BIONEXGEN Overview

The FutureCLEAs have been prepared from the most effective laccases and will be compared with the free laccase with regard to activity and catalyst lifetime (stability). For optimum results with mixtures of azo dyes it could be interesting to prepare combi-CLEAs from mixtures of laccases. The next step will be to test the most promising laccase-CLEA(s) in different reactor configurations (to be performed by the DTU group). Another possibility, being examined in collaboration with Lentikats, is to immobilize the CLEA in a polyvinyl alcohol gel in order to improve the morphology of the particles.

Further reading1. R. A. Sheldon, Cross-Linked Enzyme Aggregates as Industrial Biocatalysts, Org. Proc. Res. Dev., (2011) 15, 213-223. 2. I. Matijosyte, I. W.C.E. Arends, S. de Vries and R. A. Sheldon, Preparation and use of cross-linked enzyme aggregates (CLEAs) of laccases, J. Mol. Catal. B: Enzymatic (2010) 62, 142–148.3. H. Cabana, J. P. Jones and S. N. Agathos, Utilization of Cross-Linked Laccase Aggregates in a Perfusion Basket reactor for the Continuous elimination of endocrine-Disrupting Chemicals, Biotechnol. Bioeng (2009), 102, 1582-1592.

for its continuous utilisation but, at the same time, results in low productivities owing to the large percentage of non-catalytic mass in the catalyst. In stark contrast, immobilisation of laccase as cross-linked enzyme aggregates (CLEAs) affords an immobilised catalyst that combines enhanced operational stability and reusability with productivities close to those of the free enzyme. Hence, our solution is to use a laccase CLEA for the cost-effective remediation of these waste streams.

ResultsIn the BIONEXGEN project we are developing cost-effective, laccase-based processes for use in industrial and environmental biotechnology applications. These include selective oxidation of alcohols and polyols, including polysaccharides, and waste water treatment aimed at removal of low concentrations of e.g. phenols and aromatic azo dyes. To this end we have screened a variety of laccases, from different sources, in the degradation of typical azo dyes.

Reaction samples from the decolourization reaction of commercial textile dyes compared to the reference dyes are shown in Fig 1. It can be seen that CLEA outperforms the FE (free enzyme) in almost all reactions.

The UV spectra of measured samples with reference dye, reaction by free enzyme and reaction by CLEA are shown in Fig 2.

Laccase-CLEAs for effective treatment of aqueous effluent contaminated with dyestuffs

The ChallengeWastewater from textile, paper and printing industries and dye manufacturing plants is characterized by high chemical and biological oxygen demands and intense colour due to the extensive use of synthetic dyes. Direct discharge of these effluents into municipal wastewater is problematic as many complex aromatic azo dyes are toxic to the microorganisms present in biological waste water treatment plants. The cost of removal of these harmful compounds in waste water by conventional chemical means is prohibitive. There is, therefore, a pressing need to develop an alternative effective and economically viable way of dealing with them.

Laccase (polyphenoloxidase, EC 1.10.3.2) is a class of multicopper lignin-modifying enzymes that catalyzes the oxidation of phenolic compounds and aromatic amines. Over the last 2 decades the use of laccase has been explored for bleaching in pulp and paper and decolorization of azo dyes in the textile industry. However, the application of laccase as the free form (in solution) in the treatment of waste water is not economically viable owing to poor operational stability and lack of reusability.

The SolutionOne approach to overcoming these obstacles and, hence, enabling an economically viable technology, is to immobilise the laccase. Immobilisation of laccase on solid supports significantly enhances its stability towards denaturation under operating conditions and allows

Edition 3, Feb 2013

Developing the Next Generation of Biocatalysts for Industrial Chemical SynthesisA Framework 7 supported project, this second edition of the project newsletter provides an update on project activities; highlights relevant research and technology developments and provides introduction to three more of the project partners.

Novel application of laccase-CLEAs

In the first edition, the immobilization of enzymes through preparing cross linked aggregates was highlighted. Here, the novel application of such CLEAs in the treatment of aqueous effluent is described.

Figure 1: Decolourization reactions. Abbreviations: RBBR: Remazol Brilliant Blue R, RB5: Reactive Black 5, MO: Methyl Orange, AG25: Acid Green 25, IC: Indigo Carmine.

Figure 2: UV spectra of the measured samples, Absorbance plotted against wavelength. Black: reference dye solution, blue: reaction carried out by FE, red: reaction carried out by CLEA.

