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1.  Exoproteome analysis of Clostridium cellulovorans in natural soft-biomass degradation 
AMB Express  2015;5(1):2.
Clostridium cellulovorans is an anaerobic, cellulolytic bacterium, capable of effectively degrading various types of soft biomass. Its excellent capacity for degradation results from optimization of the composition of the protein complex (cellulosome) and production of non-cellulosomal proteins according to the type of substrates. In this study, we performed a quantitative proteome analysis to determine changes in the extracellular proteins produced by C. cellulovorans for degradation of several types of natural soft biomass. C. cellulovorans was cultured in media containing bagasse, corn germ, rice straw (natural soft biomass), or cellobiose (control). Using an isobaric tag method and a liquid chromatograph equipped with a long monolithic silica capillary column/mass spectrometer, we identified 372 proteins in the culture supernatant. Of these, we focused on 77 saccharification-related proteins of both cellulosomal and non-cellulosomal origins. Statistical analysis showed that 18 of the proteins were specifically produced during degradation of types of natural soft biomass. Interestingly, the protein Clocel_3197 was found and commonly involved in the degradation of every natural soft biomass studied. This protein may perform functions, in addition to its known metabolic functions, that contribute to effective degradation of natural soft biomass.
Electronic supplementary material
The online version of this article (doi:10.1186/s13568-014-0089-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4305082  PMID: 25642399
Clostridium cellulovorans; Cellulosome; Soft-biomass degradation; Proteome analysis; Monolithic column
2.  Simple observation of Streptococcus mutans biofilm by scanning electron microscopy using ionic liquids 
AMB Express  2015;5(1):6.
Scanning electron microscopy (SEM) has been successfully used to image biofilms because of its high resolution and magnification. However, conventional SEM requires dehydration and metal coating of biological samples before observation, and because biofilms consist mainly of water, sample dehydration may influence the biofilm structure. When coated with an ionic liquid, which is a kind of salt that exists in the liquid state at room temperature, biological samples for SEM observation do not require dehydration or metal coating because ionic liquids do not evaporate under vacuum conditions and are electrically conductive. This study investigates the ability of ionic liquids to allow SEM observation of Streptococcus mutans biofilms compared with conventional coating methods. Two hydrophilic and two hydrophobic ionic liquids, all of which are electronic conductors, are used. Compared with samples prepared by the conventional method, the ionic-liquid-treated samples do not exhibit a fibrous extracellular matrix structure and cracking on the biofilm surface. The hydrophilic ionic liquids give clearer images of the biofilm structure than those of the hydrophobic ionic liquids. This study finds that ionic liquids are useful for allowing the observation of biofilms by SEM without preparation by dehydration and metal coating.
Electronic supplementary material
The online version of this article (doi:10.1186/s13568-015-0097-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4305086  PMID: 25642403
Biofilm; Extracellular polymeric substance; Ionic liquid; Scanning electron microscopy; Streptococcus mutans
3.  Fungal strain matters: colony growth and bioactivity of the European medicinal polypores Fomes fomentarius, Fomitopsis pinicola and Piptoporus betulinus 
AMB Express  2015;5(1):4.
Polypores have been applied in traditional Chinese medicine up to the present day, and are becoming more and more popular worldwide. They show a wide range of bioactivities including anti-cancer, anti-inflammatory, antiviral and immuno-enhancing effects. Their secondary metabolites have been the focus of many studies, but the importance of fungal strain for bioactivity and metabolite production has not been investigated so far for these Basidiomycetes. Therefore, we screened several strains from three medicinal polypore species from traditional European medicine: Fomes fomentarius, Fomitopsis pinicola and Piptoporus betulinus. A total of 22 strains were compared concerning their growth rates, optimum growth temperatures, as well as antimicrobial and antifungal properties of ethanolic fruit body extracts. The morphological identification of strains was confirmed based on rDNA ITS phylogenetic analyses. Our results showed that species delimitation is critical due to the presence of several distinct lineages, e.g. within the Fomes fomentarius species complex. Fungal strains within one lineage showed distinct differences in optimum growth temperatures, in secondary metabolite production, and accordingly, in their bioactivities. In general, F. pinicola and P. betulinus extracts exerted distinct antibiotic activities against Bacillus subtilis and Staphylococcus aureus at minimum inhibitory concentrations (MIC) ranging from 31-125 μg mL−1; The antifungal activities of all three polypores against Aspergillus flavus, A. fumigatus, Absidia orchidis and Candida krusei were often strain-specific, ranging from 125-1000 μg mL−1. Our results highlight that a reliable species identification, followed by an extensive screening for a ‘best strain’ is an essential prerequisite for the proper identification of bioactive material.
Electronic supplementary material
The online version of this article (doi:10.1186/s13568-014-0093-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4305089  PMID: 25642401
Fungal strain selection; Temperature optimum; Wood-rotting fungi; Antimicrobial activity of fungal extracts; Phylogeny
4.  Feasibility study of an alkaline-based chemical treatment for the purification of polyhydroxybutyrate produced by a mixed enriched culture 
AMB Express  2015;5(1):5.
