Production of scopularide A in submerged culture with Scopulariopsis brevicaulis

Background: Marine organisms produce many novel compounds with useful biological activity, but are currently underexploited. Considerable research has been invested in the study of compounds from marine bacteria, and several groups have now recognised that marine fungi also produce an interesting range of compounds. During product discovery, these compounds are often produced only in non-agitated culture conditions, which are unfortunately not well suited for scaling up. A marine isolate of Scopulariopsis brevicaulis, strain LF580, produces the cyclodepsipeptide scopularide A, which has previously only been produced in non-agitated cultivation. Results: Scopulariopsis brevicaulis LF580 produced scopularide A when grown in batch and fed-batch submerged cultures. Scopularide A was extracted primarily from the biomass, with approximately 7% being extractable from the culture supernatant. By increasing the biomass density of the cultivations, we were able to increase the volumetric production of the cultures, but it was important to avoid nitrogen limitation. Specific production also increased with increasing biomass density, leading to improvements in volumetric production up to 29-fold, compared with previous, non-agitated cultivations. Cell densities up to 36 g L-1 were achieved in 1 to 10 L bioreactors. Production of scopularide A was optimised in complex medium, but was also possible in a completely defined medium. Conclusions: Scopularide A production has been transferred from a non-agitated to a stirred tank bioreactor environment with an approximately 6-fold increase in specific and 29-fold increase in volumetric production. Production of scopularide A in stirred tank bioreactors demonstrates that marine fungal compounds can be suitable for scalable production, even with the native production organism

Euglena gracilis growth and cell composition under different temperature, light and trophic conditions

BackgroundEuglena gracilis, a photosynthetic protist, produces protein, unsaturated fatty acids, wax esters, and a unique β-1,3-glucan called paramylon, along with other valuable compounds. The cell composition of E. gracilis was investigated in this study to understand how light and organic carbon (photo-, mixo- and heterotrophic conditions) affected growth and cell composition (especially lipids). Comparisons were primarily carried out in cultures grown at 23 °C, but the effect of growth at higher temperatures (27 or 30 °C) was also considered.Cell growthSpecific growth rates were slightly lower when E. gracilis was grown on glucose in either heterotrophic or mixotrophic conditions than when grown photoautotrophically, although the duration of exponential growth was longer. Temperature determined the rate of exponential growth in all cultures, but not the linear growth rate during light-limited growth in phototrophic conditions. Temperature had less effect on cell composition.Cell compositionAlthough E. gracilis was not expected to store large amounts of paramylon when grown phototrophically, we observed that phototrophic cells could contain up to 50% paramylon. These cells contained up to 33% protein and less than 20% lipophilic compounds, as expected. The biomass contained about 8% fatty acids (measured as fatty acid methyl esters), most of which were unsaturated. The fatty acid content of cells grown in mixotrophic conditions was similar to that observed in phototrophic cells, but was lower in cells grown heterotrophically. Heterotrophic cells contained less unsaturated fatty acids than phototrophic or mixotrophic cells. α-Linolenic acid was present at 5 to 18 mg g-1 dry biomass in cells grown in the presence of light, but at [less than] 0.5 mg g-1 biomass in cells grown in the dark. Eicosapentaenoic and docosahexaenoic acids were detected at 1 to 5 mg g-1 biomass. Light was also important for the production of vitamin E and phytol

Biotechnological versatility of riboflavin producer Ashbya gossypii Expression of Trichoderma reesei cellulases CBHI and EGI

The filamentous fungus Ashbya gossypii shows the potential for the production of, yet unexploited, valuable compounds other than riboflavin. To explore the ability of A. gossypii as a host for the expression of recombinant proteins, endoglucanase I (EGI) and cellobiohydrolase I (CBHI) from the fungus Trichoderma reesei were expressed in A. gossypii under Saccharomyces cerevisiae PGK1 promoter. The proteins were secreted into the culture medium, but there were differences in the amount or activity of the protein being produced. In one hand, CBHI activity was not detected using 4-methylumbelliferyl--Dlactoside as substrate, being only detected by Western blot. On the other hand, EGI activity was detectable, the level of activity being comparable to that produced by a S. cerevisiae strain containing the same plasmid. Thus more EGI was secreted than CBHI, or more active protein was produced. Partial characterization of CBHI and EGI expressed in A. gossypii revealed overglycosylation when compared to the native T. reesei proteins, but the glycosylation was less extensive than on cellulases expressed in S. cerevisiae

Bioconversion of D-galacturonate to keto-deoxy-L-galactonate (3-deoxy-L-threo-hex-2-ulosonate) using filamentous fungi

