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ARTICULOS PARA SEMINARIO DE BIOTECNOLOGÍA 2014

1. Bhargavi SD, Savitha J. Arsenate Resistant Penicillium coffeae: A Potential Fungus for Soil

Bioremediation. Bull Environ Contam Toxicol. 2014; 92(3):369-73.

Bioremediation is an effective method for the treatment of major metal contaminated sites. Fungi were

isolated from soil samples collected from different arsenate contaminated areas across India. An isolate,

Penicillium coffeae, exhibited resistance to arsenate up to 500 mM.

Results indicated that pretreatment of biomass with alkali (NaOH) enhanced the percentage of

adsorption to 66.8 % as compared to that of live and untreated dead biomass whose adsorption was

22.9 % and 60.2 % respectively. The physiological parameters evaluated in this study may help pilot

studies aimed at bioremediation of arsenate contaminated effluents using arsenate resistant fungus P.

coffeae.

2. Ubeda JF, Maldonado M, Briones AI, González FJ. Bio-prospecting of Distillery Yeasts as Bio-control

and Bio-remediation Agents. Curr Microbiol. 2014;68(5):594-602.

This work constitutes a preliminary study in which the capacity of non-Saccharomyces yeasts isolated

from ancient distilleries as bio-control agents against moulds and in the treatment of waste waters

contaminated by heavy metals—i.e. bio-remediation—is shown. In the first control assays, antagonist

effect between non-Saccharomyces yeasts, their extracts and supernatants against some moulds,

analysing the plausible (not exhaustive) involved factors were qualitatively verified. In addition, two

enzymatic degrading properties of cell wall plant polymers, quitinolitic and pectinolitic, were screened.

Finally, their use as agents of bio-remediation of three heavy metals (cadmium, chromium and lead)

was analysed semi-quantitatively. The results showed that all isolates belonging to Pichia species

effectively inhibited all moulds assayed. Moreover, P. kudriavzevii is a good candidate for both

biocontrol

and bio-remediation because it inhibited moulds and accumulated the major proportion of the three

tested metals.

3. Giavasis I. Bioactive fungal polysaccharides as potential functional ingredients in food and

nutraceuticals. Curr Opin Biotechnol. 2014;26C:162-173.

Fungal bioactive polysaccharides deriving mainly from the Basidiomycetes family (and some from the

Ascomycetes) and medicinal mushrooms have been well known and widely used in far Asia as part of

traditional diet and medicine, and in the last decades have been the core of intense research for the

understanding and the utilization of their medicinal properties in naturally produced pharmaceuticals.

In fact, some of these biopolymers (mainly b-glucans or heteropolysaccharides) have already made their

way to the market as antitumor, immunostimulating or prophylactic drugs. The fact that many of these

biopolymers are produced by edible mushrooms makes them also very good candidates for the

formulation of novel functional foods and nutraceuticals without any serious safety concerns, in order

to make use of their immunomodulating, anticancer, antimicrobial, hypocholesterolemic, hypoglycemic

and health-promoting properties. This article summarizes the most important properties and

applications of bioactive fungal polysaccharides and discusses the latest developments on the utilization

of these biopolymers in human nutrition.

4. Borneman AR, Pretorius IS, Chambers PJ. Comparative genomics: a revolutionary tool for wine yeast

strain development. Curr Opin Biotechnol. 2013;24(2):192-9.

The application of Next Generation sequencing to comparative genomics is enabling in-depth

characterization of genetic variation between wine yeast strains used in fermentation starter cultures.

Knowledge from this work will be harnessed in strain development programs. As a result, winemakers

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will soon have at their disposal novel, improved yeast starter cultures displaying increased reliability and

providing a means of tailoring wine sensory characteristics for new and everchanging markets.

5. Saparrat MC, Balatti PA, Arambarri AM, Martínez MJ. Coriolopsis rigida, a potential model of white-

rot fungi that produce extracellular laccases. J Ind Microbiol Biotechnol. 2014;41(4):607-17.

In the last two decades, a significant amount of work aimed at studying the ability of the white-rot

fungus Coriolopsis rigida strain LPSC no. 232 to degrade lignin, sterols, as well as several hazardous

pollutants like dyes and aliphatic and aromatic fractions of crude oil, including polycyclic aromatic

hydrocarbons, has been performed. Additionally, C. rigida in association with arbuscular mycorrhizal

fungi appears to enhance plant growth, albeit the physiological and molecular bases of this effect

remain to be elucidated. C. rigida's ability to degrade lignin andlignin-related compounds and the

capacity to transform the aromatic fraction of crude oil in the soil might be partially ascribed to its

ligninolytic enzyme system. Two extracellular laccases are the only enzymatic components of its lignin-

degrading system. We reviewed the most relevant findings regarding the activity and role of C. rigida

LPSC no. 232 and its laccases and discussed the work that remains to be done in order to assess, more

precisely, the potential use of this fungus and its extracellular enzymes as a model in several applied

processes.