This study focused on investigating the feasibility of purifying polyhydroxybutyrate (PHB) from mixed culture biomass by alkaline-based chemical treatment. The PHB-containing biomass was enriched on acetate under non-sterile conditions. Alkaline treatment (0.2 M NaOH) together with surfactant SDS (0.2 w/v% SDS) could reach 99% purity, with more than 90% recovery. The lost PHB could be mostly attributed to PHB hydrolysis during the alkaline treatment. PHB hydrolysis could be moderated by increasing the crystallinity of the PHB granules, for example, by biomass pretreatment (e.g. freezing or lyophilization) or by effective cell lysis (e.g. adjusting alkali concentration). The suitability of the purified PHB by alkaline treatment for polymer applications was evaluated by molecular weight and thermal stability. A solvent based purification method was also performed for comparison purposes. As result, PHB produced by mixed enriched cultures was found suitable for thermoplastic applications when purified by the solvent method. While the alkaline method resulted in purity, recovery yield and molecular weight comparable to values reported in literature for PHB produced by pure cultures, it was found unsuitable for thermoplastic applications. Given the potential low cost and favorable environmental impact of this method, it is expected that PHB purified by alkaline method may be suitable for other non-thermal polymer applications, and as a platform chemical.
PMCID: PMC4305094  PMID: 25642402
Polyhydroxybutyrate; Alkaline treatment; Crystallinity; Thermal stability; Mixed cultures
5.  Characterization of Aspergillus aculeatus β-glucosidase 1 accelerating cellulose hydrolysis with Trichoderma cellulase system 
AMB Express  2015;5(1):3.
Aspergillus aculeatus β-glucosidase 1 (AaBGL1), which promotes cellulose hydrolysis by Trichoderma cellulase system, was characterized and compared some properties to a commercially supplied orthologue in A. niger (AnBGL) to elucidate advantages of recombinant AaBGL1 (rAaBGL1) for synergistic effect on Trichoderma enzymes. Steady–state kinetic studies revealed that rAaBGL1 showed high catalytic efficiency towards β-linked glucooligosaccharides. Up to a degree of polymerization (DP) 3, rAaBGL1 prefered to hydrolyze β-1,3 linked glucooligosaccharides, but longer than DP 3, preferred β-1,4 glucooligosaccharides (up to DP 5). This result suggested that there were different formation for subsites in the catalytic cleft of AaBGL1 between β-1,3 and β-1,4 glucooligosaccharides, therefore rAaBGL1 preferred short chain of laminarioligosaccharides and long chain of cellooligosaccharides on hydrolysis. rAaBGL1 was more insensitive to glucose inhibition and more efficient to hydrolyze the one of major transglycosylation product, gentiobiose than AnBGL, resulting that rAaBGL1 completely hydrolyzed 5% cellobiose to glucose faster than AnBGL. These data indicate that AaBGL1 is valuable for the use of cellulosic biomass conversion.
PMCID: PMC4305095  PMID: 25642400
Aspergillus aculeatus; β-glucosidase; Biomass conversion; Trichoderma reesei; Cellulase
6.  Genetic transformation of Knufia petricola A95 - a model organism for biofilm-material interactions 
AMB Express  2014;4:80.
We established a protoplast-based system to transfer DNA to Knufia petricola strain A95, a melanised rock-inhabiting microcolonial fungus that is also a component of a model sub-aerial biofilm (SAB) system. To test whether the desiccation resistant, highly melanised cell walls would hinder protoplast formation, we treated a melanin-minus mutant of A95 as well as the type-strain with a variety of cell-degrading enzymes. Of the different enzymes tested, lysing enzymes from Trichoderma harzianum were most effective in producing protoplasts. This mixture was equally effective on the melanin-minus mutant and the type-strain. Protoplasts produced using lysing enzymes were mixed with polyethyleneglycol (PEG) and plasmid pCB1004 which contains the hygromycin B (HmB) phosphotransferase (hph) gene under the control of the Aspergillus nidulans trpC. Integration and expression of hph into the A95 genome conferred hygromycin resistance upon the transformants. Two weeks after plating out on selective agar containing HmB, the protoplasts developed cell-walls and formed colonies. Transformation frequencies were in the range 36 to 87 transformants per 10 μg of vector DNA and 106 protoplasts. Stability of transformation was confirmed by sub-culturing the putative transformants on selective agar containing HmB as well as by PCR-detection of the hph gene in the colonies. The hph gene was stably integrated as shown by five subsequent passages with and without selection pressure.
