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1.  Synthesis and assembly of fungal melanin 
Melanin is a unique pigment with myriad functions that is found in all biological kingdoms. It is multifunctional, providing defense against environmental stresses such as ultraviolet (UV) light, oxidizing agents and ionizing radiation. Melanin contributes to the ability of fungi to survive in harsh environments. In addition, it plays a role in fungal pathogenesis. Melanin is an amorphous polymer that is produced by one of two synthetic pathways. Fungi may synthesize melanin from endogenous substrate via a 1,8-dihydroxynaphthalene (DHN) intermediate. Alternatively, some fungi produce melanin from l-3,4-dihydroxyphenylalanine (l-dopa). The detailed chemical structure of melanin is not known. However, microscopic studies show that it has an overall granular structure. In fungi, melanin granules are localized to the cell wall where they are likely cross-linked to polysaccharides. Recent studies suggest the fungal melanin may be synthesized in internal vesicles akin to mammalian melanosomes and transported to the cell wall. Potential applications of melanin take advantage of melanin's radioprotective properties and propensity to bind to a variety of substances.
doi:10.1007/s00253-011-3777-2
PMCID: PMC4318813  PMID: 22173481
Fungi; Melanin; Cell wall; Vesicle; Chitin; Radioprotection
2.  [No title available] 
PMCID: PMC3968793  PMID: 24402415
3.  [No title available] 
PMCID: PMC4096848  PMID: 24292079
4.  An updated view on horseradish peroxidases: recombinant production and biotechnological applications 
Horseradish peroxidase has been the subject of scientific research for centuries. It has been used exhaustively as reporter enzyme in diagnostics and histochemistry and still plays a major role in these applications. Numerous studies have been conducted on the role of horseradish peroxidase in the plant and its catalytic mechanism. However, little progress has been made in its recombinant production. Until now, commercial preparations of horseradish peroxidase are still isolated from plant roots. These preparations are commonly mixtures of various isoenzymes of which only a small fraction has been described so far. The composition of isoenzymes in these mixed isolates is subjected to uncontrollable environmental conditions. Nowadays, horseradish peroxidase regains interest due to its broad applicability in the fields of medicine, life sciences, and biotechnology in cancer therapy, biosensor systems, bioremediation, and biocatalysis. These medically and commercially relevant applications, the recent discovery of new natural isoenzymes with different biochemical properties, as well as the challenges in recombinant production render this enzyme particularly interesting for future biotechnological solutions. Therefore, we reviewed previous studies as well as current developments with biotechnological emphasis on new applications and the major remaining biotechnological challenge—the efficient recombinant production of horseradish peroxidase enzymes.
doi:10.1007/s00253-014-6346-7
PMCID: PMC4322221  PMID: 25575885
Horseradish peroxidase; Plant peroxidase; Recombinant protein production; Diagnostics; Biosensor; Indole-3-acetic acid; Cancer treatment
5.  Sustainable source of omega-3 eicosapentaenoic acid from metabolically engineered Yarrowia lipolytica: from fundamental research to commercial production 
The omega-3 fatty acids, cis-5, 8, 11, 14, and 17-eicosapentaenoic acid (C20:5; EPA) and cis-4, 7, 10, 13, 16, and 19-docosahexaenoic acid (C22:6; DHA), have wide-ranging benefits in improving heart health, immune function, mental health, and infant cognitive development. Currently, the major source for EPA and DHA is from fish oil, and a minor source of DHA is from microalgae. With the increased demand for EPA and DHA, DuPont has developed a clean and sustainable source of the omega-3 fatty acid EPA through fermentation using metabolically engineered strains of Yarrowia lipolytica. In this mini-review, we will focus on DuPont’s technology for EPA production. Specifically, EPA biosynthetic and supporting pathways have been introduced into the oleaginous yeast to synthesize and accumulate EPA under fermentation conditions. This Yarrowia platform can also produce tailored omega-3 (EPA, DHA) and/or omega-6 (ARA, GLA) fatty acid mixtures in the cellular lipid profiles. Fundamental research such as metabolic engineering for strain construction, high-throughput screening for strain selection, fermentation process development, and process scale-up were all needed to achieve the high levels of EPA titer, rate, and yield required for commercial application. Here, we summarize how we have combined the fundamental bioscience and the industrial engineering skills to achieve large-scale production of Yarrowia biomass containing high amounts of EPA, which led to two commercial products, New Harvest™ EPA oil and Verlasso® salmon.
