Current Biotechnology - Volume 6, Issue 3, 2017

BACKGROUND: Chitin is one of the world’s most abundant biopolymers, but in contrast to cellulose its potential has so far been fairly underrated. Compared to cellulose, chitin features an additional amino-group on the hexose sugar ring that renders it a promising substrate for functional biopolymers in biomedical applications as well as biochemical synthesis of specialty and fine chemicals. Fungi with specialized life styles, such as mycoparasites and entomopathogenic fungi, have evolved an effective machinery to utilize chitin derived from other fungi and arthropods, respectively. OBJECTIVE: Fungal chitin degradation and chitin synthesis have been subject to extensive investigation in the past years to gain insight into these intricately regulated processes, which render fungi capable of harnessing the recalcitrant chitin polymer as nutritional source and building block for growth. For the development of methods to exploit chitin from these origins, the design of new products and their sustainable production through enzymatic action, identifying the regulators of the chitin anabolic and catabolic pathways will be essential. In this review our current knowledge on chitin metabolism in fungi is presented; new sources for chitin production and new products derived from enzymatic processing will be introduced.

Background: Plant natural products (PNPs) play a key role in human health and life quality through the treatment of diseases and as supplements in the food and healthcare industry. These high-value metabolites are traditionally extracted from the natural resource, but ongoing research is exploring the possibilities to construct efficient microbial cell factories that can replace current production methods. Objective: This review describes recent progress for producing PNPs in S. cerevisiae. Conclusion: Various PNPs, including stillbenoids, flavonoids, terpenoids and glucosinolates can now be produced in the baker's yeast Saccharomyces cerevisiae. As a result of the growing interest in the expression of plant biosynthetic pathways in yeast, many studies have aimed at developing platform strains delivering an improved supply of precursor metabolites. Synthetic biology tools facilitate the identification of superior enzymes, balance gene expression levels in the pathway, and enable the spatial arrangement of plant-derived enzymes in yeast to optimize the performance of the introduced pathways even further. Perspectives: The entry of CRISPR/Cas9 based technologies may allow other yeast species or filamentous fungi to be engineered for the production of PNPs.

Background: Fungi are key organisms in the biotechnological production of a plethora of products relevant for mankind. Recently several new products from fungi have entered the markets, including biosurfactants. Biosurfactants are microbial produced surface active compounds with emulsifying properties which proved to be an interesting alternative to petrochemically or palm oil derived surfactants. Methods: We have performed a review of the current literature on fungal surfactants and their application, focusing on MEL and CL variants and the microbial strains producing them. We have also included unpublished own findings to further add the newest perspectives for potential application of these biosurfactants. Results: The main fungal biosurfactants currently are generated by species from the order Saccharomycetales and Ustilaginales. These species produce a variety of glycolipids, including sophorolipids, mannosylerythritol lipids and cellobiose lipids. They have been described as promising microbial biosurfactants suitable for personal care, cosmetic, pharmaceutical or biomedical applications as well as in bioremediation technologies like solubilisation and removal of oil from contaminated soil, or in oil recovery. Some of these fungal biosurfactants are already included in cleaning agents and cosmetic products available commercially. The properties of surfactants can be modified by fermentation and feeding strategies as well as by selection of different strains or their genetic modification. By that tailor made surfactants for various applications can be designed. Conclusions: Although fungal surfactants provide a large portfolio of compounds with a performance equal or better than petrochemical derived surfactants and have shown their environmental advantages, commercialization of these molecules remains a challenge due to a higher price at the currently low production volume. More efficient production processes would support further introduction of these compounds into the market. In this review we have given a brief overview and positive resume of the currently available fungal surfactants focusing on MEL and CL, their derivatives and the biotechnological opportunities for their further commercialization.