PMCID: PMC4230810  PMID: 25401079
DNA transfer; Fungal cell-walls; Protoplasts; Hygromycin resistance; Black yeast; Sub-aerial biofilms; Stress-protective morphology; Ancestor of opportunistic pathogens & lichens
7.  Paracentrin 1, a synthetic antimicrobial peptide from the sea-urchin Paracentrotus lividus, interferes with staphylococcal and Pseudomonas aeruginosa biofilm formation 
AMB Express  2014;4:78.
The rise of antibiotic-resistance as well as the reduction of investments by pharmaceutical companies in the development of new antibiotics have stimulated the investigation for alternative strategies to conventional antibiotics. Many antimicrobial peptides show a high specificity for prokaryotes and a low toxicity for eukaryotic cells and, due to their mode of action the development of resistance is considered unlikely. We recently characterized an antimicrobial peptide that was called Paracentrin 1 from the 5-kDa peptide fraction from the coelomocyte cytosol of the Paracentrotus lividus. In this study, the chemically synthesized Paracentrin 1, was tested for its antimicrobial and antibiofilm properties against reference strains of Gram positive and Gram negative. The Paracentrin 1 was active against planktonic form of staphylococcal strains (reference and isolates) and Pseudomonas aeruginosa ATCC 15442 at concentrations ranging from 12.5 to 6.2 mg/ml. The Paracentrin 1 was able to inhibit biofilm formation of staphylococcal and Pseudomonas aeruginosa strains at concentrations ranging from 3.1 to 0.75 mg/ml. We consider the tested peptide as a good starting molecule for novel synthetic derivatives with improved pharmaceutical potential.
PMCID: PMC4230904  PMID: 25401078
AMP (Antimicrobial peptides); Biofilm; Staphylococci; Pseudomonas aeruginosa; Paracentrotus lividus
8.  NADH-dependent biosensor in Saccharomyces cerevisiae: principle and validation at the single cell level 
AMB Express  2014;4:81.
A reporter system was constructed to measure perturbations in the NADH/NAD+ co-factor balance in yeast, by using the green fluorescent protein gene under the control of the GPD2 promoter that is induced under conditions of excess of NADH. High fluorescence levels were obtained in a glycerol 3-phosphate dehydrogenase double deletion strain (gpd1Δgpd2Δ), which is deficient in the ability to regenerate NAD+ via glycerol formation. The responsiveness of the reporter system to externally induced perturbations in NADH oxidation was also evaluated in the gpd1Δgpd2Δ strain background by addition of acetoin, as well as by introduction of a set of heterologous xylose reductases (XRs) having different selectivities for NADH. Addition of acetoin during cell proliferation under oxygen-limited conditions resulted in a more than 2-fold decrease in mean fluorescence intensity as compared to the control experiment. Strains carrying XRs with different selectivities for NADH could be distinguished at the single cell level, so that the XR with the highest selectivity for NADH displayed the lowest fluorescence. In conclusion, the designed system successfully allowed for monitoring perturbations in the cellular redox metabolism caused by environmental changes, or by heterologous gene expression. The reporter system displayed high resolution in distinguishing cytosolic NADH oxidation capacity and hence has potential to be used for high-throughput screening based on the fluorescence of single cells.
PMCID: PMC4230897  PMID: 25401080
Saccharomyces cerevisiae; Redox balance; NADH biosensor; Single cell analysis
9.  Diverse allosteric and catalytic functions of tetrameric d-lactate dehydrogenases from three Gram-negative bacteria 
AMB Express  2014;4:76.
NAD-dependent d-lactate dehydrogenases (d-LDHs) reduce pyruvate into d-lactate with oxidation of NADH into NAD+. Although non-allosteric d-LDHs from Lactobacilli have been extensively studied, the catalytic properties of allosteric d-LDHs from Gram-negative bacteria except for Escherichia coli remain unknown. We characterized the catalytic properties of d-LDHs from three Gram-negative bacteria, Fusobacterium nucleatum (FNLDH), Pseudomonas aeruginosa (PALDH), and E. coli (ECLDH) to gain an insight into allosteric mechanism of d-LDHs. While PALDH and ECLDH exhibited narrow substrate specificities toward pyruvate like usual d-LDHs, FNLDH exhibited a broad substrate specificity toward hydrophobic 2-ketoacids such as 2-ketobutyrate and 2-ketovalerate, the former of which gave a 2-fold higher kcat/S0.5 value than pyruvate. Whereas the three enzymes consistently showed hyperbolic shaped pyruvate saturation curves below pH 6.5, FNLDH and ECLDH, and PALDH showed marked positive and negative cooperativity, respectively, in the pyruvate saturation curves above pH 7.5. Oxamate inhibited the catalytic reactions of FNLDH competitively with pyruvate, and the PALDH reaction in a mixed manner at pH 7.0, but markedly enhanced the reactions of the two enzymes at low concentration through canceling of the apparent homotropic cooperativity at pH 8.0, although it constantly inhibited the ECLDH reaction. Fructose 1,6-bisphosphate and certain divalent metal ions such as Mg2+ also markedly enhanced the reactions of FNLDH and PALDH, but none of them enhanced the reaction of ECLDH. Thus, our study demonstrates that bacterial d-LDHs have highly divergent allosteric and catalytic properties.