doi:10.1007/s00253-014-6318-y
PMCID: PMC4322222  PMID: 25567511
Yarrowia lipolytica; Omega-3 fatty acid; Metabolic engineering; Fermentation; Commercialization
6.  Staphylococcus epidermidis in the human skin microbiome mediates fermentation to inhibit the growth of Propionibacterium acnes: Implications of probiotics in acne vulgaris 
Increasing evidence demonstrates that commensal microorganisms in the human skin microbiome help fight pathogens and maintain homeostasis of the microbiome. However, it is unclear how these microorganisms maintain biological balance when one of them overgrows. The overgrowth of Propionibacterium acnes (P. acnes), a commensal skin bacterium, has been associated with the progression of acne vulgaris. Our results demonstrate that skin microorganisms can mediate fermentation of glycerol, which is naturally produced in skin, to enhance their inhibitory effects on P. acnes growth. The skin microorganisms, most of which have been identified as Staphylococcus epidermidis (S. epidermidis), in the microbiome of human fingerprints can ferment glycerol and create inhibition zones to repel a colony of overgrown P. acnes. Succinic acid, one of four short-chain fatty acids (SCFAs) detected in fermented media by nuclear magnetic resonance (NMR) analysis, effectively inhibits the growth of P. acnes in vitro and in vivo. Both intralesional injection and topical application of succinic acid to P. acnes-induced lesions markedly suppress the P. acnes-induced inflammation in mice. We demonstrate for the first time that bacterial members in the skin microbiome can undergo fermentation to rein in the overgrowth of P. acnes. The concept of bacterial interference between P. acnes and S. epidermidis via fermentation can be applied to develop probiotics against acne vulgaris and other skin diseases. In addition, it will open up an entirely new area of study for the biological function of the skin microbiome in promoting human health.
doi:10.1007/s00253-013-5394-8
PMCID: PMC3888247  PMID: 24265031
Acne; Fermentation; P. acnes; Probiotic; S. epidermidis; Skin Microbiome
7.  ω3 fatty acid desaturases from microorganisms: structure, function, evolution, and biotechnological use 
Applied microbiology and biotechnology  2013;97(24):10255-10262.
The biosynthesis of very-long-chain polyunsaturated fatty acids involves an alternating process of fatty acid desaturation and elongation catalyzed by complex series of enzymes. ω3 desaturase plays an important role in converting ω6 fatty acids into ω3 fatty acids. Genes for this desaturase have been identified and characterized in a wide range of microorganisms, including cyanobacteria, yeasts, molds, and microalgae. Like all fatty acid desaturases, ω3 desaturase is structurally characterized by the presence of three highly conserved histidine-rich motifs; however, unlike some desaturases, it lacks a cytochrome b5-like domain. Understanding the structure, function, and evolution of ω3 desaturases, particularly their substrate specificities in the biosynthesis of very-long-chain polyunsaturated fatty acids, lays the foundation for potential production of various ω3 fatty acids in transgenic microorganisms.
doi:10.1007/s00253-013-5336-5
PMCID: PMC3937066  PMID: 24177732
Polyunsaturated fatty acids (PUFAs); ω3 desaturase; Lipid biochemistry; Substrate specificity; Biotechnology
8.  Modifying the lipid content and composition of plant seeds: engineering the production of LC-PUFA 
Omega-3 fatty acids are characterized by a double bond at the third carbon atom from the end of the carbon chain. Latterly, long chain polyunsaturated omega-3 fatty acids such as eicosapentaenoic acid (EPA; 20:5Δ5,8,11,14,17) and docosahexanoic acid (DHA; 22:6 Δ4,7,10,13,16,19), which typically only enter the human diet via the consumption of oily fish, have attracted much attention. The health benefits of the omega-3 LC-PUFAs EPA and DHA are now well established. Given the desire for a sustainable supply of omega-LC-PUFA, efforts have focused on enhancing the composition of vegetable oils to include these important fatty acids. Specifically, EPA and DHA have been the focus of much study, with the ultimate goal of producing a terrestrial plant-based source of these so-called fish oils. Over the last decade, many genes encoding the primary LC-PUFA biosynthetic activities have been identified and characterized. This has allowed the reconstitution of the LC-PUFA biosynthetic pathway in oilseed crops, producing transgenic plants engineered to accumulate omega-3 LC-PUFA to levels similar to that found in fish oil. In this review, we will describe the most recent developments in this field and the challenges of overwriting endogenous seed lipid metabolism to maximize the accumulation of these important fatty acids.