For more information contact: Dr. Roger Sheldon, Clea Technologies BV. Email: R.Sheldon@clea.nl

BIONEXGEN Overview BIONEXGEN Overview

The University of Manchester, United KingdomThe University of Stuttgart, GermanyTechnical University of Denmark (DTU), DenmarkThe Institute of Microbiology of the Czech Academy of Sciences (IMIC), Czech RepublicUniversity of Groningen, NetherlandsCLEA Technologies BV, NetherlandsEntreChem SL, SpainUniversity of Oviedo, SpainGALAB Laboratories GmbH, GermanyLeibniz Institute of Plant Biochemistry, GermanyAustrian Centre of Industrial Biotechnology, (ACIB) AustriaRoyal Institute of Technology (KTH), Stockholm, SwedenLentiKats a.s, Czech RepublicSlovak University of Technology, SlovakiaBASF SE, GermanyUniversity College London (UCL), United KingdomChemistry Innovation Ltd, United Kingdom

For more information on the BIONEXGEN project visit:http://bionexgen-fp7.eu/

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Co-ordinated by The University of Manchester, the BIONEXGEN project consortium consists of 17 partners from 9 European countries:

In This Issue: BIONEXGEN Overview01 Case Study - Novel application of laccase-CLEAs 03 BIONEXGEN Update - Project activity

BIONEXGEN Work 04 Industrial Biotechnology in the Press

BIONEXGEN Technology Platforms07 Development of novel drugs from natural products by combinatorial biosynthesis and biocatalysis08 Pichia Expression System

Meet the BIONEXGEN Researchers10 Meet the researchers from three of the project participants

Product Areas

Industrial Amine SynthesisAmines are vital for the industrial synthesis of pharmaceuticals, bulk and speciality chemicals.

Renewable Resources in Novel Polymer ChemistryPolymers are by far the largest volume of chemical products on the market with strong market pull for bio-based polymers in many industries e.g. automotive, packaging, construction, cosmetics and detergents.

Applications of Enzymes to GlycoscienceEnzymatic methods have great synthetic appeal for this traditionally

challenging area of chemistry producing molecules which can be used for controlling health and disease and in food and feed.

Industrial Applications of OxidasesDevelopment of efficient and robust oxidative biocatalysts and the technology for performing selective oxidations that will be valuable for use in the pharmaceutical, fine chemical and food industries.

Underpinning Technology

Fermentation ScienceA focus on efficient production strains and high density fermentation techniques which are critical to economic performance.

BIONEXGEN Research is split into 8 multi-disciplinary themes:

Biocatalyst Supports and Chemocatalysts Integration Application of biocatalyst immobilisation technology to utilise biocatalysts in industrial chemical synthesis. Bioprocess and Chemical EngineeringProcess engineering research to develop and implement new biocatalytic processes in industry. Economic, Environmental and Life Cycle AnalysisDeveloping a simplified methodology for quick and reliable quantitative assessment.

BIONEXGEN Overview

At the end of the meeting it was announced that Dr. Kirk Malone (below) would be stepping down as project manager for BIONEXGEN to pursue further career options. The consortium would like to thank Kirk for his dedication, commitment and enthusiasm for the project and wish him well in his new role.

“I feel privileged to have had the opportunity to project manage BIONEXGEN, and have thoroughly enjoyed working on the project. I would like to thank all the partners for their hard work and support over the last two years, it has been a pleasure to see the project take shape and I am sure BIONEXGEN will make a lasting contribution to European Industrial Biotechnology.” ... Kirk Malone

Dr. Mark Corbett was appointed as the new project manager in February. Before taking on his new role with BIONEXGEN, Mark worked as a postdoctoral research associate with CoEBio3 at the University of Manchester for four years, collaborating on complex, multi-centre research projects. Mark’s research was around biocatalytic routes to access renewable feedstocks, firstly in partnership with major industry (Shell Global Solutions International B.V.), and more recently as part of the international Flagship Cluster on ‘Biotechnological solutions to Australia’s transport energy and greenhouse gas challenges’ funded by CSIRO.

“BIONEXGEN has enjoyed great success over its first two years, and will continue to build upon those achievements over the next twelve months. I look forward to working with all of the BIONEXGEN partners in order to deliver scientific research and industrial biotechnology of the highest quality.”

BIONEXGEN Overview

Project Activity

The end of August 2012 marked the midpoint of BIONEXGEN, and the production of the 18 month report was a chance to survey the impressive hard work and take stock of progress so far. The eagerly awaited results of the EU mid-term review were delivered at the end of the year and as anticipated, the response from the Commission was positive, noting the good performance of all partners and strong collaborations. In order to build upon the solid progress made in the first half of the project, partners were asked to provide detailed work plans and clear targets for the remainder of the scientific programme with particular focus on the core aim of BIONEXGEN to “develop the next generation of biocatalysts to be used for eco-efficient manufacturing processes in the chemical industry.”