PMCID: PMC4230899  PMID: 25401076
Allosteric regulation; NAD-dependent d-lactate dehydrogenase; Gram-negative bacteria; Escherichia coli; Fusobacterium nucleatum; Pseudomonas aeruginosa
10.  Effect of high pressure on hydrocarbon-degrading bacteria 
AMB Express  2014;4:77.
The blowout of the Deepwater Horizon in the Gulf of Mexico in 2010 occurred at a depth of 1500 m, corresponding to a hydrostatic pressure of 15 MPa. Up to now, knowledge about the impact of high pressure on oil-degrading bacteria has been scarce. To investigate how the biodegradation of crude oil and its components is influenced by high pressures, like those in deep-sea environments, hydrocarbon degradation and growth of two model strains were studied in high-pressure reactors. The alkane-degrading strain Rhodococcus qingshengii TUHH-12 grew well on n-hexadecane at 15 MPa at a rate of 0.16 h−1, although slightly slower than at ambient pressure (0.36 h−1). In contrast, the growth of the aromatic hydrocarbon degrading strain Sphingobium yanoikuyae B1 was highly affected by elevated pressures. Pressures of up to 8.8 MPa had little effect on growth of this strain. However, above this pressure growth decreased and at 12 MPa or more no more growth was observed. Nevertheless, S. yanoikuyae continued to convert naphthalene at pressure >12 MPa, although at a lower rate than at 0.1 MPa. This suggests that certain metabolic functions of this bacterium were inhibited by pressure to a greater extent than the enzymes responsible for naphthalene degradation. These results show that high pressure has a strong influence on the biodegradation of crude oil components and that, contrary to previous assumptions, the role of pressure cannot be discounted when estimating the biodegradation and ultimate fate of deep-sea oil releases such as the Deepwater Horizon event.
PMCID: PMC4230898  PMID: 25401077
Biodegradation; High pressure; Hydrocarbons; Naphthalene; n-Hexadecane
11.  Metabolism of alkenes and ketones by Candida maltosa and related yeasts 
AMB Express  2014;4:75.
Knowledge is scarce about the degradation of ketones in yeasts. For bacteria a subterminal degradation of alkanes to ketones and their further metabolization has been described which always involved Baeyer-Villiger monooxygenases (BVMOs). In addition, the question has to be clarified whether alkenes are converted to ketones, in particular for the oil degrading yeast Candida maltosa little is known. In this study we show the degradation of the aliphatic ketone dodecane-2-one by Candida maltosa and the related yeasts Candida tropicalis, Candida catenulata and Candida albicans as well as Trichosporon asahii and Yarrowia lipolytica. One pathway is initiated by the formation of decyl acetate, resulting from a Baeyer-Villiger-oxidation of this ketone. Beyond this, an initial reduction to dodecane-2-ol by a keto reductase was clearly shown. In addition, two different ways to metabolize dodec-1-ene were proposed. One involved the formation of dodecane-2-one and the other one a conversion leading to carboxylic and dicarboxylic acids. Furthermore the induction of ketone degrading enzymes by dodecane-2-one and dodec-1-ene was shown. Interestingly, with dodecane no subterminal degradation products were detected and it did not induce any enzymes to convert dodecane-2-one.
PMCID: PMC4192553  PMID: 25309846
Hydrocarbon; alkene; ketone; Candida; yeast; biotransformation
12.  Trametes meyenii possesses elevated dye degradation abilities under normal nutritional conditions compared to other white rot fungi 
AMB Express  2014;4:74.
Several species of white-rot fungi were investigated for their utility in prolonged decolouration of the recalcitrant sulfonated azo dye, amaranth. Trametes pubescens, T. multicolor, T. meyenii and T. versicolor decoloured amaranth azo-dye best on low-nitrogen agar-solidified media whereas Bjerkandera adusta and Phlebia radiata were most effective in low nitrogen medium supplemented with manganese. Trametes cotonea did not decolour effectively under any condition. The decolouring Trametes species were also effective in liquid culture whereas B. adusta and P. radiata were not. Trametes meyenii, T. pubescens and T. multicolor were equal to or better than commonly employed T. versicolor at decolouring amaranth. This is the first study to show the dye decolouration potential of T. meyenii, T. pubescens, and T. multicolor. Supplementing with Mn(II) increased assayable manganese peroxidase activity, but not long-term decolouration, indicating that laccase is the main decolourizing enzyme in these Trametes species. This appears to be because of inadequate Mn3+ chelation required by manganese peroxidase because adding relatively low amounts of malonate enhanced decolouration rates. The ability of Trametes meyenii to simultaneously decolour dye over prolonged periods of time while growing in relatively nutrient-rich medium appears to be unique amongst white-rot fungi, indicating its potential in wastewater bioremediation.