doi:10.1007/s00253-014-6217-2
PMCID: PMC4286622  PMID: 25417743
Omega-3 fatty acids; Polyunsaturated fatty acid; Triacylglycerol; Transgenic plant; Oilseed; Camelina sativa
9.  Isolation, sequencing, and heterologous expression of the Paecilomyces variotii gene encoding S-hydroxymethylglutathione dehydrogenase (fldA) 
The filamentous fungus Paecilomyces variotii NBRC 109023 (teleomorph: Byssochlamys spectabilis NBRC 109023) degrades formaldehyde at concentrations as high as 2.4 % (w/v). In many prokaryotes and in all known eukaryotes, formaldehyde degradation is catalyzed by S-hydroxymethylglutathione (S-HMGSH) dehydrogenase. We report here the isolation and characterization of the gene encoding S-HMGSH dehydrogenase activity in P. variotii. The 1.6-kb fldA gene contained 5 introns and 6 exons, and the corresponding cDNA was 1143 bp, encoding a 40-kDa protein composed of 380 amino acids. FldA was predicted to have 74.3, 73.7, 68.5, and 67.4 % amino acid identity to the S-HMGSH dehydrogenases of Hansenula polymorpha, Candida boidinii, Saccharomyces cerevisiae, and Kluyveromyces lactis, respectively. The predicted protein also showed high amino acid similarity (84∼86 %) to the products of putative fldA genes from other filamentous fungi, including Aspergillus sp. and Penicillium sp. Notably, the P. variotii fldA gene was able to functionally complement a Saccharomyces cerevisiae strain (BY4741 ∆sfa1) lacking the gene for S-HMGSH dehydrogenase. The heterologous expression construct rendered BY4741 ∆sfa1 tolerant to exogenous formaldehyde. Although BY4741 (parental wild-type strain) was unable to degrade even low concentrations of formaldehyde, BY4741 ∆sfa1 harboring Paecilomyces fldA was able to degrade 4 mM formaldehyde within 30 h. The findings from this study confirm the essential role of S-HMGSH dehydrogenase in detoxifying formaldehyde.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-014-6203-8) contains supplementary material, which is available to authorized users.
doi:10.1007/s00253-014-6203-8
PMCID: PMC4322224  PMID: 25398285
Paecilomyces variotii; Formaldehyde degradation; S-hydroxymethylglutathione dehydrogenase; fldA; Heterologous expression
10.  Bacillus subtilis as heterologous host for the secretory production of the non-ribosomal cyclodepsipeptide enniatin 
The heterologous expression of genes or gene clusters in microbial hosts, followed by metabolic engineering of biosynthetic pathways, is key to access industrially and pharmaceutically relevant compounds in an economically affordable and sustainable manner. Therefore, platforms need to be developed, which provide tools for the controlled synthesis of bioactive compounds. The Gram-positive bacterium Bacillus subtilis is a promising candidate for such applications, as it is generally regarded as a safe production host, its physiology is well investigated and a variety of tools is available for its genetic manipulation. Furthermore, this industrially relevant bacterium provides a high secretory potential not only for enzymes but also for primary and secondary metabolites. In this study, we present the first heterologous expression of an eukaryotic non-ribosomal peptide synthetase gene (esyn) coding for the biosynthesis of the small molecule enniatin in B. subtilis. Enniatin is a pharmaceutically used cyclodepsipeptide for treatment of topical bacterial and fungal infections. We generated various enniatin-producing B. subtilis strains, allowing for either single chromosomal or plasmid-based multi-copy expression of the esyn cluster under the control of an acetoin-inducible promoter system. Optimization of cultivation conditions, combined with modifications of the genetic background and multi-copy plasmid-based esyn expression, resulted in a secretory production of enniatin B. This work presents B. subtilis as a suitable host for the expression of heterologous eukaryotic non-ribosomal peptide synthetases (NRPS) clusters.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-014-6199-0) contains supplementary material, which is available to authorized users.
doi:10.1007/s00253-014-6199-0
PMCID: PMC4306738  PMID: 25398283
Heterologous expression; Iterative NRPS; Cyclodepsipeptide; acoA promoter; Acetoin; Metabolic engineering
11.  ClaR—a novel key regulator of cellobiose and lactose metabolism in Lactococcus lactis IL1403 
In a number of previous studies, our group has discovered an alternative pathway for lactose utilization in Lactococcus lactis that, in addition to a sugar-hydrolyzing enzyme with both P-β-glucosidase and P-β-galactosidase activity (BglS), engages chromosomally encoded components of cellobiose-specific PTS (PTSCel-Lac), including PtcA, PtcB, and CelB. In this report, we show that this system undergoes regulation via ClaR, a novel activator protein from the RpiR family of transcriptional regulators. Although RpiR proteins are widely distributed among lactic acid bacteria, their roles have yet to be confirmed by functional assays. Here, we show that ClaR activity depends on intracellular cellobiose-6-phosphate availability, while other sugars such as glucose or galactose have no influence on it. We also show that ClaR is crucial for activation of the bglS and celB expression in the presence of cellobiose, with some limited effects on ptcA and ptcB activation. Among 190 of carbon sources tested, the deletion of claR reduces L. lactis growth only in lactose- and/or cellobiose-containing media, suggesting a narrow specificity of this regulator within the context of sugar metabolism.
