BIONEXGEN edition3

BIONEXGEN Overview
The Future
CLEAs 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 reading
1. 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.
Results
In 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 Challenge
Wastewater 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 Solution
One 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 Synthesis
A 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 OverviewBIONEXGEN Overview
The University of Manchester, United Kingdom
The University of Stuttgart, Germany
Technical University of Denmark (DTU), Denmark
The Institute of Microbiology of the Czech Academy of Sciences (IMIC), Czech Republic
University of Groningen, Netherlands
CLEA Technologies BV, Netherlands
EntreChem SL, Spain
University of Oviedo, Spain
GALAB Laboratories GmbH, Germany
Leibniz Institute of Plant Biochemistry, Germany
Austrian Centre of Industrial Biotechnology, (ACIB) Austria
Royal Institute of Technology (KTH), Stockholm, Sweden
LentiKats a.s, Czech Republic
Slovak University of Technology, Slovakia
BASF SE, Germany
University College London (UCL), United Kingdom
Chemistry Innovation Ltd, United Kingdom
For more information on the BIONEXGEN project visit:
http://bionexgen-fp7.eu/
02 http://bionexgen-fp7.eu
Co-ordinated by The University of Manchester, the
BIONEXGEN project consortium consists of 17 partners from
9 European countries:
In This Issue:
BIONEXGEN Overview
01 Case Study - Novel application of
laccase-CLEAs
03 BIONEXGEN Update - Project activity
BIONEXGEN Work
04 Industrial Biotechnology in the Press
BIONEXGEN Technology Platforms
07 Development of novel drugs from
natural products by combinatorial
biosynthesis and biocatalysis
08 Pichia Expression System
Meet the BIONEXGEN Researchers
10 Meet the researchers from three of
the project participants
Product Areas
Industrial Amine Synthesis
Amines are vital for the industrial
synthesis of pharmaceuticals, bulk
and speciality chemicals.
Renewable Resources in Novel
Polymer Chemistry
Polymers 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
Glycoscience
Enzymatic 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 Oxidases
Development 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 Science
A 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 Engineering
Process engineering research
to develop and implement new
biocatalytic processes in industry.
Economic, Environmental and Life
Cycle Analysis
Developing 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.

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:

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
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 and
http://www.industrialbiotech-europe.eu/
Industrial Biotechnology News

BIONEXGEN OverviewBIONEXGEN 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.com
www.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

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.

BIONEXGEN Technology Platforms
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 Modification
mutS
CBS7435 AOX1 Knockout
dHIS CBS7435 HIS4 Auxotophy
mutS_dHIS mutS
AOX1 Knockout HIS4 Auxotrophy
∆KU70 CBS7435 KU70 inactive
∆KU70_dHIS ∆KU70 KU70 inactive + HIS4 Auxotrophy
PLASMID Promoter /
Terminator
for
Marker
Promoter /
Terminator for
Expression
Function /
Marker
Resistance
pPpT4_S
pAOXsyn/
pILV5/
AOD-TT
pAOXsyn/
AOX1-TTSyn
Expression
vector
Zeocin
pPpT4_Alpha_S
pAOXsyn/
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.at
Institute of Molecular Biotechnology,
Graz University of Technology

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.

Printed on FSC certified paper
Knowledge
Transfer
Network
Chemistry Innovation
This project is financially supported by the 7th Framework
Programme of the European Commission
(grant agreement number 266025)
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