6. Pan ZY, Yang ZM, Pan L, Zheng SP, Han SY, Lin Y. Displaying Candida Antarctica lipase B on the cell

surface of Aspergillus niger as a potential food-grade whole-cell catalyst. J Ind Microbiol Biotechnol.

2014 Apr;41(4):711-20.

Aspergillus niger is a recognized workhorse used to produce food processing enzymes because of its

extraordinarily high protein-producing capacity. We have developed a new cell surface display system

de novo in A. niger using expression elements from generally recognized as safe certified

microorganisms. Candida Antarctica lipase B (CALB), a widely used hydrolase, was fused to an

endogenous cell wall mannoprotein, CwpA, and functionally displayed on the cell surface. Localization

of CALB was confirmed by enzymatic assay and immunofluorescence analysis using laser scanning

confocal microscopy. After induction by maltose for 45 h, the hydrolytic activity and synthesis activity of

A. niger mycelium-surface displayed CALB (AN-CALB) reached 400 and 240 U/g dry cell, respectively.

AN-CALB was successfully used as a whole-cell catalyst for the enzymatic production of ethyl esters

from a series of fatty acids of different chain lengths and ethanol. In a solvent-free system, AN-CALB

showed great synthetic activity and afforded high substrate mole conversions, which amounted to 87 %

for ethyl hexanoate after 2 h, 89 % for ethyl laurate after 2 h, and 84 % for ethyl stearate after 3 h.

These results suggested that CwpA can act as an efficient anchoring motif for displaying enzyme on A.

niger, and AN-CALB is a robust, green, and cost-effective alternative food-grade whole-cell catalyst to

commercial lipase.

7. Pan ZY, Yang ZM, Pan L, Zheng SP, Han SY, Lin Y. Displaying Candida Antarctica lipase B on the cell

surface of Aspergillus niger as a potential food-grade whole-cell catalyst. J Ind Microbiol Biotechnol.

2014 Apr;41(4):711-20.

A novel fungal strain JAS4 was isolated from agricultural soil and was found to be highly effective in

degrading chlorpyrifos and its major degradation product 3,5,6-trichloro-2-pyridinol (TCP). The

molecular characterization based on 18S rRNA sequence analysis, revealed strain JAS4 as Ganoderma

sp. which could able to degrade chlorpyrifos and its metabolite in an aqueous medium with rate

constant of 0.8460 day1, following first order rate kinetics, and the time in which the initial insecticide

concentration was reduced by 50% (DT50) was 0.81 days. Studies on biodegradation in soil with

nutrients showed that JAS4 strain exhibited efficient degradation of insecticide with a rate constant of

0.9 day1, and DT50 was 0.73 day. In contrast, degradation of insecticide in soil without nutrients was

characterized by a rate constant of 0.7576 day1 and the DT50 was 0.91 day.

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8. Kolvenbach BA, Helbling DE, Kohler HP, Corvini P. Emerging chemicals and the evolution of

biodegradation capacities and pathways in bacteria. Curr Opin Biotechnol. 2014, 27:8–14.

The number of new chemicals produced is increasing daily by the thousands, and it is inevitable that

many of these chemicals will reach the environment. Current research provides an understanding of

how the evolution of promiscuous enzymes and the recruitment of enzymes available from the

metagenome allows for the assembly of these pathways. Nevertheless, physicochemical constraints

including bioavailability, bioaccessibility, and the structural variations of similar chemicals limit the

evolution of biodegradation pathways. Similarly, physiological constraints related to kinetics and

substrate utilization at low concentrations likewise limit chemical-enzyme interactions and

consequently evolution. Considering these new data, the biodegradation decalogue still proves valid

while at the same time the underlying mechanisms are better understood.

9. Dufossé L, Fouillaud M, Caro Y, Mapari SAS, Sutthiwong N. Filamentous fungi are large-scale

producers of pigments and colorants for the food industry. Curr Opin Biotechnol. 2014;26:56-61.

With globalization in the research trends, healthier life styles, and the growing market for the natural

food colorants in the economically fast-growing countries all over the world, filamentous fungi are being

investigated as readily available sources of chemically diverse colorants. With two selected examples,

polyketide-Monascus-like pigments from the new fungal production strains, and the promising and yet

unexplored hydroxy-anthraquinoid colorants, the present review highlights exciting recent findings,

which may pave the way for alternative and/or additional biotechnological processes for the industrial

production of natural food colorants of improved functionality. As an additional aspect, marine fungi

are discussed as potential sources of novel pigments of numerous color hues and atypical chemical

structures.