doi:10.1007/s00253-014-6067-y
PMCID: PMC4286628  PMID: 25239037
Lactose assimilation; Cellobiose assimilation; Phosphotransferase system (PTS); Genes regulation; Lactococcus lactis
12.  The biosurfactant viscosin transiently stimulates n-hexadecane mineralization by a bacterial consortium 
Pseudomonas produces powerful lipopeptide biosurfactants including viscosin, massetolide A, putisolvin, and amphisin, but their ability to stimulate alkane mineralization and their utility for bioremediation have received limited attention. The four Pseudomonas lipopeptides yielded emulsification indices on hexadecane of 20–31 % at 90 mg/l, which is comparable to values for the synthetic surfactant Tween 80. Viscosin was the optimal emulsifier and significantly stimulated n-hexadecane mineralization by diesel-degrading bacterial consortia but exclusively during the first 2 days of batch culture experiments. Growth of the consortia, as determined by OD600 measurements and quantification of the alkB marker gene for alkane degradation, was arrested after the first day of the experiment. In contrast, the control consortia continued to grow and reached higher OD600 values and higher alkB copy numbers during the next days. Due to the short-lived stimulation of n-hexadecane mineralization, the stability of viscosin was analyzed, and it was observed that added viscosin was degraded by the bacterial consortium during the first 2 days. Hence, viscosin has a potential as stimulator of alkane degradation, but its utility in bioremediation may be limited by its rapid degradation and growth-inhibiting properties.
doi:10.1007/s00253-014-6054-3
PMCID: PMC4306737  PMID: 25216581
Alkanes; Biodegradation; Lipopeptides; Pseudomonas
13.  Sustainable conversion of coffee and other crop wastes to biofuels and bioproducts using coupled biochemical and thermochemical processes in a multi-stage biorefinery concept 
Applied Microbiology and Biotechnology  2014;98(20):8413-8431.
The environmental impact of agricultural waste from the processing of food and feed crops is an increasing concern worldwide. Concerted efforts are underway to develop sustainable practices for the disposal of residues from the processing of such crops as coffee, sugarcane, or corn. Coffee is crucial to the economies of many countries because its cultivation, processing, trading, and marketing provide employment for millions of people. In coffee-producing countries, improved technology for treatment of the significant amounts of coffee waste is critical to prevent ecological damage. This mini-review discusses a multi-stage biorefinery concept with the potential to convert waste produced at crop processing operations, such as coffee pulping stations, to valuable biofuels and bioproducts using biochemical and thermochemical conversion technologies. The initial bioconversion stage uses a mutant Kluyveromyces marxianus yeast strain to produce bioethanol from sugars. The resulting sugar-depleted solids (mostly protein) can be used in a second stage by the oleaginous yeast Yarrowia lipolytica to produce bio-based ammonia for fertilizer and are further degraded by Y. lipolytica proteases to peptides and free amino acids for animal feed. The lignocellulosic fraction can be ground and treated to release sugars for fermentation in a third stage by a recombinant cellulosic Saccharomyces cerevisiae, which can also be engineered to express valuable peptide products. The residual protein and lignin solids can be jet cooked and passed to a fourth-stage fermenter where Rhodotorula glutinis converts methane into isoprenoid intermediates. The residues can be combined and transferred into pyrocracking and hydroformylation reactions to convert ammonia, protein, isoprenes, lignins, and oils into renewable gas. Any remaining waste can be thermoconverted to biochar as a humus soil enhancer. The integration of multiple technologies for treatment of coffee waste has the potential to contribute to economic and environmental sustainability.
doi:10.1007/s00253-014-5991-1
PMCID: PMC4192581  PMID: 25204861
Coffee waste; Multi-stage biorefinery; Oleaginous yeast triglycerides; Renewable biofuel; Bioprocessing
14.  Pyrosequence analyses of bacterial communities during simulated in situ bioremediation of polycyclic aromatic hydrocarbon-contaminated soil 
Applied microbiology and biotechnology  2012;97(18):8381-8391.