With this goal in mind, a strategic prioritisation and focussing event was hosted by the University of Manchester at the beginning of December. Over the course of two days each Work Package held sessions for detailed discussion of individual projects, and these were followed by update presentations by Work Package leaders and researchers to the whole consortium. After thought-provoking question and answer sessions and exchange of scientific ideas, discussions turned to how the research and development work will deliver the remaining aims of BIONEXGEN.

Additional focus on commercialisation of outputs will be provided by the newly established Exploitation Committee, chaired by Dr. John Whittall, CoEBio3’s Research Exploitation Manager, and consisting of representatives of the industrial and academic partners along with Chemistry Innovation KTN. This committee will be tasked with maximising the exploitation of foreground IP and research output by project partners and also to develop strategies to increase industry awareness beyond the consortium.

“The Exploitation Committee will have a membership centred on the industrial partners and will identify and promote research outcomes of BIONEXGEN. By using CoEBio3 networks and activities we will link innovative ideas with a wider stakeholder audience with the objective of increasing uptake of the foreground in the wider market place and future product developments.” ... John Whittall.

The meeting reception was held in the impressive surroundings of The Manchester Museum. This commenced with networking in the Pre-historic Life gallery, watched over by Stan, the Tyrannosaurus rex, followed by an atmospheric dinner amongst the skeletons and other exhibits of the Living Worlds gallery. The spectacular specimens of ancient biology on display were an appropriate contrast to the cutting edge science being discussed.

The BIONEXGEN newsletter edition 3 03

BIONEXGEN Update

At the midpoint of the project, activity within BIONEXGEN is gaining a rapid pace. The EU mid-term review was received positively and plans are in place for commercialisation opportunities with a newly established Exploitation Committee.

BIONEXGEN partners, watched over by Stan, the T-Rex, at the projects 18 month meeting reception.

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BIONEXGEN Work

R. Sardzík, A. P Green, N. Laurent, P. Both, C. Fontana, J. Voglmeir, M. J Weissenborn, R. Haddoub, P. Grassi, S. M. Haslam, G. Widmalm, S. L. Flitsch, Chemoenzymatic Synthesis of O-Mannosylpeptides in Solution and on Solid Phase, J. Am. Chem. Soc., 2012, 134 (10), pp 4521–4524.

Abstract: O-Mannosyl glycans are known to play an important role in regulating the function of α-dystroglycan (α-DG), as defective glycosylation is associated with various phenotypes of congenital muscular dystrophy. Despite the well-established biological significance of these glycans, questions regarding their precise molecular function remain unanswered. Further biological investigation will require synthetic methods for the generation of pure samples of homogeneous glycopeptides with diverse sequences. Here we describe the first total syntheses of glycopeptides containing the tetrasaccharide NeuNAcα2-3Galβ1-4GlcNAcβ1-2Manα, which is reported to be the most abundant O-mannosyl glycan on α-DG. Our approach is based on biomimetic stepwise assembly from the reducing end and also gives access to the naturally occurring mono-, di-, and trisaccharide substructures. In addition to the total synthesis, we have developed a “one-pot” enzymatic cascade leading to the rapid synthesis of the target tetrasaccharide. Finally, solid-phase synthesis of the desired glycopeptides directly on a gold microarray platform is described.

In the Press

This section highlights the technically relevant publications from within the consortium and a few of the recent global announcements of Industrial Biotechnology and its commercialisation in the chemical industry

D.Gerstorferova, B. Fliedrova, P. Halada, P. Marhol, V. Kren, L. Weignerova, Recombinant α-l-rhamnosidase from Aspergillus terreus in selective trimming of rutin, Process Biochemistry, 2012, 47, pp 828–835.

