Combined 6-benzylaminopurine and H2O2 stimulate the astaxanthin biosynthesis in Xanthophyllomyces dendrorhous.

Astaxanthin is one of the most attractive carotenoids due to its high antioxidant activity and beneficial biological properties, while Xanthophyllomyces dendrorhous is one of its main microbial sources. Since astaxanthin is synthesized as a response to oxidative stress, several oxidative agents have been evaluated to increase X. dendrorhous astaxanthin yields. However, the extent of the stimulation is determined by the cellular damage caused by the applied oxidative agent. Phytohormones have also been reported as stimulants of astaxanthin biosynthesis acting directly on its metabolic pathway and indirectly promoting cellular resistance to reactive oxygen species. We reasoned that both oxidative agents and phytohormones lead to increased astaxanthin synthesis, but the latter could mitigate the drawbacks of the former. Thus, here, the stimulation on astaxanthin biosynthesis, as well as the cellular and transcriptional responses of wild type X. dendrorhous to phytohormones (6-benzylaminopurine, 6-BAP; abscisic acid, ABA; and indole-3-acetic acid, IAA), and oxidative agents (glutamate, menadione, H2O2, and/or Fe2+) were evaluated as a single or combined treatments. ABA and 6-BAP were the best individual stimulants leading to 2.24- and 2.60-fold astaxanthin biosynthesis increase, respectively. Nevertheless, the effect of combined 6-BAP and H2O2 led to a 3.69-fold astaxanthin synthesis increase (0.127 ± 0.018 mg astaxanthin/g biomass). Moreover, cell viability (> 82.75%) and mitochondrial activity (> 82.2%) remained almost intact in the combined treatment (6-BAP + H2O2) compared to control (< 52.17% cell viability; < 85.3% mitochondrial activity). On the other hand, mRNA levels of hmgR, idi, crtYB, crtR, and crtS, genes of the astaxanthin biosynthetic pathway, increased transiently along X. dendrorhous fermentation due to stimulations assayed in this study. KEY POINTS: • Combined 6-BAP and H2O2 is the best treatment to increase astaxanthin yields in X. dendrorhous. • 6-BAP preserves cell integrity under oxidative H2O2 stress conditions. • 6-BAP and H2O2 increase transcriptional responses of hmgR, idi, and crt family genes transiently.

Metabolic engineering for high yield synthesis of astaxanthin in Xanthophyllomyces dendrorhous.

Astaxanthin is a carotenoid with a number of assets useful for the food, cosmetic and pharmaceutical industries. Nowadays, it is mainly produced by chemical synthesis. However, the process leads to an enantiomeric mixture where the biologically assimilable forms (3R, 3'R or 3S, 3'S) are a minority. Microbial production of (3R, 3'R) astaxanthin by Xanthophyllomyces dendrorhous is an appealing alternative due to its fast growth rate and easy large-scale production. In order to increase X. dendrorhous astaxanthin yields, random mutant strains able to produce from 6 to 10 mg/g dry mass have been generated; nevertheless, they often are unstable. On the other hand, site-directed mutant strains have also been obtained, but they increase only the yield of non-astaxanthin carotenoids. In this review, we insightfully analyze the metabolic carbon flow converging in astaxanthin biosynthesis and, by integrating the biological features of X. dendrorhous with available metabolic, genomic, transcriptomic, and proteomic data, as well as the knowledge gained with random and site-directed mutants that lead to increased carotenoids yield, we propose new metabolic engineering targets to increase astaxanthin biosynthesis.

Cell surface display of proteins on filamentous fungi.

Protein display approaches have been useful to endow the cell surface of yeasts with new catalytic activities so that they can act as enhanced whole-cell biocatalysts. Despite their biotechnological potential, protein display technologies remain poorly developed for filamentous fungi. The lignocellulolytic character of some of them coupled to the cell surface biosynthesis of valuable molecules by a single or a cascade of several displayed enzymes is an appealing prospect. Cell surface protein display consists in the co-translational fusion of a functional protein (passenger) to an anchor one, usually a cell-wall-resident protein. The abundance, spacing, and local environment of the displayed enzymes-determined by the relationship of the anchor protein with the structure and dynamics of the engineered cell wall-are factors that influence the performance of display-based biocatalysts. The development of protein display strategies in filamentous fungi could be based on the field advances in yeasts; however, the unique composition, structure, and biology of filamentous fungi cell walls require the customization of the approach to those microorganisms. In this prospective review, the cellular bases, the design principles, and the available tools to foster the development of cell surface protein display technologies in filamentous fungi are discussed.