Barcoded amplicon pyrosequencing was used to generate libraries of partial 16S rRNA genes from two columns designed to simulate in situ bioremediation of polycyclic aromatic hydrocarbons (PAHs) in weathered, contaminated soil. Both columns received a continuous flow of artificial groundwater but one of the columns additionally tested the impact of biostimulation with oxygen and inorganic nutrients on indigenous soil bacterial communities. The penetration of oxygen to previously anoxic regions of the columns resulted in the most significant community changes. PAH-degrading bacteria previously determined by stable-isotope probing (SIP) of the untreated soil generally responded negatively to the treatment conditions, with only members of the Acidovorax and a group of uncharacterized PAH-degrading Gammaproteobacteria maintaining a significant presence in the columns. Additional groups of sequences associated with the Betaproteobacterial family Rhodocyclaceae (including those associated with PAH degradation in other soils), and the Thiobacillus, Thermomonas, and Bradyrhizobium genera were also present in high abundance in the biostimulated column. Similar community responses were previously observed during biostimulated ex situ treatment of the same soil in aerobic, slurry-phase bioreactors. While the low relative abundance of many SIP-determined groups in the column libraries may be a reflection of the slow removal of PAHs in that system, the similar response of known PAH-degraders in a higher-rate bioreactor system suggests that alternative PAH-degrading bacteria, unidentified by SIP of the untreated soil, may also be enriched in engineered systems.
doi:10.1007/s00253-012-4531-0
PMCID: PMC3600395  PMID: 23132343
pyrosequencing; polycyclic aromatic hydrocarbons; bioremediation
15.  Non-coding RNAs in the development and pathogenesis of eukaryotic microbes 
Applied microbiology and biotechnology  2013;97(18):7989-7997.
RNA has long been regarded as the important intermediary in the central dogma of gene expression. Recently, the importance of RNAs in the regulation of gene expression became evident with the identification and characterization of non-protein coding transcripts named noncoding RNA (ncRNA). The ncRNAs, small and long, are ubiquitously present in all three domains of life and are being recognized for their important roles in genome defense and development. Some of the ncRNAs have been associated with diseases and therefore they offer diagnostic and therapeutic potential. In this mini-review, we have highlighted some recent research on the ncRNAs identified in eukaryotic microbes, with special emphasis on fungi that are pathogenic to humans or plants when possible. It is our contention that further elucidation and understanding of ncRNAs will advance our understanding of the development and pathogenesis of eukaryotic microbes and offer alternatives in the diagnosis and treatment of the diseases caused by these pathogens.
doi:10.1007/s00253-013-5160-y
PMCID: PMC3791853  PMID: 23948725
non-coding RNA; RNAi; lncRNA; fungi; eukaryotic microbes; regulatory RNA
16.  Biochemical and structural characterization of a thermostable β-glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis 
Lactose is a major disaccharide by-product from the dairy industries, and production of whey alone amounts to about 200 million tons globally each year. Thus, it is of particular interest to identify improved enzymatic processes for lactose utilization. Microbial β-glucosidases (BGL) with significant β-galactosidase (BGAL) activity can be used to convert lactose to glucose (Glc) and galactose (Gal), and most retaining BGLs also synthesize more complex sugars from the monosaccharides by transglycosylation, such as galacto-oligosaccharides (GOS), which are prebiotic compounds that stimulate growth of beneficial gut bacteria. In this work, a BGL from the thermophilic and halophilic bacterium Halothermothrix orenii, HoBGLA, was characterized biochemically and structurally. It is an unspecific β-glucosidase with mixed activities for different substrates and prominent activity with various galactosidases such as lactose. We show that HoBGLA is an attractive candidate for industrial lactose conversion based on its high activity and stability within a broad pH range (4.5–7.5), with maximal β-galactosidase activity at pH 6.0. The temperature optimum is in the range of 65–70 °C, and HoBGLA also shows excellent thermostability at this temperature range. The main GOS products from HoBGLA transgalactosylation are β-d-Galp-(1→6)-d-Lac (6GALA) and β-d-Galp-(1→3)-d-Lac (3GALA), indicating that d-lactose is a better galactosyl acceptor than either of the monosaccharides. To evaluate ligand binding and guide GOS modeling, crystal structures of HoBGLA were determined in complex with thiocellobiose, 2-deoxy-2-fluoro-d-glucose and glucose. The two major GOS products, 3GALA and 6GALA, were modeled in the substrate-binding cleft of wild-type HoBGLA and shown to be favorably accommodated.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-014-6015-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s00253-014-6015-x
PMCID: PMC4322223  PMID: 25173693
β-glucosidase; β-galactosidase; Halothermophile; Halothermothrix; Lactose conversion; Galacto-oligosaccharides; Biochemical characterization; Structural analysis
17.  Accurate comparison of antibody expression levels by reproducible transgene targeting in engineered recombination-competent CHO cells 
Applied Microbiology and Biotechnology  2014;98(23):9723-9733.