Background: Itaconic acid is a C5 dicaboxylic acid that can serve as a building block to be used in industry to synthesize polymers that are currently based on petroleum-based components. Methods: An overview of the recent literature on microbial itaconic acid production is given in this minireview. The biosynthetic pathways as they are known in Aspergillus terreus and Ustilago maydis are described. Major advances have been made in the development of different microorganisms to serve as potential novel itaconic acid production hosts. Although fermentation strategies are discussed, our main focus is therefore on metabolic engineering strategies for optimal itaconic acid biosynthesis. Results: Itaconic acid is naturally produced by Aspergillus terreus, certain Ustilago and Candida species and Pseudozyma antarctica. Also in mammalian cells itaconic acid is found during macrophage activation. The biosynthetic pathway in A. terreus was well studied and the crucial enzyme for itaconic acid synthesis was found to be cis-aconitate decarboxylase (CadA) that converts cis-aconitate into itaconate. On one hand, optimization of itaconic acid production was done by optimizing fermentation processes and by applying metabolic engineering strategies to the natural producers, most of this was done with A. terreus. On the other hand, the identification of CadA allowed the exploration of heterologous expression of the gene in different hosts. Since citric acid is the metabolic precursor for itaconic acid biosynthesis, many research efforts have focused on Aspergillus niger as a potential itaconic acid producer. The results of this research showed that besides the heterologous expression of cadA, transport between different compartments and re-routing of the central carbon metabolism are important factors for the efficient biosynthesis of itaconic acid. Conclusion: Several microorganisms have been investigated in the past years as potential itaconic acid producing hosts. Titers obtained by metabolic engineering of non-producing hosts range between 14.5 mg/L and 7.8 g/L. Although substantial progress has been made, the titers are not yet competitive with the titers obtained with the natural producer A. terreus.

Background: Marine-derived fungi are an underexploited source of lipids for health and nutrition. Little is known about the impact of sugar type on the lipid diversity produced by a marine-derived fungi. Objective: Traditionally glucose is used as the main sugar source for fungal growth. Thus, this study aimed to reveal the glycolipid alteration induced by glucose replacement by galactose. Methods: The marine-derived Acremonium sp. MMS540 fungal strain was cultivated on three different media with for each the use of glucose or galactose as sugar source, using an OSMAC (One Strain Many Compounds) strategy. For the six different culture conditions, the total lipid and lipid class content of the fungal extracts were evaluated. Fatty acids composition was determined by GC-MS. Finally an LC-MS lipidomic approach was used to evaluate glycolipid modification induced by this sugar replacement. Results: The replacement of glucose by galactose did not alter drastically the total lipid production. However, their GC-MS profiling reveal major modification in the main detected fatty acid ratio (Δ 916:1, 16:0, Δ 9-1218:2, Δ 918:1, 18:0) with a decrease of the minor fatty acids diversity. Complementarily, the LC-MS profiles revealed that in the case of Acremonium sp. MMS540, this culture medium modification alter lipid and glycolipid chemical diversity. Conclusion: This study revealed the link between the sugar uptake in fungi and the associated glycolipid production. This emphasis that very minor changes may drastically alter not only the secondary natural products in fungi but also its lipid related production.

Background: Glucose oxidase (GoxA) catalyzes the reaction from β-D-glucose to gluconic acid. It has a wide range of applications, for example as a sugar sensor for diabetes monitoring or as a prominent additive in food industry. The fungus Aspergillus niger naturally expresses and secretes GoxA. Currently, GoxA is produced by A. niger on yeast peptone dextrose media or by yeasts on media containing sugars in high concentration. Objective: Trichoderma reesei is a well-studied, saprotrophic fungus that is used for industry-scale enzyme production due to its high secretory capacity. GoxA production in T. reesei could combine two promising aspects: high expression and secretion on the one hand, and the utilization of a sustainable and inexpensive carbon source, such as wheat straw or chitin, on the other hand. Method: To evaluate if this is a feasible concept for GoxA production we applied four different expression systems: the constitutive promoter of the pyruvate kinase-encoding gene pki1 of T. reesei, the inducible promoters of the xylanase II-encoding gene xyn2 and of the cellobiohydrolase I-encoding gene cbh1, which is considered as one of the strongest promoters known in T. reesei, and finally, the promoter of the N-acetylglucosaminidase-encoding gene nag1 of Trichoderma harzianum. Result: We discovered that an engineered variant of the cbh1 promoter led to higher yields of GoxA than the wild-type promoter did. This could be demonstrated in shake flask and bioreactor cultivation experiments. The obtained yields (between 28.90 U/ml and 39.00 U/ml) from wheat straw even exceeded the ones reported for A. niger.