Abstract: α-l-Rhamnosidase is a biotechnologically important enzyme used for derhamnosylation of many natural compounds. The extracellular α-l-rhamnosidase was purified from the culture of Aspergillus terreus grown on l-rhamnose-rich medium. This enzyme was found to be thermo- and alkali-tolerant, able to operate at 70 °C and pH 8.0. The α-l-rhamnosidase cDNA was cloned from A. terreus, sequenced, and expressed in the yeast Pichia pastoris as a fully functional protein. The recombinant protein was purified to apparent homogeneity and biochemically characterized. Both the native and the recombinant α-l-rhamnosidases catalyzed the conversion of rutin into quercetin-3-glucopyranoside (isoquercitrin), a pharmacologically significant flavonoid usable in nutraceutics. This procedure has high volumetric productivity (up to 300 g/L) and yields the product void of unwanted quercetin. The significant advantage of our expression system consists in shorter production times, up to fourfold increase in enzyme yields and the absence of unwanted β-d-glucosidase as compared to the native production system. Thanks to its unique properties, this enzyme is applicable in a selective synthesis/hydrolysis of various rhamnose containing structures.

“The significant advantage of our expression system consists in shorter production times, up to fourfold increase in enzyme yields and the absence of unwanted β-d-glucosidase”

BIONEXGEN recent publications:

The BIONEXGEN newsletter edition 3 05

BIONEXGEN Work

S. H. Malca, D. Scheps, L. Kühnel, E. Venegas-Venegas, A. Seifert, B. M. Nestl, B. Hauer, Bacterial CYP153A monooxygenases for the synthesis of omega-hydroxylated fatty acids, Chem. Commun., 2012, 48, pp 5115-5117.

Abstract: CYP153A from Marinobacter aquaeolei has been identified as a fatty acid ω-hydroxylase with a broad substrate range. Two hotspots predicted to influence substrate specificity and selectivity were exchanged. Mutant G307A is 2- to 20-fold more active towards fatty acids than the wild-type. Residue L354 is determinant for the enzyme ω-regioselectivity.

L. Wessjohann, T. Vogt, J. Kufka, R. Klein, Alkylierende Enzyme. Prenyl- und Methyltransferasen in Natur und Synthese, Biospektrum 2012, 18, pp 22-25

Abstract: Late stage enzymatic prenylation and methylation are means to diversify (natural) compounds and to specify their functions. In eukaryotes and microbes, these steps are performed by large enzyme families, the prenyl and methyl transferases, which modify various types of small molecules, like isoprenoids, phenolics or alkaloids, but also DNA and proteins. We investigate the theoretical basis of these processes and possible commercial applications in synthetic chemistry.

Díaz-Rodríguez, I. Lavandera, S. Kanbak-Aksu, R. A. Sheldon, V. Gotor, V. Gotor-Fernández, From Diols to Lactones under Aerobic Conditions Using a Laccase/TEMPO Catalytic System in Aqueous Medium, Adv. Synth. Catal. 2012, 354, 3405 – 3408

Abstract: An efficient catalytic system to oxidize quantitatively aliphatic diols using Trametes versicolor laccase and TEMPO has been developed in aqueous medium. Oxidations have occurred in a non-stereoselective fashion but with complete regio- and/or monoselectivity, obtaining lactones with excellent purity after simple extraction. This catalytic system has been demonstrated to be scalable, compatible with the presence of a variety of functionalities, and also allowed the successful enzyme recycling using a laccase-cross-linked enzyme aggregates (CLEA) preparation.

R. K. Gudiminchi, M. Geier, A. Glieder, A. Camattari, Screening for Cytochrome P450 Expression in Pichia pastoris whole-cells by P450-Carbon Monoxide Complex Determination, Biotechnol. J., 2013, 8, 146–152, 146

Abstract: Cytochrome P450 (CYP) enzymes are useful biocatalysts for the pharmaceutical and biotechnological industries. A high-throughput method for quantification of CYP expression in yeast is needed in order to fully exploit the yeast expression system. Carbon monoxide (CO) difference spectra of whole cells have been successfully used for the quantification of heterologous CYP expressed in Escherichia coli in the 96-well format; however, very few researchers have shown whole-cell CO difference spectra with yeast cells using 1-cm path length. Spectral interference from the native hemoproteins often obscures the P450 peak, challenging functional CYP quantification in whole yeast cells. For the first time, we describe the high-throughput determination of CO difference spectra using whole cells in the 96-well format for the quantification of CYP genes expressed in Pichia pastoris. Very little interference from the hemoproteins of P. pastoris enabled CYP quantification even at relatively low expression levels. P. pastoris strains carrying a single copy or three copies of both hCPR and CYP2D6 integrated into the chromosomal DNA were used to establish the method in 96-well format, allowing to detect quantities of CYP2D6 as low as 6 nmol gCDW(-1 ) and 12 pmol per well. Finally, the established method was successfully demonstrated and used to screen P. pastoris clones expressing Candida CYP52A13.

