1. BIONEXGEN Overview
(e.g. expression, protein production,
substrate tolerance of the biocatalyst).
This progression to scale-up enables key
data to be obtained which can be fed back
into life-cycle analysis to benchmark new
biocatalytic processes.
Inevitably at the end of a project such as
BIONEXGEN there are many unanswered
questions and new challenges. For
example, the work-package devoted to
(chiral) amine synthesis has led to the
discovery and development of two new
biocatalyst platforms [(R)-selective amine
oxidase and (R)-/(S)-imine reductases]
which now require further research in
order to improve their activity, stability
etc. such that they can soon be applied
in industrial biocatalysis. These new
biocatalytic platforms will be taken forward
within future projects through collaboration
with industry. Additionally, a new EU-FP7
I believe that BIONEXGEN has been a
major success, delivering on almost all
aspects of the project which were initially
envisaged and planned. During the lifetime
of the project new products have been
created (e.g. immobilized biocatalysts
from the SMEs Lentikat’s a.s. and CLEA
Technologies BV) and a new spin-out
company has been spawned offering
screening kits for biocatalysis (Discovery
Biocatalysts Ltd). Patents have been
filed together with a large number of
publications in high-impact journals. PhD
students and postdoctoral fellows have
received specialist training such that they
are now well placed to enter the chemical
industry equipped with the requisite set
of skills. Selected biocatalytic processes
have been moved from the laboratory
to initial scale-up in order to understand
the factors which need to be further
improved for commercial application
Edition 6, Jan 2014
Developing the Next Generation of Biocatalysts for Industrial Chemical Synthesis
A Framework 7 supported project this 6th edition of the newsletter provides an update on project activities and highlights
some of the exploitable results from the project.
project entitled BIOOX, which commenced
on 1st October 2013, will focus on the
application at scale of oxygen-dependent
biocatalysts for targeted chemical
synthesis. Some of the important enabling
studies for BIOOX were carried out within
BIONEXGEN thereby ensuring a smooth
transition and translation of emerging
technology into ongoing research
programmes.
I would like to finish by thanking all of
those who participated in BIONEXGEN,
helping to make it a highly enjoyable and
stimulating research programme. I am also
very grateful for the guidance and advice
provided by the Project Officer and the
European Commission.
Nicholas J Turner, Coordinator (The
University of Manchester) 21 Jan 2014
BIONEXGEN – Perspectives and Outlook
Dr Rachel Heath, BIONEXGEN Researcher
BIONEXGEN was funded by the EU-FP7 programme as a ‘Flagship Project’
with the overall aim of developing novel biocatalytic processes for chemical
manufacture in the future. By bringing together some of the leading academic
and industrial research groups from across Europe, and organizing these
groups into focused work-packages, we envisaged that we could tackle some
of the key underpinning problems and challenges that currently limit the more
widespread application of biotechnology in the chemical industry. Projects
within BIONEXGEN addressed the full range of activities required to implement
a new biocatalytic process including enzyme discovery, biocatalyst engineering,
high-throughput screening, enzyme immobilization, preparative scale
transformations and life-cycle analysis. Within BIONEXGEN there was a degree
of focus on specific platform chemicals (e.g. amines, fatty acids, carbohydrates)
but these merely acted as illustrative of where new advances in technology
and innovation are required in order to accelerate the uptake of industrial
biocatalysis in industry.

2. BIONEXGEN OverviewBIONEXGEN Overview
Introduction
The BIONEXGEN newsletter edition 6 03
From the BIONEXGEN project
manager...
In March 2011, one month after
the start of the project, the kick-
off event for BIONEXGEN was
held in Brussels. The meeting
was opened by Alfredo Aguilar
Romanillos, Head of Unit, and Maria
Fernandez Gutierrez, the Project
Officer, representing Unit E2 –
Biotechnologies, DG RTD of the
European Commission. In opening,
it was highlighted that BIONEXGEN
was unique in scope, receiving the
largest budget to date of any FP7
project in the KBBE area, bringing
together 17 academic, SME, and
large Industrial partners to develop
the Next Generation of Biocatalysts
for Industrial chemical Synthesis.