PMCID: PMC4230814  PMID: 25401075
White-rot fungi; Dye decolouration; Trametes; Laccase; Manganese peroxidase; Manganic chelation
13.  Biodegradation of variable-chain-length n-alkanes in Rhodococcus opacus R7 and the involvement of an alkane hydroxylase system in the metabolism 
AMB Express  2014;4:73.
Rhodococcus opacus R7 is a Gram-positive bacterium isolated from a polycyclic aromatic hydrocarbon contaminated soil for its versatile metabolism; indeed the strain is able to grow on naphthalene, o-xylene, and several long- and medium-chain n-alkanes. In this work we determined the degradation of n-alkanes in Rhodococcus opacus R7 in presence of n-dodecane (C12), n-hexadecane (C16), n-eicosane (C20), n-tetracosane (C24) and the metabolic pathway in presence of C12. The consumption rate of C12 was 88%, of C16 was 69%, of C20 was 51% and of C24 it was 78%. The decrement of the degradation rate seems to be correlated to the length of the aliphatic chain of these hydrocarbons. On the basis of the metabolic intermediates determined by the R7 growth on C12, our data indicated that R. opacus R7 metabolizes medium-chain n-alkanes by the primary alcohol formation. This represents a difference in comparison with other Rhodococcus strains, in which a mixture of the two alcohols was observed. By GC-MSD analysis we also identified the monocarboxylic acid, confirming the terminal oxidation.
Moreover, the alkB gene cluster from R. opacus R7 was isolated and its involvement in the n-alkane degradation system was investigated by the cloning of this genomic region into a shuttle-vector E. coli-Rhodococcus to evaluate the alkane hydroxylase activity. Our results showed an increased biodegradation of C12 in the recombinant strain R. erythropolis AP (pTipQT1-alkR7) in comparison with the wild type strain R. erythropolis AP. These data supported the involvement of the alkB gene cluster in the n-alkane degradation in the R7 strain.
PMCID: PMC4230829  PMID: 25401074
Rhodococcus; n-alkanes degradation; Alkane hydroxylase; AlkB; Enzymatic expression
14.  Stepwise enhancement of catalytic performance of haloalkane dehalogenase LinB towards β-hexachlorocyclohexane 
AMB Express  2014;4:72.
Two haloalkane dehalogenases, LinBUT and LinBMI, each with 296 amino acid residues, exhibit only seven amino acid residue differences between them, but LinBMI’s catalytic performance towards β-hexachlorocyclohexane (β-HCH) is considerably higher than LinBUT’s. To elucidate the molecular basis governing this difference, intermediate mutants between LinBUT and LinBMI were constructed and kinetically characterized. The activities of LinBUT-based mutants gradually increased by cumulative mutations into LinBUT, and the effects of the individual amino acid substitutions depended on combination with other mutations. These results indicated that LinBUT’s β-HCH degradation activity can be enhanced in a stepwise manner by the accumulation of point mutations.
PMCID: PMC4230811  PMID: 25401073
β-Hexachlorocyclohexane; Xenobiotics; Biodegradation; Haloalkane dehalogenase; Protein evolution
15.  The use of nano-sized acicular material, sliding friction, and antisense DNA oligonucleotides to silence bacterial genes 
AMB Express  2014;4:70.
Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons. We have used nano-sized acicular material to establish a novel method for bacterial transformation. Here, we generated penetrons that carried antisense DNA adsorbed on nano-sized acicular material (α-sepiolite) by providing sliding friction onto the surface of agar gel; we then investigated whether penetron formation was applicable to gene silencing techniques. Antisense DNA was artificially synthesized as 15 or 90mer DNA oligonucleotides based on the sequences around the translation start codon of target mRNAs. Mixtures of bacterial cells with antisense DNA adsorbed on α-sepiolite were stimulated by sliding friction on the surface of agar gel for 60 s. Upon formation of Escherichia coli penetrons, β-lactamase and β-galactosidase expression was evaluated by counting the numbers of colonies formed on LB agar containing ampicillin and by measuring β-galactosidase activity respectively. The numbers of ampicillin resistant colonies and the β-galactosidase activity derived from penetrons bearing antisense DNA (90mer) was repressed to 15% and 25%, respectively, of that of control penetrons which lacked antisense DNA. Biphenyl metabolite, ring cleavage yellow compound produced by Pseudomonas pseudoalcaligenes penetron treated with antisense oligonucleotide DNA targeted to bphD increased higher than that lacking antisense DNA. This result indicated that expression of bphD in P. pseudoalcaligenes penetrons was repressed by antisense DNA that targeted bphD mRNA. Sporulation rates of Bacillus subtilis penetrons treated with antisense DNA (15mer) targeted to spo0A decreased to 24.4% relative to penetrons lacking antisense DNA. This novel method of gene silencing has substantial promise for elucidation of gene function in bacterial species that have been refractory to experimental introduction of exogenous DNA.