Background: Volatile organic compounds (VOCs) are gaseous at room temperature, readily dissipate throughout the environment, and may be of anthropogenic or biogenic origin. Despite an increasing scientific interest in the role VOCs play in interspecific interactions, there remains a limited understanding of the impact of VOCs on fungi living in a shared space. Objective: In this study, we aimed to determine the sensitivity of the model organism Saccharomyces cerevisiae (yeast) in response to exposure to VOCs, collectively or singularly produced by bacteria, fungi, plants, and in industrial processes, and containing various chemical functional groups. Methods: Using a serial dilution spot assay with yeast wild-type strain BY4741, 27 compounds were screened at 10 ppm for 48 hr to determine their impact on yeast growth. Results: We found that gas-phase formaldehyde, three common microbial VOCs, 1-octanol, 1-octen-3- one, and trans-2-octenal, and a common plant VOC, trans-2-hexen-1-al, completely inhibited yeast growth at 10 ppm, while 1-octen-3-ol, 2-methylpropanal and benzene were significantly limiting. Additionally, we identified 2 common microbial VOCs, 3-methyl-1-butanol and 3-octanone, that significantly increased yeast growth. Conclusion: This study demonstrates that yeast provides a useful tool to study the effect of VOCs in shared spaces, serving as a model for other eukaryotic species in the built environment.

Background: There is a growing interest in utilizing endophytes as biofertilizers or biological controls. Beneficial effects may be obtained by synthesizing phytohormones, enzymes and antagonistic substances, fixing nitrogen and carbon dioxide, inducing defence mechanisms and competing colonizing sites and nutrients. Endophytes enhance plant growth and health through plant growth promoting rhizobacteria. Endophytes enter plant tissues through root zone or aerial portions, via germinating radicles, secondary roots, stomata, or foliar route. Endophyte-plant-polymer degrading enzymes such as cellulases and pectinases play a role for their internal colonization and can be detected by immunological or in situ hybridization or tagging with reporter genes. Endophytes interact biochemically and genetically with their host plant and synthesize osmolytes, osmoprotectants, antioxidants, allowing the plants to mitigate the impacts of abiotic stress. Plant genes are modulated by endophyte, and the genes so expressed provide clues as to the effects of endophytes in plants. Objective: The present review describes bioprospecting of endophyte-plant interactions and discusses the way forward to understand their molecular mechanisms. Conclusion: Endophytes are useful models to study the genetic expression of microbe inside the plants, which are well-regulated and flexible. This helps in developing effective endophyte bioinoculants for abiotic stress and crop disease management.