Expectations for BIONEXGEN were
therefore high, in recognition of
the ambitious targets set out in
the proposal and the potential to
deliver the highest quality research
with significant tangible benefits
to the EU Industrial Biotechnology
community.
In December 2013, at the Crowne Plaza
Brussels Airport, BIONEXGEN hosted
events showcasing the impact of EU
funding on Industrial Biotechnology
research in Europe. Drawing speakers,
exhibitors, and delegates from academic
and industrial research, IB end-users,
funding bodies, and industry groups,
this was an opportunity to present the
benefits and outputs of FP7 projects,
but also to look forward to the future
of Industrial Biotechnology innovation,
with the EU as a global leader in the
field. The next generation of biocatalysis
for industrial chemical synthesis was a
technical dissemination session, featuring
work from past and current projects
including BIONEXGEN, KYROBIO
(Grant No. 289646), AMBIOCAS (Grant
No. 245144), BIOTRAINS (Grant No.
238531), CHEM21 (IMI Joint Technology
Initiative), BIOINTENSE (Grant No.
312148), and P4FIFTY (Marie Curie
ITN 289217). Industrial Biotechnology
for Europe featured presentations and
discussions on the role and impact of EU
funding in developing European Industrial
Biotechnology. This session drew from
across the range of the European IB
community, from academia, to SMEs,
and industrial end-users, with invited
participants from The University of
Manchester, BASF SE, C-Tech Innovation
Ltd, Bio-Prodict BV, Prozomix Ltd, and
CLEA Technologies BV. The events
brought together many representatives of
the community in an excellent reflection
of the strong research and development
network fostered around many related
and interconnected FP7 and EU projects,
and the role of BIONEXGEN as flagship
amongst these. As the project reaches a
conclusion, it is finally possible to survey
its many outputs and declare the success
of BIONEXGEN.
BIONEXGEN finishes at the end of
January 2014, and it is very clear that the
initial ambition of the project has been
realised. The impact of BIONEXGEN
can be judged from the body of literature
highlighted here and which continues to
be published under the auspices of the
project, through enhanced cross-sector
research collaborations which will continue
beyond the lifetime of this project, and
most immediately by the exploitable
outputs and new products, including those
discussed in this publication.
Mark Corbett, Project Manager (The
University of Manchester)
BIONEXGEN 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:
Contents:
01 BIONEXGEN – Perspectives and
Outlook
02 Introduction
03 BIONEXGEN Overview
04 Exploitable Results
06 In the Press
10 Complete list of publication
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.

3. and microorganisms and can offer this
expertise to other academic/industrial
partners. Bioreactor optimization units are
available at SUT for the development of
protocols for every stage of biocatalyst
production, including fermentation
processes and production of recombinant
enzymes. The Laboratories of Applied
Biocatalysis at offer the additional benefits
of scale-up and large-scale verification
of all technologies including downstream
processing, and fermentors up to 400 l
with appropriate downstream units are
available for these purposes. Partnership
with SUT offers the facilities for production
of industrial samples in kg amounts and
full pilot plant process evaluation.
Contact:
Ing. Martin Rebros, PhD.
Institute of Biotechnology and Food
Science
Slovak University of Technology
Radlinskeho 9
812 37 Bratislava
Slovakia
Phone: +421 2 59 325 480
Email: martin.rebros@stuba.sk
aqueous solutions (wastewater) especially
for use in textile industry were developed
during the BIONEXGEN project. Dye
decolorization by laccase (from Trametes
versicolor) immobilized into PVA matrix as
Lentikats Biocatalyst providing as high as
83% removal efficiency.
Rhamnosidases are biotechnologically
important enzymes used for
derhamnosylation of natural glycosides
containing terminal α-L-rhamnose.
Applications of interest in the food
industry include the debittering of
grapefruit and other citrus juices by
hydrolysis of the rhamnoside naringin,
and aroma enhancement of wines and
juices by hydrolysis of terpenyl glycoside
precursors.