PMCID: PMC4230895  PMID: 25401071
Antisense oligonucleotide DNA; BphD; Gene silencing; β-Galactosidase; β-Lactamase; Sepiolite; Sliding friction; Spo0A
16.  Co-production of bioethanol and probiotic yeast biomass from agricultural feedstock: application of the rural biorefinery concept 
AMB Express  2014;4:64.
Microbial biotechnology and biotransformations promise to diversify the scope of the biorefinery approach for the production of high-value products and biofuels from industrial, rural and municipal waste feedstocks. In addition to bio-based chemicals and metabolites, microbial biomass itself constitutes an obvious but overlooked by-product of existing biofermentation systems which warrants fuller attention. The probiotic yeast Saccharomyces boulardii is used to treat gastrointestinal disorders and marketed as a human health supplement. Despite its relatedness to S. cerevisiae that is employed widely in biotechnology, food and biofuel industries, the alternative applications of S. boulardii are not well studied. Using a biorefinery approach, we compared the bioethanol and biomass yields attainable from agriculturally-sourced grass juice using probiotic S. boulardii (strain MYA-769) and a commercial S. cerevisiae brewing strain (Turbo yeast). Maximum product yields for MYA-769 (39.18 [±2.42] mg ethanol mL−1 and 4.96 [±0.15] g dry weight L−1) compared closely to those of Turbo (37.43 [±1.99] mg mL−1 and 4.78 [±0.10] g L−1, respectively). Co-production, marketing and/or on-site utilisation of probiotic yeast biomass as a direct-fed microbial to improve livestock health represents a novel and viable prospect for rural biorefineries. Given emergent evidence to suggest that dietary yeast supplementations might also mitigate ruminant enteric methane emissions, the administration of probiotic yeast biomass could also offer an economically feasible way of reducing atmospheric CH4.
PMCID: PMC4230830  PMID: 25401067
Bioethanol; Biomass; Biorefinery; Cholesterol; Probiotic; Saccharomyces boulardii
17.  Characterisation of a recombinant β-xylosidase (xylA) from Aspergillus oryzae expressed in Pichia pastoris 
AMB Express  2014;4:68.
β-xylosidases catalyse the hydrolysis of short chain xylooligosaccharides from their non-reducing ends into xylose. In this study we report the heterologous expression of Aspergillus oryzae β-xylosidase (XylA) in Pichia pastoris under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The recombinant enzyme was optimally active at 55°C and pH 4.5 with Km and Vmax values of 1.0 mM and 250 μmol min−1 mg−1 respectively against 4-nitrophenyl β-xylopyranoside. Xylose was a competitive inhibitor with a Ki of 2.72 mM, whereas fructose was an uncompetitive inhibitor reducing substrate binding affinity (Km) and conversion efficiency (Vmax). The enzyme was characterised to be an exo-cutting enzyme releasing xylose from the non-reducing ends of β-1,4 linked xylooligosaccharides (X2, X3 and X4). Catalytic conversion of X2, X3 and X4 decreased (Vmax and kcat) with increasing chain length.
PMCID: PMC4230903  PMID: 25401069
Aspergillus oryzae; Xylose; β-xylosidase; Enzyme kinetics; Protein expression
18.  Diversity and biochemical features of culturable fungi from the coastal waters of Southern China 
AMB Express  2014;4:60.
Fungi play a major role in various biogeochemical cycles of terrestrial and marine ecosystems. However, fungi in marine environments remain to be one of the most under-studied microbial groups. This study investigates the diversity of planktonic fungi from the coastal habitat off Pearl River Delta (China) using culture-dependent approach. A total of 22 fungi and 9 yeast isolates were recovered from 30 seawater and 2 sediment samples. Microscopic and ITS rRNA gene sequence analyses revealed that most of the fungi belonged to the phylum Ascomycota and Basidiomycota with a very small percentage (3%) of the subphylum Mucoromycotina of the Phylum Zygomycota. Most of these fungal isolates exhibited considerable production of extracellular enzymes, cellulase, lipase and laccase. Fungal isolates of two genera Mucor and Aspergillus sp. demonstrated pelletization capability over a wide range of pH, suggesting them as potential agents towards algae harvesting and wastewater treatment.
PMCID: PMC4230900  PMID: 25401065
Marine-derived fungi; Diversity; Hydrolytic enzymes; Pelleterization
19.  Reduced catabolic protein expression in Clostridium butyricum DSM 10702 correlate with reduced 1,3-propanediol synthesis at high glycerol loading 
AMB Express  2014;4:63.