<p>Abstract</p> <p>Background</p> <p>The D-galacturonic acid derived from plant pectin can be converted into a variety of other chemicals which have potential use as chelators, clarifiers, preservatives and plastic precursors. Among these is the deoxy-keto acid derived from L-galactonic acid, keto-deoxy-L-galactonic acid or 3-deoxy-L-<it>threo</it>-hex-2-ulosonic acid. The keto-deoxy sugars have been found to be useful precursors for producing further derivatives. Keto-deoxy-L-galactonate is a natural intermediate in the fungal D-galacturonate metabolic pathway, and thus keto-deoxy-L-galactonate can be produced in a simple biological conversion.</p> <p>Results</p> <p>Keto-deoxy-L-galactonate (3-deoxy-L-<it>threo</it>-hex-2-ulosonate) accumulated in the culture supernatant when <it>Trichoderma reesei </it>Δ<it>lga1 </it>and <it>Aspergillus niger </it>Δ<it>gaaC </it>were grown in the presence of D-galacturonate. Keto-deoxy-L-galactonate accumulated even if no metabolisable carbon source was present in the culture supernatant, but was enhanced when D-xylose was provided as a carbon and energy source. Up to 10.5 g keto-deoxy-L-galactonate l^-1was produced from 20 g D-galacturonate l^-1and <it>A. niger </it>Δ<it>gaaC </it>produced 15.0 g keto-deoxy-L-galactonate l^-1from 20 g polygalacturonate l^-1, at yields of 0.4 to 1.0 g keto-deoxy-L-galactonate [g D-galacturonate consumed]^-1. Keto-deoxy-L-galactonate accumulated to concentrations of 12 to 16 g l^-1intracellularly in both producing organisms. This intracellular concentration was sustained throughout production in <it>A. niger </it>Δ<it>gaaC</it>, but decreased in <it>T. reesei</it>.</p> <p>Conclusions</p> <p>Bioconversion of D-galacturonate to keto-deoxy-L-galactonate was achieved with both <it>A. niger </it>Δ<it>gaaC </it>and <it>T. reesei </it>Δ<it>lga1</it>, although production (titre, volumetric and specific rates) was better with <it>A. niger </it>than <it>T. reesei</it>. <it>A. niger </it>was also able to produce keto-deoxy-L-galactonate directly from pectin or polygalacturonate demonstrating the feasibility of simultaneous hydrolysis and bioconversion. Although keto-deoxy-L-galactonate accumulated intracellularly, concentrations above ~12 g l^-1were exported to the culture supernatant. Lysis may have contributed to the release of keto-deoxy-L-galactonate from <it>T. reesei </it>mycelia.</p

High throughput, small scale methods to characterise the growth of marine fungi

Various marine fungi have been shown to produce interesting, bioactive compounds, but scaling up the production of these compounds can be challenging, particularly because little is generally known about how the producing organisms grow. Here we assessed the suitability of using 100-well BioScreen plates or 96-well plates incubated in a robot hotel to cultivate eight filamentous marine fungi, six sporulating and two non-sporulating, to obtain data on growth and substrate (glucose, xylose, galactose or glycerol) utilisation in a high throughput manner. All eight fungi grew in both cultivation systems, but growth was more variable and with more noise in the data in the Cytomat plate hotel than in the BioScreen. Specific growth rates between 0.01 (no added substrate) and 0.07 h-1 were measured for strains growing in the BioScreen and between 0.01 and 0.27 h-1 for strains in the plate hotel. Three strains, Dendryphiella salina LF304, Penicillium chrysogenum KF657 and Penicillium pinophilum LF458, consistently had higher specific growth rates on glucose and xylose in the plate hotel than in the BioScreen, but otherwise results were similar in the two systems. However, because of the noise in data from the plate hotel, the data obtained from it could only be used to distinguish between substrates which did or did not support growth, whereas data from BioScreen also provided information on substrate preference. Glucose was the preferred substrate for all strains, followed by xylose and galactose. Five strains also grew on glycerol. Therefore it was important to minimise the amount of glycerol introduced with the inoculum to avoid misinterpreting the results for growth on poor substrates. We concluded that both systems could provide physiological data with filamentous fungi, provided sufficient replicates are included in the measurements

Transcriptional monitoring of steady state and effects of anaerobic phases in chemostat cultures of the filamentous fungus Trichoderma reesei