Intensification of the most important
characteristic of a quality wine, its
aromatic fragrance, can be achieved using
immobilised Rhamnosidases developed
during the BIONEXGEN project, in
collaboration with SUT. Recombinant
α-L-rhamnosidase from Aspergilus
terreus expressed in Pichia pastoris and
immobilized as Lentikats Biocatalyst was
developed to provide an inexpensive and
stable enzymatic solution for wine aroma
enhancement.
Contact us
LentiKatˊs a.s.
Pod Vinicí 83
471 27 Stráž pod Ralskem
Czech Republic
Phone: +420 255 710 680
Fax: +420 255 710 699
Email: info@lentikats.eu
Web: www.lentikats.eu
Exploitable Results
04 http://bionexgen-fp7.eu The BIONEXGEN newsletter edition 6 05
Bio-based polymers by lipase catalysis
At KTH the collaboration between
biochemists and polymer chemists has
created a research environment (the
BioPol group) in the interdisciplinary areas
of biocatalysis and polymer chemistry.
Research concerns the development
of strategies that promote efficient and
selective enzyme catalysed synthesis of
functional polymer resins and the design of
efficient curing technologies where these
polymer resins are transferred into tailored
materials.
In BIONEXGEN the BioPol group
combined activities in biocatalysis, curing
technology and material characterisation
aiming for functional polymers and
polymer cross-linked films from bio-based
monomers. Functional polyesters and
poly(ester-amide)s were prepared with
lipase catalysis and cured into cross-linked
NOVEL POLYMER CHEMISTRY
Example of a chemo-enzymatic route to cross-linked polymer films developed in BIONEXGEN
O
O
N
O
O
O
O
m
O
O
O
m
p
O
O
O
O
O
O
OO
HS
SH
SH
HS
H
Bulk
Photoinitiator
UV-irradiation
O
O
N
O
O
O
O
m
O
HO
O
m
p
HO
N
OH
H
O
O
O
O
O
m
OH
10-
undec
en-1-
ol
amide-diol
O
O
O
O
dimethyl succinate
O
O
O
O
dimethyl adipate
O
O
O
O
dimethyl sebacate
(spacer)
HO
NH2
O
OH
O
ethanolamine
+
methyl 10-hydroxydecanoate
Immobilized CalB
Immobilized CalB
(m=1) (m=3) (m=7)
DP=3-4
Contact:
Dr Mats Martinelle
KTH – Royal Institute of Technology,
School of Biotechnology, Division of
Industrial Biotechnology, AlbaNova
University Center , SE-106 91 Stockholm,
Sweden
Phone: + 46 8 5537 8384
Email: matsm@biotech.kth.se
polymer films using thiol-ene chemistry.
The BioPol group has in BIONEXGEN
(and in other projects) successfully
demonstrated several examples of
chemo-enzymatic routes toward polymer
materials applied to coatings. The BioPol
group at KTH has a research platform
available to meet the future challenges and
development requirements in the area of
bio-based polymer materials.
BIOPROCESS & CHEMICAL
ENGINEERING
IMPROVED BIOCATALYST &
ENZYME SUPPORT
Slovak University of Technology
The Laboratories of Applied Biocatalysis
at the Institute of Biotechnology and Food
Science, Slovak University of Technology
(SUT) offer a range of expertise across
the field of biocatalysis. One of the main
areas studied intensively in BIONEXGEN
were applications of immobilized cells
and enzymes as applied biocatalysts.
Immobilization of recombinant Escherichia
coli overexpressing Monoamine Oxidase
(MAO) as whole cell biocatalysts, along
with immobilization of cell-free enzyme
extracts were studied in collaboration
with partners LK (LentiKats a.s.) and
UNIMAN (University of Manchester). This
led to the development of MAO LentiKats
biocatalysts which can be extensively
recycled over the course of multiple
reactions.