Traffic of chitin synthase 1 (CHS-1) to the Spitzenkörper and developing septa in hyphae of Neurospora crassa: actin dependence and evidence of distinct microvesicle populations.

We describe the subcellular location of chitin synthase 1 (CHS-1), one of seven chitin synthases in Neurospora crassa. Laser scanning confocal microscopy of growing hyphae showed CHS-1-green fluorescent protein (GFP) localized conspicuously in regions of active wall synthesis, namely, the core of the Spitzenkörper (Spk), the apical cell surface, and developing septa. It was also present in numerous fine particles throughout the cytoplasm plus some large vacuoles in distal hyphal regions. Although the same general subcellular distribution was observed previously for CHS-3 and CHS-6, they did not fully colocalize. Dual labeling showed that the three different chitin synthases were contained in different vesicular compartments, suggesting the existence of a different subpopulation of chitosomes for each CHS. CHS-1-GFP persisted in the Spk during hyphal elongation but disappeared from the septum after its development was completed. Wide-field fluorescence microscopy and total internal reflection fluorescence microscopy revealed subapical clouds of particles, suggestive of chitosomes moving continuously toward the Spk. Benomyl had no effect on CHS-1-GFP localization, indicating that microtubules are not strictly required for CHS trafficking to the hyphal apex. Conversely, actin inhibitors caused severe mislocalization of CHS-1-GFP, indicating that actin plays a major role in the orderly traffic and localization of CHS-1 at the apex.

An F-Actin Mega-Cable Is Associated With the Migration of the Sperm Nucleus During the Fertilization of the Polarity-Inverted Central Cell of Agave inaequidens.

Asparagaceae's large embryo sacs display a central cell nucleus polarized toward the chalaza, which means the sperm nucleus that fuses with it during double fertilization migrates an atypical long distance before karyogamy. Because of the size and inverted polarity of the central cell in Asparagaceae, we hypothesize that the second fertilization process is supported by an F-actin machinery different from the short-range F-actin structures observed in Arabidopsis and other plant models. Here, we analyzed the F-actin dynamics of Agave inaequidens, a classical Asparagaceae, before, during, and after the central cell fertilization. Several parallel F-actin cables, spanning from the central cell nucleus to the micropylar pole, and enclosing the vacuole, were observed. As fertilization progressed, a thick F-actin mega-cable traversing the vacuole appeared, connecting the central cell nucleus with the micropylar pole near the egg cell. This mega-cable wrapped the sperm nucleus in transit to fuse with the central cell nucleus. Once karyogamy finished, and the endosperm started to develop, the mega-cable disassembled, but new F-actin structures formed. These observations suggest that Asparagaceae, and probably other plant species with similar embryo sacs, evolved an F-actin machinery specifically adapted to support the migration of the fertilizing sperm nucleus within a large-sized and polarity-inverted central cell.

Response to Edaphoclimatic Conditions and Crop Management of the Bacterial Microbiome of Musa acuminata Rhizosphere Profiled by 16S rRNA Gene Amplicon Sequencing.

Bacterial rhizospheric microbiomes of Musa acuminata cultivated in farms close to the west and east Mexican coasts and with different climate, soils, and crop management practices were characterized by 16S rRNA gene amplicon sequencing. Results showed that rhizospheric microbiome composition changed along with seasonal weather but were mostly indifferent to soil type.

Functional stratification of the Spitzenkörper of Neurospora crassa.