P. Zajkoska, M. Rebroš, M. Rosenberg, Biocatalysis with immobilized Escherichia coli, Appl Microbiol Biotechnol, DOI 10.1007/s00253-012-4651-6

Abstract: Immobilization is one of the great tools for developing economically and ecologically available biocatalysts and can be applied for both enzymes and whole cells. Much research dealing with the immobilization of Escherichia coli has been published in the past two decades. E.coli in the form of immobilized biocatalyst catalyzes many interesting reactions and has been used mainly in laboratories, but also on an industrial scale, leading to the production of valuable substances. It has the potential to be applied in many fields of modern biotechnology. This paper aims to give a general overview of immobilization techniques and matrices suitable mostly for entrapment, encapsulation, and adsorption, which have been most frequently used for the immobilization of E. coli. An extensive analysis reviewing the history and current state of immobilized E.coli catalyzing different types of biotransformations is provided. The review is organized according to the enzymes expressed in immobilized E.coli, which were grouped into main enzyme classes. The industrial applications of immobilized E.coli biocatalyst are also discussed.

BIONEXGEN Work

Networks in Industrial Biotechnology and Bioenergy (NIBB)

The Biotechnology and Biological Sciences Research Council (BBSRC) in association with Engineering and Physical Sciences Research Council (EPSRC), the Technology Strategy Board (TSB), Bioscience KTN, Chemistry Innovation KTN and Health KTN wish to support a number of cross-disciplinary, community-building networks as part of its two-component strategy in supporting Industrial Biotechnology and Bioenergy (IBBE). The expansion of IBBE approaches and expertise will underpin the development of a sustainable UK Bioeconomy with the potential to maintain future energy security, create innovative bio-based products, and increase the efficiency of a wide range of manufacturing processes, generating sustainable economic growth and creating jobs. A brief description of IBBE, as it relates to this call, is provided in the call text: http://www.bbsrc.ac.uk/web/FILES/Guidelines/nibb-call-text.pdf

Once established, the networks will foster collaborative activities between academic researchers and business at all levels to identify and develop new approaches to tackle major research challenges and help deliver key benefits in IBBE through the application of a range of approaches, including genomic, systems and synthetic biology as well as the underpinning sciences such as biochemistry, enzymology, metabolism and microbiology. The networks will be cross-disciplinary, working across the boundaries of biology, chemistry and engineering. Participation from other disciplines including mathematics, computational modelling, environmental science, economics and social science is strongly encouraged. Closing date for expression of interest is 16th April 2013

ERANET in Industrial Biotechnology: 4th Call Announcement

The ERA-IB-2 consortium is preparing the 4th Call for Proposals in collaboration with EuroTransBio. The aim is to launch this 4th Call for Proposals in February 2013. Funding possibilities will be offered to excellent innovative industrially relevant R&D and applied research projects.

The deadline for submitting pre-proposals is 26 March 2013 and full proposals must be submitted by 28 June 2013. Projects are expected to start early in 2014. Further information about the 4th Call for Proposals will be available in January 2013 at the ERA-IB website

www.era-ib.net

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BIO-TIC a SusChem-Inspired FP7 Project that is looking for your input.

The BIO-TIC project is ‘a solutions approach’ centred on an extensive roadmap development process that will comprehensively examine the many barriers to innovation in industrial biotechnology across Europe and formulate action plans and recommendations to overcome them.

BIO-TIC aims to establish an overview of the barriers to biotechnology innovation and design a clear action plan. This solid roadmapping exercise requires the involvement of stakeholders from industry as well as from knowledge organisations and other stakeholders including governments and NGOs.

The final aim of the project will be to draw up a set of recommendations for overcoming the identified innovation hurdles within a selection of European business and societal opportunities. The process used to develop the roadmap and recommendations will engage with all the relevant value-chain partners, promoting and facilitating active discussion groups across all industrial biotechnology sectors and leaving a partnering platform that will make a major contribution to continuing accelerated take-up of industrial biotechnology once the project is completed.

More information can be found at http://bionexgen-fp7.eu andhttp://www.industrialbiotech-europe.eu/

Industrial Biotechnology News

BIONEXGEN Overview BIONEXGEN Technology Platforms

Development of novel drugs from natural products by combinatorial biosynthesis and biocatalysis

EntreChem is a start-up company whose areas of activity are the discovery and development of new drugs

from natural products, especially from bacterial sources and the design and implementation of novel biocatalytic routes for pharmaceutical intermediates and fine chemicals. The technology involves the use of both isolated enzymes and whole cell biocatalysts, and also the use of genetically engineered organisms to produce novel drugs. The company brings together a multidisciplinary team of organic chemists and molecular biologists in order to increase the chemical diversity of libraries obtained by combinatorial biosynthesis and to broaden the biocatalysis toolbox to perform transformations difficult to do chemically.