Higher initial glycerol loadings (620 mM) have a negative effect on growth and 1,3-propanediol (1,3-PDO) synthesis in Clostridium butyricum DSM 10702 relative to lower initial glycerol concentrations (170 mM). To help understand metabolic shifts associated with elevated glycerol, protein expression levels were quantified by LC/MS/MS analyses. Thirty one (31) proteins involved in conversion of glycerol to 1,3-PDO and other by-products were analyzed by multiple reaction monitoring (MRM). The analyses revealed that high glycerol concentrations reduced cell growth. The expression levels of most proteins in glycerol catabolism pathways were down-regulated, consistent with the slower growth rates observed. However, at high initial glycerol concentrations, some of the proteins involved in the butyrate synthesis pathways such as a putative ethanol dehydrogenase (CBY_3753) and a 3-hydroxybutyryl-CoA dehydrogenase (CBY_3045) were up-regulated in both exponential and stationary growth phases. Expression levels of proteins (CBY_0500, CBY_0501 and CBY_0502) involved in the reductive pathway of glycerol to 1,3-PDO were consistent with glycerol consumption and product concentrations observed during fermentation at both glycerol concentrations, and the molar yields of 1,3-PDO were similar in both cultures. This is the first report that correlates expression levels of glycerol catabolism enzymes with synthesis of 1,3-PDO in C. butyricum. The results revealed that significant differences in the expression of a small subset of proteins were observed between exponential and stationary growth phases at both low and high glycerol concentrations.
PMCID: PMC4230902  PMID: 25401066
Clostridium butyricum; 1,3-propanediol synthesis; Glycerol catabolism; Proteomics; Multiple reaction monitoring
20.  Subpopulation-proteomics reveal growth rate, but not cell cycling, as a major impact on protein composition in Pseudomonas putida KT2440 
AMB Express  2014;4:71.
Population heterogeneity occurring in industrial microbial bioprocesses is regarded as a putative effector causing performance loss in large scale. While the existence of subpopulations is a commonly accepted fact, their appearance and impact on process performance still remains rather unclear. During cell cycling, distinct subpopulations differing in cell division state and DNA content appear which contribute individually to the efficiency of the bioprocess. To identify stressed or impaired subpopulations, we analyzed the interplay of growth rate, cell cycle and phenotypic profile of subpopulations by using flow cytometry and cell sorting in conjunction with mass spectrometry based global proteomics. Adjusting distinct growth rates in chemostats with the model strain Pseudomonas putida KT2440, cells were differentiated by DNA content reflecting different cell cycle stages. The proteome of separated subpopulations at given growth rates was found to be highly similar, while different growth rates caused major changes of the protein inventory with respect to e.g. carbon storage, motility, lipid metabolism and the translational machinery.
In conclusion, cells in various cell cycle stages at the same growth rate were found to have similar to identical proteome profiles showing no significant population heterogeneity on the proteome level. In contrast, the growth rate clearly determines the protein composition and therefore the metabolic strategy of the cells.
PMCID: PMC4230896  PMID: 25401072
Heterogeneity; Subpopulations; Pseudomonas putida; Proteome; Flow cytometry; Cell cycle
21.  (S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase (FadB’) from fatty acid degradation operon of Ralstonia eutropha H16 
AMB Express  2014;4:69.
In this study (S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase (H16_A0461/FadB’, gene ID: 4247876) from one of two active fatty acid degradation operons of Ralstonia eutropha H16 has been heterologously expressed in Escherichia coli, purified as protein possessing a His-Tag and initially characterized. FadB’ is an enzyme with two catalytic domains exhibiting a single monomeric structure and possessing a molecular weight of 86 kDa. The C-terminal part of the enzyme harbors enoyl-CoA hydratase activity and is able to convert trans-crotonyl-CoA to 3-hydroxybutyryl-CoA. The N-terminal part of FadB’ comprises an NAD+ binding site and is responsible for 3-hydroxyacyl-CoA dehydrogenase activity converting (S)-3-hydroxybutyryl-CoA to acetoacetyl-CoA. Enoyl-CoA hydratase activity was detected spectrophotometrically with trans-crotonyl-CoA. (S)-3-Hydroxyacyl-CoA dehydrogenase activity was measured in both directions with acetoacetyl-CoA and 3-hydroxybutyryl-CoA. FadB’ was found to be strictly stereospecific to (S)-3-hydroxybutyryl-CoA and to prefer NAD+. The Km value for acetoacetyl-CoA was 48 μM and Vmax 149 μmol mg−1 min−1. NADP(H) was utilized at a rate of less than 10% in comparison to activity with NAD(H). FadB’ exhibited optimal activity at pH 6–7 and the activity decreased at alkaline and acidic pH values. Acetyl-CoA, propionyl-CoA and CoA were found to have an inhibitory effect on FadB’. This study is a first report on biochemical properties of purified (S)-stereospecific 3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase with the inverted domain order from R. eutropha H16. In addition to fundamental information about FadB’ and fatty acid metabolism, FadB’ might be also interesting for biotechnological applications.
PMCID: PMC4230905  PMID: 25401070
Fatty acid metabolism; 3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase, Ralstonia eutropha H16
22.  Identification of a gene, FMP21, whose expression levels are involved in thermotolerance in Saccharomyces cerevisiae 
AMB Express  2014;4:67.