Over the years, Chinese hamster ovary (CHO) cells have emerged as the major host for expressing biotherapeutic proteins. Traditional methods to generate high-producer cell lines rely on random integration(s) of the gene of interest but have thereby left the identification of bottlenecks as a challenging task. For comparison of different producer cell lines derived from various transfections, a system that provides control over transgene expression behavior is highly needed. This motivated us to develop a novel “DUKX-B11 F3/F” cell line to target different single-chain antibody fragments into the same chromosomal target site by recombinase-mediated cassette exchange (RMCE) using the flippase (FLP)/FLP recognition target (FRT) system. The RMCE-competent cell line contains a gfp reporter fused to a positive/negative selection system flanked by heterospecific FRT (F) variants under control of an external CMV promoter, constructed as “promoter trap”. The expression stability and FLP accessibility of the tagged locus was demonstrated by successive rounds of RMCE. As a proof of concept, we performed RMCE using cassettes encoding two different anti-HIV single-chain Fc fragments, 3D6scFv-Fc and 2F5scFv-Fc. Both targeted integrations yielded homogenous cell populations with comparable intracellular product contents and messenger RNA (mRNA) levels but product related differences in specific productivities. These studies confirm the potential of the newly available “DUKX-B11 F3/F” cell line to guide different transgenes into identical transcriptional control regions by RMCE and thereby generate clones with comparable amounts of transgene mRNA. This new host is a prerequisite for cell biology studies of independent transfections and transgenes.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-014-6011-1) contains supplementary material, which is available to authorized users.
doi:10.1007/s00253-014-6011-1
PMCID: PMC4231286  PMID: 25158835
Site-directed integration; Cell engineering; Flippase; Specific productivity; RMCE target site
18.  Manipulation of fungal development as source of novel secondary metabolites for biotechnology 
Applied Microbiology and Biotechnology  2014;98(20):8443-8455.
Fungal genomics revealed a large potential of yet-unexplored secondary metabolites, which are not produced during vegetative growth. The discovery of novel bioactive compounds is increasingly gaining importance. The high number of resistances against established antibiotics requires novel drugs to counteract increasing human and animal mortality rates. In addition, growth of plant pathogens has to be controlled to minimize harvest losses. An additional critical issue is the post-harvest production of deleterious mycotoxins. Fungal development and secondary metabolite production are linked processes. Therefore, molecular regulators of development might be suitable to discover new bioactive fungal molecules or to serve as targets to control fungal growth, development, or secondary metabolite production. The fungal impact is relevant as well for our healthcare systems as for agriculture. We propose here to use the knowledge about mutant strains discovered in fungal model systems for a broader application to detect and explore new fungal drugs or toxins. As examples, mutant strains impaired in two conserved eukaryotic regulatory complexes are discussed. The COP9 signalosome (CSN) and the velvet complex act at the interface between development and secondary metabolism. The CSN is a multi-protein complex of up to eight subunits and controls the activation of CULLIN-RING E3 ubiquitin ligases, which mark substrates with ubiquitin chains for protein degradation by the proteasome. The nuclear velvet complex consists of the velvet-domain proteins VeA and VelB and the putative methyltransferase LaeA acting as a global regulator for secondary metabolism. Defects in both complexes disturb fungal development, light perception, and the control of secondary metabolism. The potential biotechnological relevance of these developmental fungal mutant strains for drug discovery, agriculture, food safety, and human healthcare is discussed.
doi:10.1007/s00253-014-5997-8
PMCID: PMC4192562  PMID: 25142695
Velvet domain proteins; COP9 signalosome CSN; Secondary metabolites; Fungal development
19.  Roles of type II thioesterases and their application for secondary metabolite yield improvement 
Applied Microbiology and Biotechnology  2014;98(18):7735-7746.
A large number of antibiotics and other industrially important microbial secondary metabolites are synthesized by polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). These multienzymatic complexes provide an enormous flexibility in formation of diverse chemical structures from simple substrates, such as carboxylic acids and amino acids. Modular PKSs and NRPSs, often referred to as megasynthases, have brought about a special interest due to the colinearity between enzymatic domains in the proteins working as an “assembly line” and the chain elongation and modification steps. Extensive efforts toward modified compound biosynthesis by changing organization of PKS and NRPS domains in a combinatorial manner laid good grounds for rational design of new structures and their controllable biosynthesis as proposed by the synthetic biology approach. Despite undeniable progress made in this field, the yield of such “unnatural” natural products is often not satisfactory. Here, we focus on type II thioesterases (TEIIs)—discrete hydrolytic enzymes often encoded within PKS and NRPS gene clusters which can be used to enhance product yield. We review diverse roles of TEIIs (removal of aberrant residues blocking the megasynthase, participation in substrate selection, intermediate, and product release) and discuss their application in new biosynthetic systems utilizing PKS and NRPS parts.
doi:10.1007/s00253-014-5952-8
PMCID: PMC4147253  PMID: 25081554
Type II thioesterase; Polyketide synthase; Nonribosomal peptide synthetase; PKS; NRPS; Synthetic biology
20.  Identification of microRNAs specific for high producer CHO cell lines using steady-state cultivation 
Applied Microbiology and Biotechnology  2014;98(17):7535-7548.