BACKGROUND: Chemostat cultures are commonly used in production of cellular material for systems-wide biological studies. We have used the novel TRAC (transcript analysis with aid of affinity capture) method to study expression stability of approximately 30 process relevant marker genes in chemostat cultures of the filamentous fungus Trichoderma reesei and its transformant expressing laccase from Melanocarpus albomyces. Transcriptional responses caused by transient oxygen deprivations and production of foreign protein were also studied in T. reesei by TRAC. RESULTS: In cultures with good steady states, the expression of the marker genes varied less than 20% on average between sequential samples for at least 5 or 6 residence times. However, in a number of T. reesei cultures continuous flow did not result in a good steady state. Perturbations to the steady state were always evident at the transcriptional level, even when they were not measurable as changes in biomass or product concentrations. Both unintentional and intentional perturbations of the steady state demonstrated that a number of genes involved in growth, protein production and secretion are sensitive markers for culture disturbances. Exposure to anaerobic conditions caused strong responses at the level of gene expression, but surprisingly the cultures could regain their previous steady state quickly, even after 3 h O(2 )depletion. The main effect of producing M. albomyces laccase was down-regulation of the native cellulases compared with the host strain. CONCLUSION: This study demonstrates the usefulness of transcriptional analysis by TRAC in ensuring the quality of chemostat cultures prior to costly and laborious genome-wide analysis. In addition TRAC was shown to be an efficient tool in studying gene expression dynamics in transient conditions

Investigation of protein secretion and secretion stress in Ashbya gossypii

Background: Ashbya gossypii is a filamentous Saccharomycete used for the industrial production of riboflavin that has been recently explored as a host system for recombinant protein production. To gain insight into the protein secretory pathway of this biotechnologically relevant fungus, we undertook genome-wide analyses to explore its secretome and its transcriptional responses to protein secretion stress. Results: A computational pipeline was used to predict the inventory of proteins putatively secreted by A. gossypii via the general secretory pathway. The proteins actually secreted by this fungus into the supernatants of submerged cultures in minimal and rich medium were mapped by two-dimensional gel electrophoresis, revealing that most of the A. gossypii secreted proteins have an isoelectric point between 4 and 6, and a molecular mass above 25 kDa. These analyses together indicated that 1-4% of A. gossypii proteins are likely to be secreted, of which less than 33% are putative hydrolases. Furthermore, transcriptomic analyses carried out in A. gossypii cells under recombinant protein secretion conditions and dithiothreitol-induced secretion stress unexpectedly revealed that a conventional unfolded protein response (UPR) was not activated in any of the conditions, as the expression levels of several well-known UPR target genes (e.g. IRE1, KAR2, HAC1 and PDI1 homologs) remained unaffected. However, several other genes involved in protein unfolding, endoplasmatic reticulum-associated degradation, proteolysis, vesicle trafficking, vacuolar protein sorting, secretion and mRNA degradation were up-regulated by dithiothreitol-induced secretion stress. Conversely, the transcription of several genes encoding secretory proteins, such as components of the glycosylation pathway, was severely repressed by dithiothreitol Conclusions: This study provides the first insights into the secretion stress response of A. gossypii, as well as a basic understanding of its protein secretion potential, which is more similar to that of yeast than to that of other filamentous fungi. Contrary to what has been widely described for yeast and fungi, a conventional UPR was not observed in A. gossypii, but alternative protein quality control mechanisms enabled it to cope with secretion stress. These data will help provide strategies for improving heterologous protein secretion in A. gossypii.This work was financially supported by Fundacao para a Ciencia e a Tecnologia, Portugal, through: PhD grant SFRH/BD/30229/2006 to OR, MIT-Portugal Program PhD grant SFRH/BD/39112/2007 to TQA, Project AshByofactory (PTDC/EBB-EBI/101985/2008 - FCOMP-01-0124-FEDER-009701), Project RECI/BBB-EBI/0179/2012 - FCOMP-01-0124-FEDER-027462, Strategic Project PEst-OE/EQB/LA0023/2013 and Project BioInd (NORTE-07-0124-FEDER000028) co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER. We thank Dominik Mojzita and Mari Hakkinen from VTT Finland for their assistance with the microarray sample preparation, hybridization and data acquisition

Characterization of the Ashbya gossypii secreted N-glycome and genomic insights into its N-glycosylation pathway

