the fungus was between 38.5 °C and 41.5 °C, while the optimal pH was 6.6-6.8. Like other dark fermentation systems, hydrogen production is dependent on the hydrogen partial pressure and pH. Shaking the bottles or stirring the fermenters led to an increase in hydrogen and a decrease in lactate and ethanol production. Regulation of the pH to 6.8 in the fermenter nearly doubled the amount of produced hydrogen. CONCLUSIONS: Novel insights into the metabolism of Neocallimastix cameroonii were gained, with hydrogen being the preferred way of electron disposal over lactate and ethanol. In addition, our study highlights the potential application of the fungus for hydrogen production from un-pretreated biomass. Finally, we established the first cultivation of an anaerobic fungus in a stirred tank reactor system.

Simultaneous Metabarcoding and Quantification of Neocallimastigomycetes from Environmental Samples: Insights into Community Composition and Novel Lineages.

Anaerobic fungi from the herbivore digestive tract (Neocallimastigomycetes) are primary lignocellulose modifiers and hold promise for biotechnological applications. Their molecular detection is currently difficult due to the non-specificity of published primer pairs, which impairs evolutionary and ecological research with environmental samples. We developed and validated a Neocallimastigomycetes-specific PCR primer pair targeting the D2 region of the ribosomal large subunit suitable for screening, quantifying, and sequencing. We evaluated this primer pair in silico on sequences from all known genera, in vitro with pure cultures covering 16 of the 20 known genera, and on environmental samples with highly diverse microbiomes. The amplified region allowed phylogenetic differentiation of all known genera and most species. The amplicon is about 350 bp long, suitable for short-read high-throughput sequencing as well as qPCR assays. Sequencing of herbivore fecal samples verified the specificity of the primer pair and recovered highly diverse and so far unknown anaerobic gut fungal taxa. As the chosen barcoding region can be easily aligned and is taxonomically informative, the sequences can be used for classification and phylogenetic inferences. Several new Neocallimastigomycetes clades were obtained, some of which represent putative novel lineages such as a clade from feces of the rodent Dolichotis patagonum (mara).

Global Transcriptome Profile of the Oleaginous Yeast Saitozyma podzolica DSM 27192 Cultivated in Glucose and Xylose.

Unlike conventional yeasts, several oleaginous yeasts, including Saitozyma podzolica DSM 27192, possess the innate ability to grow and produce biochemicals from plant-derived lignocellulosic components such as hexose and pentose sugars. To elucidate the genetic basis of S. podzolica growth and lipid production on glucose and xylose, we performed comparative temporal transcriptome analysis using RNA-seq method. Approximately 3.4 and 22.2% of the 10,670 expressed genes were differentially (FDR < 0.05, and log2FC > 1.5) expressed under batch and fed batch modes, respectively. Our analysis revealed that a higher number of sugar transporter genes were significantly overrepresented in xylose relative to glucose-grown cultures. Given the low homology between proteins encoded by most of these genes and those of the well-characterised transporters, it is plausible to conclude that S. podzolica possesses a cache of putatively novel sugar transporters. The analysis also suggests that S. podzolica potentially channels carbon flux from xylose via both the non-oxidative pentose phosphate and potentially via the first steps of the Weimberg pathways to yield xylonic acid. However, only the ATP citrate lyase (ACL) gene showed significant upregulation among the essential oleaginous pathway genes under nitrogen limitation in xylose compared to glucose cultivation. Combined, these findings pave the way toward the design of strategies or the engineering of efficient biomass hydrolysate utilization in S. podzolica for the production of various biochemicals.