MicroRNAs are short non-coding RNAs that play an important role in the regulation of gene expression. Hence, microRNAs are considered as potential targets for engineering of Chinese hamster ovary (CHO) cells to improve recombinant protein production. Here, we analyzed and compared the microRNA expression patterns of high, low, and non-producing recombinant CHO cell lines expressing two structurally different model proteins in order to identify microRNAs that are involved in heterologous protein synthesis and secretion and thus might be promising targets for cell engineering to increase productivity. To generate reproducible and comparable data, the cells were cultivated in a bioreactor under steady-state conditions. Global microRNA expression analysis showed that mature microRNAs were predominantly upregulated in the producing cell lines compared to the non-producer. Several microRNAs were significantly differentially expressed between high and low producers, but none of them commonly for both model proteins. The identification of target messenger RNAs (mRNAs) is essential to understand the biological function of microRNAs. Therefore, we negatively correlated microRNA and global mRNA expression data and combined them with computationally predicted and experimentally validated targets. However, statistical analysis of the identified microRNA-mRNA interactions indicated a considerable false positive rate. Our results and the comparison to published data suggest that the reaction of CHO cells to the heterologous protein expression is strongly product- and/or clone-specific. In addition, this study highlights the urgent need for reliable CHO-specific microRNA target prediction tools and experimentally validated target databases in order to facilitate functional analysis of high-throughput microRNA expression data in CHO cells.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-014-5911-4) contains supplementary material, which is available to authorized users.
doi:10.1007/s00253-014-5911-4
PMCID: PMC4139590  PMID: 25052466
Chinese hamster ovary cells; Productivity; Chemostat; Microarray; miRNA expression profiling; miRNA target identification
21.  Biotransformation and recovery of the isoflavones genistein and daidzein from industrial antibiotic fermentations 
Applied microbiology and biotechnology  2013;97(14):6427-6437.
The objective of this study was to follow the metabolic fate of isoflavone glucosides from the soybean meal in a model industrial fermentation to determine if commercially useful isoflavones could be harvested as coproducts from the spent broth at the end of the fermentation. The isoflavone aglycones, genistein and daidzein, together make up 0.1 - 0.2% of the soybean meal by weight but serve no known function in the manufacturing process. After feeding genistein to washed cells of the erythromycin-producing organism, Saccharopolyspora erythraea, the first biotransformation product (Gbp1) was determined by x-ray crystallography to be genistein-7-O-α-rhamnoside (rhamnosylgenistein). Subsequent feeding of rhamnosylgenistein to growing cells of S. erythraea led to the production of a second biotransformation product, Gbp2. Chromatographic evidence suggested that Gbp2 accumulated in the spent broth of the erythromycin fermentation. When the spent broth was hydrolyzed with acid or industrial enzyme preparations the isoflavone biotransformation products were returned back to their parental forms, genistein and daidzein, which were then recovered as coproducts. Desirable features of this method are that it does not require modification of the erythromycin manufacturing process or genetic engineering of the producing organism to be put into practice. A preliminary investigation of five additional antibiotic fermentations of industrial importance were also found to have isoflavone coproduct potential.
doi:10.1007/s00253-013-4839-4
PMCID: PMC3700627  PMID: 23604533
Isoflavones; genistein; daidzein; coproduct; soybean; fermentation; Saccharopolyspora erythraea; 7-O-rhamnosylgenistein
22.  An Aspergillus nidulans β-mannanase with high transglycosylation capacity revealed through comparative studies within glycosidase family 5 
Applied Microbiology and Biotechnology  2014;98(24):10091-10104.
β-Mannanases are involved in the conversion and modification of mannan-based saccharides. Using a retaining mechanism, they can, in addition to hydrolysis, also potentially perform transglycosylation reactions, synthesizing new glyco-conjugates. Transglycosylation has been reported for β-mannanases in GH5 and GH113. However, although they share the same fold and catalytic mechanism, there may be differences in the enzymes’ ability to perform transglycosylation. Three GH5 β-mannanases from Aspergillus nidulans, AnMan5A, AnMan5B and AnMan5C, which belong to subfamily GH5_7 were studied. Comparative studies, including the GH5_7 TrMan5A from Trichoderma reesei, showed some differences between the enzymes. All the enzymes could perform transglycosylation but AnMan5B stood out in generating comparably higher amounts of transglycosylation products when incubated with manno-oligosaccharides. In addition, AnMan5B did not use alcohols as acceptor, which was also different compared to the other three β-mannanases. In order to map the preferred binding of manno-oligosaccharides, incubations were performed in H218O. AnMan5B in contrary to the other enzymes did not generate any 18O-labelled products. This further supported the idea that AnMan5B potentially prefers to use saccharides as acceptor instead of water. A homology model of AnMan5B showed a non-conserved Trp located in subsite +2, not present in the other studied enzymes. Strong aglycone binding seems to be important for transglycosylation with saccharides. Depending on the application, it is important to select the right enzyme.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-014-5871-8) contains supplementary material, which is available to authorized users.