10. Liu L, Redden H, Alper HS. Frontiers of yeast metabolic engineering: Diversifying beyond ethanol and

saccharomyces. Curr Opin Biotechnol. 2013;24(6):1023-30.

Microbial systems provide an attractive, renewable route to produce desired organic molecules such as

fuels and chemicals. While attention within the field of metabolic engineering has mostly focused on

Escherichia coli, yeast is a potent host and growing host for industrial products and has many

outstanding, biotechnologically desirable native traits. Thus, there has been a recent shift in focus

toward yeast as production hosts to replace E. coli. As such, products have diversified in yeast beyond

simply ethanol. Additionally, nonconventional yeasts have been considered to move beyond

Saccharomyces cerevisiae. This review highlights recent advances in metabolic engineering of yeasts for

producing value-added chemical compounds including alcohols, sugar derivatives, organic acids, fats,

terpenes, aromatics, and polyketides. Furthermore, we will also discuss the future direction of

metabolic engineering of yeasts.

11. Svobodová K, Mikesková H, Petráčková D. Fungal microsomes in a biotransformation perspective:

protein nature of membrane-associated reactions. Appl Microbiol Biotechnol. 2013;97(24):10263-73.

Microsomal fraction of fungal cells grabs the attention of many researchers for it contains enzymes that

play a role in biotechnologically relevant processes. Microsomal enzymes, namely, CYP450s, were

shown to metabolize a wide range of xenobiotic compounds, including PAHs, PCBs, dioxins, and

endocrine disruptors, and take part in other fungal biotransformation reactions. However, little is

known about the nature and regulation of these membrane-associated reactions. Advanced proteomic

and post-genomic techniques make it possible to identify larger numbers of microsomal proteins and

thus add to a deeper study of fungal intracellular processes. In this work, proteins that were identified

through a shotgun proteomic approach in fungal microsomes under various culture conditions are

reviewed. However, further research is still needed to fully understand the role of microsomes in fungal

biodegradation and biotransformation reactions.

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12. Ali MI, Ahmed S, Robson G, Javed I, Ali N, Atiq N, Hameed A. Isolation and molecular characterization

of polyvinyl chloride (PVC) plastic degrading fungal isolates. J Basic Microbiol. 2014;54(1):18-27.

The recalcitrant nature of polyvinyl chloride creates serious environmental concerns during

manufacturing and waste disposal. The present study was aimed to isolate and screen different soil

fungi having potential to biodegrade PVC films. After 10 months of soil burial experiment, it was

observed that a number of fungal strains were flourishing on PVC films. On morphological as well as on

18rRNA gene sequence and phylogenetic basis they were identified as Phanerochaete chrysosporium

PV1, Lentinus tigrinus PV2, Aspergillus niger PV3, and Aspergillus sydowii PV4. The biodegradation

ability of these fungal isolates was further checked in shake flask experiments by taking thin films of PVC

(C source) in mineral salt medium. A significant change in color and surface deterioration of PVC films

was confirmed through visual observation and Scanning electron microscopy. During shake flask

experiments, P. chrysosporium PV1 produced maximum biomass of about 2.57 mg ml�1 followed by A.

niger PV3. P. chrysosporium PV1 showed significant reduction (178,292 Da�1) in Molecular weight of

the PVC film than control (200,000 Da�1) by gel permeation chromatography. Furthermore more

Fourier transform infrared spectroscopy and nuclear magnetic resonance also revealed structural

changes in the PVC. It was concluded that isolated fungal strains have significant potential for

biodegradation of PVC plastics.

13. Nielsen J, Larsson C, van Maris A, Pronk J. Metabolic engineering of yeast for production of fuels and

chemicals. Curr Opin Biotechnol. 2013;24(3):398-404.

Microbial production of fuels and chemicals from renewable carbohydrate feedstocks offers sustainable

and economically attractive alternatives to their petroleum-based production. The yeast Saccharomyces

cerevisiae offers many advantages as a platform cell factory for such applications. Already applied on a

huge scale for bioethanol production, this yeast is easy to genetically engineer, its physiology,

metabolism and genetics have been intensively studied and its robustness enables it to handle harsh

industrial conditions. Introduction of novel pathways and optimization of its native cellular processes by

metabolic engineering are rapidly expanding its range of cellfactory applications. Here we review recent

scientific progress in metabolic engineering of S. cerevisiae for the production of bioethanol, advanced

biofuels, and chemicals.