Background: Scenedesmus sp. AMDD (S-AMDD) has been the focus of several studies to assess its potential as a feedstock for biofuels and bioremediation, while the evaluation of its potential suitability as a novel animal feed ingredient has only just begun. In an initial study, S-AMDD demonstrated rapid growth rate and biomass productivity during exponential growth phase and the resulting biomass appeared to have good potential for animal nutrition based on its attractive proximate composition, favorable essential amino acid, fatty acid and elemental profiles and lack of contaminating heavy metals. However, the total carbohydrate and fibre fractions of whole-cell and lipid-extracted S-AMDD were relatively high which could limit their digestion, particularly when fed to monogastric animals. The difference in the capacity to digest and metabolically utilize diets rich in cellulosic material (e.g., fibre) is vast between various farmed animal species. As such, knowledge on the nutritional value of novel ingredients for ruminant animals can rarely be immediately extrapolated to monogastrics and vice versa. Simulated fermentation using rumen-derived digestive fluids or in vitro digestibility using purified monogastric- derived enzymes can provide valuable information. Although not fully conclusive, results from these types of rapid assays are generally inexpensive, require smaller amounts of sample, utilize fewer or no experimental animals, avoid feed refusal issues associated with ingredient off-flavours or odours and can be effective tools for research and for routine industrial use. Objective: The present study is the second in a series of projects designed to evaluate the nutritional value of S-AMDD for animal feed applications. The main objective was to generate novel digestibility data of whole-cell and lipid-extracted S-AMDD for both ruminant and monogastric animals including ruminal organic matter digestibility (OMD), apparent metabolizable energy (aME) content, methane (CH4) production, dilute pepsin digestibility (DPD) and two-phase gastric/pancreatic digestibility of protein (GPDProtein) and energy (GPDEnergy). Methods: Simulated ruminal OMD, aME contents and CH4 production of experimental test diets containing graded levels of whole-cell and lipid-extracted S-AMDD were estimated using a modified batchculture in vitro fermentation system with total gas capture using lactating dairy cattle as rumen fluid donors. In vitro monogastric DPD and two-phase GPD were measured by incubation of whole-cell and lipid- extracted S-AMDD samples in porcine pepsin and porcine pancreatin, containing amylase, lipase and protease enzyme solutions. Results: Simulated ruminal fermentations using lactating dairy cattle as rumen fluid donors indicate that both whole-cell and lipid-extracted S-AMDD have excellent potential for use in ruminant animal feeds. Dietary inclusion of whole-cell S-AMDD at 50% forage protein replacement (equivalent to 20% of the total diet) or lipid-extracted S-AMDD at 100% forage protein replacement (equivalent to 32% of the total diet) did not significantly affect OMD or aME content of the control diet. However, OMD was marginally compromised with 100% forage protein replacement with whole-cell S-AMDD (equivalent to 40% of the total diet) relative to the 25 and 50% replacement levels, although not significantly different from the control diet. Diets containing lipid-extracted S-AMDD significantly reduced CH4 production by approximately 50% compared to the control diet (47% reduction) and those containing whole-cell SAMDD (51% reduction). Since OMD and aME content of diets containing lipid-extracted S-AMDD were unaffected relative to the control diet and whole-cell S-AMDD-containing diets, it seems clear that lipid-extracted S-AMDD contains anti-methanogenic ‘non-fatty acid' substances that have the ability to suppress rumen methanogenic bacteria without disturbing ruminal digestion. This area warrants further exploration in vivo, especially considering the large volume of algal feed that could be produced without occupying significant land resources. In vitro monogastric digestibility using porcine enzymes indicates that lipid-extracted S-AMDD has potential for use in monogastric animal feeds. Protein and energy digestibility of this product were moderately high (75-84% and 70%, respectively), which resulted in relatively high contents of DP (30%) and DE (14 MJ kg-1). On the other hand, the digestibility of whole-cell S-AMDD was low at 52-61% and 50%, respectively resulting in lower levels of DP (15%) and DE (12 MJ kg-1). Despite the encouraging results for lipid-extracted S-AMDD, the digestibility (particularly of energy) remains marginal for monogastric animals and requires improvement through additional cost-effective cell rupture technologies or the production of algal protein concentrates..

Background: The two pathological hallmarks associated with Alzheimer’s disease (AD) include the accumulation of senile plaques and the generation of neurofibrillary tangles. Although it is a known fact that both amyloid beta (Aβ) and Tau exist together in mitochondria, to date, there is no reasonable explanation for the Aβ and Tau interaction in particular. Objective: The cross-seeding interactions between Aβ and Tau were studied using the potential of mean force (PMF) analysis. Methods: The Aβ- Aβ homo-dimer; Tau-Tau homo-dimer and Aβ-Tau hetero-dimer were constructed using molecular docking tools. We have used molecular dynamics (MD) simulation with the umbrella sampling methodology to examine the cross-sequence interactions between homo-dimers and the hetero- dimer by computing PMF. Results: We observed the global minimum and energy barrier to be quite higher for both the homodimers relative to the hetero-dimer, thus indicating that Aβ (25-35) has a high affinity to form dimer complex with Tau (273-284) monomer. We also observed a relatively higher range of interacting residues and interface area between the monomeric units in hetero-dimer (Aβ-Tau) the homo-dimer (Aβ- Aβ) and (Tau-Tau) showed a less number of the same. Conclusion: From the results we may therefore conclude that Aβ fragments can form complexes with the Tau monomers which consequently advance to form aggregates. This aggregation may be favored by interactions between the hydrophobic residues and charged residues present in both the fragments.