EntreChems technological competence can be assigned to two closely related fields:

Genetic engineering of antitumoral and antibiotic producing strains: EntreChem has experience in the manipulation of genes governing secondary metabolic pathways which offer a promising alternative for preparation of complex natural products and their analogs biosynthetically. Gene clusters encoding many natural products have been cloned and characterized, and it is now possible to introduce specific structural alterations

into a natural product in the presence of abundant functional groups by rational manipulation of the gene cluster governing its biosynthesis. The resulting molecules can be produced recombinantly by large-scale fermentation. Biocatalysis: A number of enzymatic methods have been developed to selectively modify multifunctional compounds by acylation, alkoxycarbonylation and aminoacylation, providing new methods for the synthesis of novel analogues of the parent compounds. Along the same lines, orthogonal protection strategies enable attachment and cleavage of different protecting groups to multifunctional molecules, and it is a valuable tool for the synthesis of a wide range of hydroxy- and amino compounds. The adequate arrangements of protecting groups attached to the diamines allow us to prepare valuable orthogonally protected cycloalkane-1,2-diamines in an enantiomerically pure form. The protecting groups bear easily removable substituent’s, which allowed us to differentiate both amino groups for the modular synthesis of a wide and novel range of derivatives.

EntreChem´s role within BIONEXGEN is to test the possibility of using enzymes derived from secondary metabolite pathways of actinomycetes for applications out of the pathways where they are normally active. Not all enzymes present in metabolic pathways are amenable to such an attempt, since most depend strongly on very specific metabolic intermediates and will not be viable if tested out of context. However, some enzymes, like halogenases and monooxygenases could be active if isolated and expressed individually, and moreover, perhaps their activity displays flexibility that allows them to convert non-natural substrates. In this way we could add new tools to the biocatalysis toolbox in order to help the synthetic chemist to develop new, greener processes or to make molecules very difficult to make otherwise.

For more information please contact:Francisco Morís: fmv@entrechem.comwww.entrechem.com

New Business Models in High Value Manufacturing

In line with their High Value Manufacturing Strategy, the Technology Strategy Board (TSB) is to invest up to £500k in feasibility studies to stimulate new business models supporting innovations in high value manufacturing. They are seeking feasibility studies across the whole manufacturing lifecycle.

The competition is open to enterprises of any size. A small or medium-sized enterprise (SME) can participate either as a single company or in collaboration with one other qualifying organisation, while large companies must participate in collaboration with an SME. Projects must be business-led. They propose to fund projects which are preparatory to industrial research. These are eligible for up to 75% public funding and the total project cost will not exceed £33k. Further information can be found on the TSB Competition website: http://www.innovateuk.org/content/competition/new-business-models-in-high-value-manufacturing.ashx

Important dates: Competition opens, 11 March 2013; Briefing event for potential applicants, 26 March 2013; Deadline for applications noon 24 April 2013.

This section of the newsletter looks at technology platforms being utilised in the project. Firstly we can read how the use of isolated enzymes, whole cell biocatalysts and genetically engineered organisms can be used to produce novel drugs and broaden the biocatalysis toolbox. Secondly we look at the Pichia expression system to highlight the opportunities for efficiently using the Pichia expression system.

In the laboratory at EntreChem SL, Spain

The BIONEXGEN newsletter edition 3 07

BIONEXGEN Overview

Isolated enzymes and whole cell biocatalysts, along with genetically engineered organisms are technologies used to produce novel drugs and broaden the biocatalysis toolbox to perform transformations too difficult to achieve by chemical means.

Pichia Expression System

Pichia pastoris has become one of the major eukaryotic hosts for recombinant protein production, mainly because of its strong and tightly regulated AOX1 promoter, ease of manipulation, growth to high cell-densities in inexpensive media, and ability to perform complex post-translational modifications.

Basic expression systems for recombinant protein production in Pichia pastoris have been commercially available in the recent past. Despite the availability of strains and vectors, the utilization of such systems are hindered by constrains related to the commercialization phase, when the provider of strains and vectors controls partially the market accessibility of the product expressed in Pichia pastoris. To promote a widespread and transparent diffusion of the P. pastoris expression system, researchers within The Austrian Centre of Industrial Biotechnology (ACIB), have developed a new independent and well characterized expression platform with improved vectors and production strains, based on wild-type strains. In particular, FTO (Freedom To Operate) in the commercialization phase is a particularly crucial aspect of the development.