The riboflavin producer Ashbya gossypii is a filamentous hemiascomycete, closely related to the yeast Saccharomyces cerevisiae, that has been used as a model organism to study fungal developmental biology. It has also been explored as a host for the expression of recombinant proteins. However, although N-glycosylation plays important roles in protein secretion, morphogenesis, and the development of multicellular organisms, the N-glycan structures synthesised by A. gossypii had not been elucidated. In this study, we report the first characterization of A. gossypii N-glycans and provide valuable insights into their biosynthetic pathway. By combined matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry profiling and nuclear magnetic resonance (NMR) spectroscopy we determined that the A. gossypii secreted N-glycome is characterized by high-mannose type structures in the range Man4–18GlcNAc2, mostly containing neutral core-type N-glycans with 8–10 mannoses. Cultivation in defined minimal media induced the production of acidic mannosylphosphorylated N-glycans, generally more elongated than the neutral N-glycans. Truncated neutral N-glycan structures similar to those found in other filamentous fungi (Man4–7GlcNAc2) were detected, suggesting the possible existence of trimming activity in A. gossypii. Homologs for all of the S. cerevisiae genes known to be involved in the endoplasmatic reticulum and Golgi N-glycan processing were found in the A. gossypii genome. However, processing of N-glycans by A. gossypii differs considerably from that by S. cerevisiae, allowing much shorter N-glycans. Genes for two putative N-glycan processing enzymes were identified, that did not have homologs in S. cerevisiae.We thank Fundacao para a Ciencia e a Tecnologia (FCT), Portugal, for financial support through the project AshByofactory (PTDC/EBB-EBI/101985/2008-FCOMP-01-0124-FEDER-009701) and MIT-Portugal Program (Ph.D. grant SFRH/BD/39112/2007 to Tatiana Q. Aguiar). We also thank Dr. Olli Aitio (University of Helsinki) for helpful assistance in the interpretation of the NMR data

Low oxygen levels as a trigger for enhancement of respiratory metabolism in Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>The industrially important yeast <it>Saccharomyces cerevisiae </it>is able to grow both in the presence and absence of oxygen. However, the regulation of its metabolism in conditions of intermediate oxygen availability is not well characterised. We assessed the effect of oxygen provision on the transcriptome and proteome of <it>S. cerevisiae </it>in glucose-limited chemostat cultivations in anaerobic and aerobic conditions, and with three intermediate (0.5, 1.0 and 2.8% oxygen) levels of oxygen in the feed gas.</p> <p>Results</p> <p>The main differences in the transcriptome were observed in the comparison of fully aerobic, intermediate oxygen and anaerobic conditions, while the transcriptome was generally unchanged in conditions receiving different intermediate levels (0.5, 1.0 or 2.8% O₂) of oxygen in the feed gas. Comparison of the transcriptome and proteome data suggested post-transcriptional regulation was important, especially in 0.5% oxygen. In the conditions of intermediate oxygen, the genes encoding enzymes of the respiratory pathway were more highly expressed than in either aerobic or anaerobic conditions. A similar trend was also seen in the proteome and in enzyme activities of the TCA cycle. Further, genes encoding proteins of the mitochondrial translation machinery were present at higher levels in all oxygen-limited and anaerobic conditions, compared to fully aerobic conditions.</p> <p>Conclusion</p> <p>Global upregulation of genes encoding components of the respiratory pathway under conditions of intermediate oxygen suggested a regulatory mechanism to control these genes as a response to the need of more efficient energy production. Further, cells grown in three different intermediate oxygen levels were highly similar at the level of transcription, while they differed at the proteome level, suggesting post-transcriptional mechanisms leading to distinct physiological modes of respiro-fermentative metabolism.</p

Transcriptome of Saccharomyces cerevisiae during production of D-xylonate

BACKGROUND: Production of D-xylonate by the yeast S. cerevisiae provides an example of bioprocess development for sustainable production of value-added chemicals from cheap raw materials or side streams. Production of D-xylonate may lead to considerable intracellular accumulation of D-xylonate and to loss of viability during the production process. In order to understand the physiological responses associated with D-xylonate production, we performed transcriptome analyses during D-xylonate production by a robust recombinant strain of S. cerevisiae which produces up to 50 g/L D-xylonate. RESULTS: Comparison of the transcriptomes of the D-xylonate producing and the control strain showed considerably higher expression of the genes controlled by the cell wall integrity (CWI) pathway and of some genes previously identified as up-regulated in response to other organic acids in the D-xylonate producing strain. Increased phosphorylation of Slt2 kinase in the D-xylonate producing strain also indicated that D-xylonate production caused stress to the cell wall. Surprisingly, genes encoding proteins involved in translation, ribosome structure and RNA metabolism, processes which are commonly down-regulated under conditions causing cellular stress, were up-regulated during D-xylonate production, compared to the control. The overall transcriptional responses were, therefore, very dissimilar to those previously reported as being associated with stress, including stress induced by organic acid treatment or production. Quantitative PCR analyses of selected genes supported the observations made in the transcriptomic analysis. In addition, consumption of ethanol was slower and the level of trehalose was lower in the D-xylonate producing strain, compared to the control. CONCLUSIONS: The production of organic acids has a major impact on the physiology of yeast cells, but the transcriptional responses to presence or production of different acids differs considerably, being much more diverse than responses to other stresses. D-Xylonate production apparently imposed considerable stress on the cell wall. Transcriptional data also indicated that activation of the PKA pathway occurred during D-xylonate production, leaving cells unable to adapt normally to stationary phase. This, together with intracellular acidification, probably contributes to cell death. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-763) contains supplementary material, which is available to authorized users