14. Vogl T, Hartner FS, Glieder A. New opportunities by synthetic biology for biopharmaceutical

production in Pichia pastoris. Curr Opin Biotechnol. 2013;24(6):1094-101.

Biopharmaceuticals are an integral part of modern medicine and pharmacy. Both, the development and

the biotechnological production of biopharmaceuticals are highly cost-intensive and require suitable

expression systems. In this review we discuss established and emerging tools for reengineering the

methylotrophic yeast Pichia pastoris for biopharmaceutical production. Recent advancements of this

industrial expression system through synthetic biology include synthetic promoters to avoid methanol

induction and to fine-tune protein production. New platform strains and molecular cloning tools as well

as in vivo glycoengineering to produce humanized glycoforms have made P. pastoris an important host

for biopharmaceutical production.

15. Li Q, Pei J, Zhao L, Xie J, Cao F, Wang G. Overexpression and Characterization of Laccase from

Trametes versicolor in Pichia pastoris. Applied Biochemistry and Microbiology. 2014; 50( 2):140–147.

A laccase_encoding gene of Trametes versicolor, lccA, was cloned and expressed in Pichia pastoris X33. The lccA gene consists of a 1560 bp open reading frame encoding 519 amino acids, which was classified into family copper blue oxidase. To improve the expression level of recombinant laccase in P. pastoris, conditions of the fermentation were optimized by the single factor experiments. The optimal fermentation conditions for the laccase production in shake flask cultivation using BMGY medium were obtained: the optimal initial pH 7.0, the presence of 0.5 mM Cu2+, 0.6% methanol added into the

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culture every 24 h. The laccase activity was up to 11, 972 U/L under optimal conditions after 16 days of induction in a medium with 4% peptone. After 100 h of large scale production in 5 L fermenter the enzyme activity reached 18, 123 U/L. The recombinant laccase was purified by ultrafiltration and (NH4)2SO4 precipitation showing a single band on SDS–PAGE, which had a molecular mass of 58 kDa. The optimum pH and temperature for the laccase were pH 2.0 and 50°C with 2,2'–azino–bis(3–ethylbenzothiazoline–6–sulfonic acid) (ABTS) as a substrate. The recombinant laccase was stable over a pH range of 2.0–7.0. The Km and the Vmax value of LccA were 0.43 mM and 82.3 U/mg for ABTS, respectively.

16. De Freitas AC, Escaramboni B, Carvalho AFA, De Lima VMG, De Oliva-Neto P. Production and

application of amylases of rhizopus oryzae and rhizopus microsporus var. oligosporus from industrial

waste in acquisition of glucose. Chemical Papers [Internet]. 2014;68(4):442-50.

Amylases from Rhizopus oryzae and Rhizopus microsporus var. oligosporus were obtained using agro-industrial wastes as substrates in submerged batch cultures. The enzymatic complex was partially characterised for use in the production of glucose syrup. Type II wheat flour proved better than cassava bagasse as sole carbon source for amylase production. The optimum fermentation condition for both microorganisms was 96 hours at 30◦C and the amylase thus produced was used for starch hydrolysis. The product of the enzymatic hydrolysis indicated that the enzyme obtained was glucoamylase, only glucose as final product was attained for both microorganisms. R. oligosporus was of greater interest than R. oryzae for amylase production, taking into account enzyme activity, cultivation time, thermal stability and pH range. Glucose syrup was produced using concentrated enzyme and 100 g L−1 starch in a 4 hours reaction at 50◦C. The bioprocess studied can contribute to fungus glucoamylase production and application.

17. Chairin T, Nitheranont N, Watanabe A, Asada Y, 2,Khanongnuch C, Lumyong S. Purification and characterization of the extracellular laccase produced by Trametes polyzona WR710–1 under solid-state fermentation. J. Basic Microbiol. 2014; 54: 35–43. Laccase from Trametes polyzona WR710–1 was produced under solid-state fermentation using the peel

from the Tangerine orange (Citrus reticulata Blanco) as substrate, and purified to homogeneity. This

laccase was found to be a monomeric protein with a molecular mass of about 71 kDa estimated by SDS–

PAGE. The optimum pH was 2.0 for ABTS, 4.0 for L-DOPA, guaiacol, and catechol, and 5.0 for 2,6-DMP.