GS-1 (ncu04189) is a protein required for the synthesis of beta-1,3-glucan in Neurospora crassa. As chitin, beta-1,3-glucan is a morphogenetically relevant component of the fungal cell wall. Previously, we showed that chitin synthases are delivered to the growing hyphal tip of N. crassa by secretory microvesicles that follow an unconventional route and accumulate in the core of the Spitzenkörper (Spk). Tagged with the green fluorescent protein (GFP), GS-1 accumulated in the hyphal apex forming a dynamic and pleomorphic ring-like structure ('Spitzenring') that corresponded to the Spk outer macrovesicular stratum and surrounded the inner core of chitin synthase-containing microvesicles. TIRF microscopy revealed that GS-1-GFP reached the hyphal apex as a population of heterogeneous-size particles that moved along defined paths. On sucrose density gradients, GS-1-associated particles mainly sedimented in a high density range 1.1272-1.2124 g ml(-1). Clearly, GS-1 and chitin synthases of N. crassa are contained in two different types of secretory vesicles that accumulate in different strata of the Spk, a differentiation presumably related to the spatial control of cell-wall synthesis.

Simple Whole-Mount Staining Protocol of F-Actin for Studies of the Female Gametophyte in Agavoideae and Other Crassinucellate Ovules.

During plant sexual reproduction, F-actin takes part in the elongation of the pollen tube and the movement of sperm cells along with it. Moreover, F-actin is involved in the transport of sperm cells throughout the embryo sac when double fertilization occurs. Different techniques for analysis of F-actin in plant cells have been developed: from classical actin-immunolocalization in fixed tissues to genetically tagged actin with fluorescent proteins for live imaging of cells. Despite the implementation of live cell imaging tools, fixed plant tissue methods for cytoskeletal studies remain an essential tool for genetically intractable systems. Also, most of the work on live imaging of the cytoskeleton has been conducted on cells located on the plant's surface, such as epidermal cells, trichomes, and root hairs. In cells situated in the plant's interior, especially those from plant species with thicker organ systems, it is necessary to utilize conventional sectioning and permeabilization methods to allow the label access to the cytoskeleton. Studies about the role of F-actin cytoskeleton during double fertilization in plants with crassinucellate ovules (e.g., Agave, Yucca, Polianthes, Prochnyantes, and Manfreda) remain scarce due to the difficulties to access the female gametophyte. Here, we have developed a straightforward method for analysis of F-actin in the female gametophyte of different Agavoideae sub-family species. The procedure includes the fixation of whole ovules with formaldehyde, followed by membrane permeabilization with cold acetone, a prolonged staining step with rhodamine-phalloidin, and Hoechst 33342 as a counterstain and two final steps of dehydration of samples in increasing-concentration series of cold isopropanol and clarification of tissues with methyl salicylate. This technique allows the analysis of a large number of samples in a short period, cell positioning relative to neighbor cells is maintained, and, with the help of a confocal microscope, reconstruction of a single 3D image of F-actin structures into the embryo sac can be obtained.

Cytosolic calcium localization and dynamics during early endosperm development in the genus Agave (Asparagales, Asparagaceae).

Calcium is a secondary messenger that regulates and coordinates the cellular responses to environmental cues. Despite calcium being a key player during fertilization in plants, little is known about its role during the development of the endosperm. For this reason, the distribution, abundance, and dynamics of cytosolic calcium during the first stages of endosperm development of Agave tequilana and Agave salmiana were analyzed. Cytosolic calcium and actin filaments detected in the embryo sacs of Agave tequilana and A. salmiana revealed that they play an important role during the division and nuclear migration of the endosperm. After fertilization, a relatively high concentration of cytosolic calcium was located in the primary nucleus of the endosperm, as well as around migrating nuclei during the development of the endosperm. Cytosolic calcium participates actively during the first mitosis of the endosperm mother cell and interacts with the actin filaments that generate the motor forces during the migration of the nuclei through the large cytoplasm of the central cell.

Off the wall: The rhyme and reason of Neurospora crassa hyphal morphogenesis.

The fungal cell wall building processes are the ultimate determinants of hyphal shape. In Neurospora crassa the main cell wall components, ß-1,3-glucan and chitin, are synthesized by enzymes conveyed by specialized vesicles to the hyphal tip. These vesicles follow different secretory routes, which are delicately coordinated by cargo-specific Rab GTPases until their accumulation at the Spitzenkörper. From there, the exocyst mediates the docking of secretory vesicles to the plasma membrane, where they ultimately get fused. Although significant progress has been done on the cellular mechanisms that carry cell wall synthesizing enzymes from the endoplasmic reticulum to hyphal tips, a lot of information is still missing. Here, the current knowledge on N. crassa cell wall composition and biosynthesis is presented with an emphasis on the underlying molecular and cellular secretory processes.