doi:10.1007/s00253-014-5871-8
PMCID: PMC4237917  PMID: 24950755
β-mannanase; GH5; Transglycosylation; H218O; Acceptor; MALDI-TOF MS
23.  Investigating bacterial populations in styrene-degrading biofilters by 16S rDNA tag pyrosequencing 
Microbial biofilms are essential components in the elimination of pollutants within biofilters, yet still little is known regarding the complex relationships between microbial community structure and biodegradation function within these engineered ecosystems. To further explore this relationship, 16S rDNA tag pyrosequencing was applied to samples taken at four time points from a styrene-degrading biofilter undergoing variable operating conditions. Changes in microbial structure were observed between different stages of biofilter operation, and the level of styrene concentration was revealed to be a critical factor affecting these changes. Bacterial genera Azoarcus and Pseudomonas were among the dominant classified genera in the biofilter. Canonical correspondence analysis (CCA) and correlation analysis revealed that the genera Brevundimonas, Hydrogenophaga, and Achromobacter may play important roles in styrene degradation under increasing styrene concentrations. No significant correlations (P > 0.05) could be detected between biofilter operational/functional parameters and biodiversity measurements, although biological heterogeneity within biofilms and/or technical variability within pyrosequencing may have considerably affected these results. Percentages of selected bacterial taxonomic groups detected by fluorescence in situ hybridization (FISH) were compared to results from pyrosequencing in order to assess the effectiveness and limitations of each method for identifying each microbial taxon. Comparison of results revealed discrepancies between the two methods in the detected percentages of numerous taxonomic groups. Biases and technical limitations of both FISH and pyrosequencing, such as the binding of FISH probes to non-target microbial groups and lack of classification of sequences for defined taxonomic groups from pyrosequencing, may partially explain some differences between the two methods.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-014-5868-3) contains supplementary material, which is available to authorized users.
doi:10.1007/s00253-014-5868-3
PMCID: PMC4286631  PMID: 24950754
Biofiltration; Styrene; Pyrosequencing; FISH
24.  A contribution to set a legal framework for biofertilisers 
Applied Microbiology and Biotechnology  2014;98(15):6599-6607.
The extensive research, production and use of microorganisms to improve plant nutrition have resulted in an inconsistent definition of the term “biofertiliser” which, in some cases, is due to the different microbial mechanisms involved. The rationale for adopting the term biofertiliser is that it derives from “biological fertiliser”, that, in turn, implies the use of living microorganisms. Here, we propose a definition for this kind of products which is distinguishing them from biostimulants or other inorganic and organic fertilisers. Special emphasis is given to microorganism(s) with multifunctional properties and biofertilisers containing more than one microorganism. This definition could be included in legal provisions regulating registration and marketing requirements. A set of rules is also proposed which could guarantee the quality of biofertilisers present on the market and thus foster their use by farmers.
doi:10.1007/s00253-014-5828-y
PMCID: PMC4108841  PMID: 24903811
PGPR; Mycorrhizal fungi; Rhizosphere; Regulation; Production standards
25.  High cell density cultivation of a recombinant E. coli strain expressing a key enzyme in bioengineered heparin production 
A bioengineered heparin, as a replacement for animal-derived heparin, is under development that relies on the fermentative production of heparosan by Escherichia coli K5 and its subsequent chemoenzymatic modification using biosynthetic enzymes. A critical enzyme in this pathway is the mammalian 6-O-sulfotransferase, and particularly 6-OST-1, which specifically sulfonates the glucosamine residue in a heparin precursor. This mammalian enzyme, previously cloned and expressed in E. coli, is required in kilogram amounts if an industrial process for bioengineered heparin is to be established. In this study high cell density cultivation techniques were exploited to obtain recombinant 6-OST-1. Physiological studies were performed in shake flasks to establish optimized growth and production conditions. Induction strategies were tested in fed-batch experiments to improve yield and productivity. High cell density cultivation in 7-L culture, together with a coupled inducer strategy using isopropyl β-D-1-thiogalactopyranoside and galactose, afforded 482 mg·L−1 of enzyme with a biomass yield of 16.2 mg·gcdw−1 and a productivity of 10.5 mg·L−1·h−1.
doi:10.1007/s00253-012-4682-z
PMCID: PMC3628930  PMID: 23318839
bioengineered heparin; high cell density cultivations; heparosan; sulfotransferase

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