The Km value of the enzyme for the substrate ABTS was 0.15 mM, its corresponding Vmax value was

1.84 mM min�1, and the kcat/Km value was about 3960 s�1 mM�1. The enzyme activity was stable

between pH 6.0 and 8.0, at temperatures of up to 40 °C. The laccase was inhibited by more than 50% in

the presence of 20 mM NaCl, by 95% at 5 mM of Fe2þ, and it was completely inhibited by 0.1 mM

NaN3. The N-terminal amino acid sequence of this laccase is AVTPVADLQISNAGISPDTF, which is highly

similar to those of laccases from other white-rot basidiomycetes.

18. Dragosits M, Pflügl S, Kurz S, Razzazi-Fazeli E, Wilson IB, Rendic D. Recombinant Aspergillus β-

galactosidases as a robust glycomic and biotechnological tool. Appl Microbiol Biotechnol. 2013 Sep

15. [Epub ahead of print] PubMed PMID: 24037406.

Galactosidases are widespread enzymes that are used for manifold applications, including production of

prebiotics, biosynthesis of different transgalactosylated products, improving lactose tolerance and in

various analytical approaches. The nature of these applications often require galactosidases to be

present in a purified form with clearly defined properties, including precisely determined substrate

specificities, low sensitivity to inhibitors, and high efficiency and stability under distinct conditions. In

this study, we present the recombinant expression and purification of two previously uncharacterized β-

galactosidases from Aspergillus nidulans as well as one β-galactosidase from Aspergillus niger. All

enzymes were active toward p-nitrophenyl-β-D galactopyranoside as substrate and displayed similar

temperature and pH optima. The purified recombinant galactosidases digested various complex

substrates containing terminal galactose β-1,4 linked to either N-acetylglucosamine or fucose, such as

N-glycans derived from bovine fibrin and Caenorhabditis elegans . In our comparative study of the

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recombinant galactosidases with the commercially available galactosidase from Aspergillus oryzae , all

enzymes also displayed various degrees of activity toward complex oligosaccharides containing β-1,3-

linked terminal galactose residues. All recombinant enzymes were found to be robust in the presence of

various organic solvents, temperature variations, and freeze/thaw cycles and were also tested for their

ability to synthesize galactooligosaccharides. Furthermore, the use of fermentors considerably

increased the yield of recombinant galactosidases. Taken together, we demonstrate that purified

recombinant galactosidases from A. niger and from A. nidulans are suitable for various glycobiological

and biotechnological applications.

19. Wiemann P, Keller NP. Strategies for mining fungal natural products. J Ind Microbiol Biotechnol.

2014;41(2):301-13.

Fungi are well known for their ability to produce a multitude of natural products. On the one hand their

potential to provide beneficial antibiotics and immunosuppressants has been maximized by the

pharmaceutical industry to service the market with cost-efficient drugs. On the other hand

identification of trace amounts of known mycotoxins in food and feed samples is of major importance

to ensure consumer health and safety. Although several fungal natural products, their biosynthesis and

regulation are known today, recent genome sequences of hundreds of fungal species illustrate that the

secondary metabolite potential of fungi has been substantially underestimated. Since expression of

genes and subsequent production of the encoded metabolites are frequently cryptic or silent under

standard laboratory conditions, strategies for activating these hidden new compounds are essential.

This review will cover the latest advances in fungal genome mining undertaken to unlock novel

products.

20. Singh S, Singh S, Bali V, Sharma L, Mangla J. Production of fungal amylases using cheap, readily

available agriresidues, for potential application in textile industry. Biomed Res Int. 2014;2014:215748.

The study aimed at isolation and screening of fungal amylase producer, optimization of solid state

fermentation conditions for maximum amylase production by the best amylase producer, and

characterization of the crude amylases, so produced. Aspergillus fumigatus NTCC1222 showed the

highest amylase activity (164.1 U/mL) in secondary screening under SSF conditions and was selected for

further studies. The test strain showed maximum amylase production (341.7 U/mL) and supernatant

protein concentration (9.7mg/mL) for incubation period (6 days), temperature (35∘C), initial pH (6.0),

nutrient salt solution asmoistening agent, and beef extract as nitrogen source. Pomegranate peel

produced maximum amylase activity, but wheat bran (only slightly lesser amylase activity as compared

to that of pomegranate peel) was chosen for further studies, keeping in mind the seasonal availability of

pomegranate peel. TLC confirmed the amylase produced to be -type and 60kDa was the molecular

weight of the partially purified amylase.The enzyme showed maximum enzyme activity at pH 6.0,

temperature of 55∘C, and incubation time of 60 minutes. UV (616.0 U/mL) and chemical (814.2 U/mL)

mutation enhanced amylase activity as compared to wild test strain. The study indicates that Aspergillus

fumigatus NTCC1222 can be an important source of amylase and the crude enzyme, hence obtained,

can be cost effectively applied in multiple sections of textile wet processing.