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BIONEXGEN Technology Platforms

The BIONEXGEN newsletter edition 3 09

This Pichia Pool consists of strains derived by strain CBS7435 and subsequent modifications, as shown in the table below:

In terms of vectors, a wide choice is available, shown below:

Strain Parental strain ModificationmutS CBS7435 AOX1 KnockoutdHIS CBS7435 HIS4 AuxotophymutS_dHIS mutS AOX1 Knockout HIS4 Auxotrophy∆KU70 CBS7435 KU70 inactive∆KU70_dHIS ∆KU70 KU70 inactive + HIS4 Auxotrophy

PLASMID Promoter /Terminator forMarker

Promoter /Terminator forExpression

Function /Marker

Resistance

pPpT4_SpAOXsyn/

pILV5/AOD-TT

pAOXsyn/AOX1-TTSyn

Expression vector

Zeocin

pPpT4_Alpha_SpAOXsyn/AOX1-TTSyn

pILV5/AOD-TT

pAOXsyn/AOX1-TTSyn

Secretion vector

Zeocin

pPpT4_G AP_S pILV5/AOD-TT

pGAP/AOX1-TTSyn

Expression vector

Zeocin

pPpT4_G AP_Alpha_S

pILV5/AOD-TT

pGAP/AOX1-TTSyn

Secretion vector

Zeocin

pPpK an_S pILV5/AOD-TT

pAOXsyn/AOX1-TTSyn

Expression vector

Kanamycin

pPpK an_Alpha_S

pILV5/AOD-TT

pAOXsyn/AOX1-TTSyn

Secretion vector

Kanamycin

pPpB1_S pADH1/ADH-TT

pAOXsyn/AOX1-TTSyn

Expression vector

Zeocin

pPpB1_G AP_S pADH1/ADH-TT

pGAP/AOX1-TTSyn

Expression vector

Zeocin

pPpARG_S wtARG4/wtARG4TT

pAOXsyn/AOX1-TTSyn

Expression vector (auxotrophy selection)

Ampicillin (in E. coli)

pE H A O X1HIS4Bgl

wtARG4/wtARG4TT

pAOX/AOX1-TT

Expression vector (auxotrophy selection)

Ampicillin (in E. coli)

This pool is made available to enable co-operation projects and further advancements of Pichia pastoris as an industrial expression system.For more information please contact: Dr. Andrea Camattari: andrea.camattari@tugraz.atInstitute of Molecular Biotechnology,Graz University of Technology

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LentiKat´s

The Joint-Stock Company LentiKat’s is focused on Lentikats Biotechnology - an original technology for the immobilization of bacteria, enzymes or microorganisms in polyvinyl alcohol (PVA) matri. Lentikats Biotechnology was discovered by Professor Klaus-Dieter Vorlop, University in Braunschweig (Germany), but it was a Czech company MEGA a.s. that first started using the technology in industrial applications, followed by LentiKat’s in 2006.

The LentiKat’s company objective is to promote this revolutionary technology in various industrial applications all over the world. This mainly concerns manufacturing, development, and application of the Lentikats Biocatalysts (LB) for industrial use in the following fields: pharmaceutical, food industry, distilleries and wastewater treatment.

Within the BIONEXGEN project, LentiKat´s is involved in optimization of an immobilization procedure for different enzymes into PVA matrix, optimizing of size and shape of PVA capsules, LB preparation in lab and industrial scale as well as optimization of operation conditions and bioreactor design for use in various fields of industry in the batch and continuous operation modes. Examples include the use of LB in different biotransformations with organic solvents, use of laccase for removing of dyes, monoamineoxidase for oxidation of different secondary monoamines, α-L-rhamnosidase for production of isoquercitrin. Lentikat’s works primarily with WP6 and WP7 BIONEXGEN partners, as well as processing data bases in cooperation with Slovak University of Technology team in WP8.

The research and development BIONEXGEN team at LentiKat´s consists of:

Dr. Radek Stloukal (project scientific and technical supervisor) - R&D Director, one of the founding members of the Lentikats Biotechnology. Expert in applied biotechnology, he has experience in implementation of immobilized systems in commercial scale and development of different immobilized systems based on enzymes, bacteria or fungi for different environmental, food and pharmaceutical industry applications.

Dr. Michal Kosar (scientific and technical specialist) - Segment manager and expert in applied biotechnology in the pharmaceutical and food industry. He is responsible for industrial applications of immobilized enzymes and microorganisms for pharma-food industry.