Elucidation of the mechanism of high temperature tolerance in yeasts is important for the molecular breeding of high temperature-tolerant yeasts that can be used in bioethanol production. We identified genes whose expression is correlated with the degree of thermotolerance in Saccharomyces cerevisiae by DNA microarray analysis. Gene expression profiles of three S. cerevisiae strains showing different levels of thermotolerance were compared, and we chose three of them as candidate genes. Among these genes, FMP21 was investigated as a thermotolerance-related gene in S. cerevisiae by comparing the growth at high temperature with the gene expression in eight strains. The expression ratio of FMP21 at 37°C was correlated with the doubling time ratio at a coefficient of determination of 0.787. The potential involvement of the Fmp21 in the thermotolerance of yeasts was evaluated. The FMP21 deletion variant showed a decreased respiratory growth rate and increased thermosensitivity. Furthermore, the overexpression of FMP21 improved thermotolerance in yeasts. In conclusion, the function of Fmp21 is important for thermotolerance in yeasts.
PMCID: PMC4143573  PMID: 25177541
Thermotolerance; Yeast; Gene expression; Growth rate; FMP21
23.  Overexpression of the Aspergillus niger GatA transporter leads to preferential use of D-galacturonic acid over D-xylose 
AMB Express  2014;4:66.
Pectin is a structural heteropolysaccharide of the primary cell walls of plants and as such is a significant fraction of agricultural waste residues that is currently insufficiently used. Its main component, D-galacturonic acid, is an attractive substrate for bioconversion. The complete metabolic pathway is present in the genome of Aspergillus niger, that is used in this study. The objective was to identify the D-galacturonic acid transporter in A. niger and to use this transporter to study D-galacturonic acid metabolism.
We have functionally characterized the gene An14g04280 that encodes the D-galacturonic acid transporter in A. niger. In a mixed sugar fermentation it was found that the An14g04280 overexpression strain, in contrast to the parent control strain, has a preference for D-galacturonic acid over D-xylose as substrate. Overexpression of this transporter in A. niger resulted in a strong increase of D-galacturonic acid uptake and induction of the D-galacturonic acid reductase activity, suggesting a metabolite controlled regulation of the endogenous D-galacturonic acid catabolic pathway.
PMCID: PMC4143577  PMID: 25177540
D-galacturonic acid; Pectin; Sustainable resources; Aspergillus niger; Transmembrane transport
24.  Heterologous expression of Gaeumannomyces graminis lipoxygenase in Aspergillus nidulans 
AMB Express  2014;4:65.
Aspergillus sp. contain ppo genes coding for Ppo enzymes that produce oxylipins from polyunsaturated fatty acids. These oxylipins function as signal molecules in sporulation and influence the asexual to sexual ratio of Aspergillus sp. Fungi like Aspergillus nidulans and Aspergillus niger contain just ppo genes where the human pathogenic Aspergillus flavus and Aspergillus fumigatus contain ppo genes as well as lipoxygenases. Lipoxygenases catalyze the synthesis of oxylipins and are hypothesized to be involved in quorum-sensing abilities and invading plant tissue. In this study we used A. nidulans WG505 as an expression host to heterologously express Gaeumannomyces graminis lipoxygenase. The presence of the recombinant LOX induced phenotypic changes in A. nidulans transformants. Also, a proteomic analysis of an A. nidulans LOX producing strain indicated that the heterologous protein was degraded before its glycosylation in the secretory pathway. We observed that the presence of LOX induced the specific production of aminopeptidase Y that possibly degrades the G. graminis lipoxygenase intercellularly. Also the presence of the protein thioredoxin reductase suggests that the G. graminis lipoxygenase is actively repressed in A. nidulans.
PMCID: PMC4230170  PMID: 25401068
Aspergillus; Lipoxygenase; Gaeumannomyces graminis; Protease; Proteomics; Thioredoxin reductase
25.  Deletion of glucose oxidase changes the pattern of organic acid production in Aspergillus carbonarius 
AMB Express  2014;4:54.
Aspergillus carbonarius has potential as a cell factory for the production of different organic acids. At pH 5.5, A.carbonarius accumulates high amounts of gluconic acid when it grows on glucose based medium whereas at low pH, it produces citric acid. The conversion of glucose to gluconic acid is carried out by secretion of the enzyme, glucose oxidase. In this work, the gene encoding glucose oxidase was identified and deleted from A. carbonarius with the aim of changing the carbon flux towards other organic acids. The effect of genetic engineering was examined by testing glucose oxidase deficient (Δgox) mutants for the production of different organic acids in a defined production medium. The results obtained showed that the gluconic acid accumulation was completely inhibited and increased amounts of citric acid, oxalic acid and malic acid were observed in the Δgox mutants.
PMCID: PMC4230901  PMID: 25401063
Aspergillus carbonarius; Citric acid; Glucose oxidase; Gluconic acid; Malic acid

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