Rhizospheric Microbiome Profiling of Capsicum annuum L. Cultivated in Amended Soils by 16S and Internal Transcribed Spacer 2 rRNA Amplicon Metagenome Sequencing.

Rhizospheric microbiomes of Capsicum annuum L. cultivated either conventionally or amended with a synthetic microbial consortium or a root exudate inductor, were characterized by 16S/internal transcribed spacer 2 (ITS2) rRNA amplicon metagenome sequencing. The most abundant taxa found, although differently represented in each treatment, were Gammaproteobacteria, Alphaproteobacteria, Actinobacteria, and Bacilli, as well as Chytridiomycetes and Mortierellomycotina.

Mechanism of versatile peroxidase inactivation by Ca(2+) depletion.

Versatile peroxidase (VP) from Bjerkandera adusta, as other class II peroxidases, is inactivated by Ca(2+) depletion. In this work, the spectroscopic characterizations of Ca(2+)-depleted VP at pH 4.5 (optimum for activity) and pH 7.5 are presented. Previous works on other ligninolytic peroxidases, such as lignin peroxidase and manganese peroxidase, have been performed at pH 7.5; nevertheless, at this pH these enzymes are inactive independently of their Ca(2+) content. At pH 7.5, UV-Vis spectra indicate a heme-Fe(3+) transition from 5-coordinated high-spin configuration in native peroxidase to 6-coordinated low-spin state in the inactive Ca(2+)-depleted form. This Fe(3+) hexa-coordination has been proposed as the origin of inactivation. However, our results at pH 4.5 show that Ca(2+)-depleted enzyme has a high spin Fe(3+). EPR measurements on VP confirm the differences in the Fe(3+) spin states at pH 4.5 and at 7.5 for both, native and Ca(2+)-depleted enzymes. In addition, EPR spectra recorded after the addition of H(2)O(2) to Ca(2+)-depleted VP show the formation of compound I with the radical species delocalized on the porphyrin ring. The lack of radical delocalization on an amino acid residue exposed to solvent, W170, as determined in native enzyme at pH 4.5, explains the inability of Ca(2+)-depleted VP to oxidize veratryl alcohol. These observations, in addition to a notorious redox potential decrease, suggest that Ca(2+)-depleted versatile peroxidase is able to form the active intermediate compound I but its long range electron transfer has been disrupted.

Tryptophan-based radical in the catalytic mechanism of versatile peroxidase from Bjerkandera adusta.

Versatile peroxidase (VP) from Bjerkandera adusta is a structural hybrid between lignin (LiP) and manganese (MnP) peroxidase. This hybrid combines the catalytic properties of the two above peroxidases, being able to oxidize typical LiP and MnP substrates. The catalytic mechanism is that of classical peroxidases, where the substrate oxidation is carried out by a two-electron multistep reaction at the expense of hydrogen peroxide. Elucidation of the structures of intermediates in this process is crucial for understanding the mechanism of substrate oxidation. In this work, the reaction of H(2)O(2) with the enzyme in the absence of substrate has been investigated with electron paramagnetic resonance (EPR) spectroscopy. The results reveal an EPR signal with partially resolved hyperfine structure typical of an organic radical. The yield of this radical is approximately 30%. Progressive microwave power saturation measurements indicate that the radical is weakly coupled to a paramagnetic metal ion, suggesting an amino acid radical in moderate distance from the ferryl heme. A tryptophan radical was identified as a protein-based radical formed during the catalytic mechanism of VP from Bjerkandera adusta through X-band and high-field EPR measurements at 94 GHz, aided by computer simulations for both frequency bands. A close analysis of the theoretical model of the VP from Bjerkandera sp. shows the presence of a tryptophan residue near to the heme prosthetic group, which is solvent-exposed as in the case of LiP and other VPs. The catalytic role of this residue in a long-range electron-transfer pathway is discussed.