Jarmila Watzkova and Lenka Janoskova (scientific and technical specialists) - R&D Project managers, experts in applied biotechnology of the Lentikats Biotechnology and its optimization for specific applications.

Jana Hofova (administrative/legal issues/finance responsible person) - Sales & Office coordinator of the LentiKat´s company.

Meet the BIONEXGEN researchers

This section of the newsletter will introduce the BIONEXGEN researchers working around Europe. In this edition we meet the researchers from LentiKat’s, KTH Royal Institute of Technology and Technical University of Denmark.

The BIONEXGEN Researchers

Laboratory testing of the Lentikats Biocatalyst with immobilized laccase (medium: Satturn Blue L4G: azo dye Direct Blue 78 in the phosphate buffer) in batch and continuous design

R&D BIONEXGEN team of LentiKat´s (from left): Dr. Radek Stloukal, Jarmila Watzkova, Jana Hofova, Lenka Janoskova and Dr. Michal Kosar

The BIONEXGEN Researchers

KTH, Royal Institute of Technology

BIONEXGEN research at KTH is led by Dr. Mats Martinelle at the Department of Industrial Biotechnology. The main research areas are enzymology and enzyme engineering, biocatalysis towards functional polymers and chemo-enzymatic routes to renewable polymer materials. Biocatalytic transformations using renewable compounds are a priority area in the group.

At KTH ongoing activity between biochemists and polymer chemists has created the ‘BioPol’ group with a fruitful collaboration in the interdisciplinary area between biocatalysis and polymer materials. Research challenges for the BioPol group concern the development of strategies that promote efficient and selective enzyme catalyzed synthesis of functional polymer resins in one-pot conditions, as well as addressing curing technologies where the functional polymers resins are transferred into tailored materials with novel properties. The BioPol group have demonstrated several examples of biocatalytic routes combined with curing technologies towards polymer materials applied to coatings.

Within BIONEXGEN the BioPol group is concerned with biocatalysis towards novel polymers and polymer films based on renewable resources. Collaboration partners in the project are University of Stuttgart, Germany and the Austrian Centre of Industrial Biotechnology, Austria. The team at KTH consists of biochemists: PhD-student Peter Hendil-Forssell, Prof. Karl Hult, Dr. Mats Martinelle and polymer chemists PhD-student Mauro Claudino, Prof. Eva Malmström Jonsson, Prof. Mats Johansson.

Technical University of Denmark

The Centre for Process Engineering and Technology is in the Department of Chemical and Biochemical Engineering at the

Technical University of Denmark (DTU) headed by John Woodley. The Centre’s vision is to provide the support required to implement new chemical and fuel production processes in industry with a focus on sustainable innovative processes. Consequently, the Centre works at the interface of several disciplines which include biotechnology, process engineering, chemistry and systems engineering.

John Woodley (left) is currently Professor of Chemical Engineering at DTU, a position he took up in 2007 after 20 years at University College London (UCL, London, UK). He leads a

collaborative research team working on the scale-up and implementation of process technology suitable for new biocatalytic and chemo-enzymatic processes involving pilot-scale and laboratory experimental evaluation, process modelling and flow-sheeting. He has published around 160

papers and 300 conference presentations. He has industrial experience from ICI and international consultancy and sits on a number of scientific advisory and editorial boards. He is a Fellow of the Institution of Chemical Engineers (UK) and a Fellow of the Royal Academy of Engineering (UK).

Hemalata Ramesh (left) is doing her PhD. at the Department of Chemical and Biochemical Engineering at DTU, after completing her Master’s in Biotechnology. She is a part of

BIONEXGEN and her project aims at using oxidases (which have been identified as the next generation biocatalysts) for developing processes which involve requirement for oxygen supply. During the course of her PhD, she will also develop a toolbox for assessing stability for oxidases.

DTU is heading two work packages in BIONEXGEN which are dealing with process engineering (WP7) and economic and environmental analysis (WP 8) of select processes within BIONEXGEN. DTU will develop processes for particular biocatalytic reactions using new tools. Additionally, a simplified methodology that will enable quick assessment of the environmental impact of the processes will be developed.

From left to right: Peter Hendil-Forssell, Prof. Karl Hult, Dr. Mats Martinelle, Mauro Claudino and Prof. Mats Johansson.

The BIONEXGEN newsletter edition 3 11

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Chemistry Innovation

This project is financially supported by the 7th Framework Programme of the European Commission

(grant agreement number 266025)

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