Activities during BIONEXGEN also
included immobilization of recombinant
α-L-rhamnosidase produced in Pichia
pastoris. Together with IMIC (Institute
of Microbiology, Academy of Sciences
of the Czech Republic) and LK a
number of immobilized biocatalysts in
form of LentiKats were developed for
applications in food (wine aroma release)
and pharmaceutical manufacturing
(isoquercitrin production).
SUT has extensive experience in
the field of immobilization protocol
optimization for both isolated enzymes
Immobilization into Polyvinylalcohol
Matrix, LentiKatˊs a.s.
The Applications
- Decolouration of Industrial Wastewater by
Laccase Immobilized into PVA matrix
- Wine Aroma Release by Immobilized
Rhamosidase into PVA matrix
Industrial Solution
Lentikats Biotechnology can be utilized for
immobilization of any enzymes and cells
for industrial applications in food and the
pharmaceutical industry, biofuel production
and waste water treatment. A key output of
the BIONEXGEN project the development
and testing of industrially available
enzymes laccase from Trametes versicolor
and recombinant α-L-rhamnosidase
from Aspergilus terreus expressed in
Pichia pastoris. In comparison with
other immobilization methods, Lentikats
Biotechnology offers several important
benefits, such as relatively simple
production procedure and easy separation
of the Lentikats Biocatalyst from the
reaction media, high enzyme activity
yields after immobilization and also high
catalytic biocatalyst activity. The Lentikats
Biocatalyst possesses excellent physical
mechanical characteristics (elasticity,
low abrasion) that provide for long-term
stability and biocatalyst lifetime. Moreover,
the PVA is broadly biologically un-
degradable and possesses no detectable
toxicity. It is an inexpensive matrix for
immobilization of any biologically active
materials with no apparent side effects
on the biochemical process. From the
technical point of view due to the high
concentration of microorganisms or
enzymes in the Lentikats Biocatalyst and
the possibilities for reuse of the Lentikats
Biocatalyst, a significantly shorter retention
time can be adopted, which minimizes the
possibility of process contamination with a
subsequent increase of the process yield.
Consequently, Lentikats Biotechnology
industrial applications require smaller
reactors, thus leading to a reduction in
investment costs and increased revenue.
Lentikats Biotechnology has several
advantages over the other currently
available techniques and a large variety of
applications. Application of this technology
for existing or newly designed production
processes leads to a substantial increase
in process yields and a reduction in
operational and investment costs.
Laccases belong to the interesting group
of multi copper enzymes with the ability
to oxidize phenolic and non-phenolic
compounds, including some environmental
pollutants. Because of their efficiency,
laccases are of interest for a variety of
chemical industries, especially the textile
industry, where they are able to catalyze
dye decolorization for the treatment of
manufacturing byproducts.
Laccases immobilized into PVA matrix for
removing/selective oxidation of dyes from

4. BIONEXGEN Overview
This section highlights the technically relevant publications from within the
consortium over the past year.
In the Press
In the Press
The BIONEXGEN newsletter edition 6 0706 http://bionexgen-fp7.eu
Redesign of a Phenylalanine Aminomutase into a Phenylalanine Ammonia Lyase
Bartsch, S.; Wybenga, G. G.; Jansen, M.; Heberling, M. M.; Wu, B.; Dijkstra, B. W.;
Janssen, D. B. Redesign of a Phenylalanine Aminomutase into a Phenylalanine
Ammonia Lyase. ChemCatChem 2013, 5, 1797–1802.
Abstract: An aminomutase, naturally catalyzing the interconversion of (S)-α-
phenylalanine and (R)-β-phenylalanine, was converted into an ammonia lyase catalyzing
the nonoxidative deamination of phenylalanine to cinnamic acid by a rational single-point
mutation. It could be shown by crystal structures and kinetic data that the flexibility of the
lid that covers the active site decides whether the enzyme acts as a lyase or a mutase.
An Arg92Ser mutation destabilized the closed conformation of the lid structure and
converted the mutase into a lyase that exhibited up to 44-fold increased reaction rates in
the enantioselective deamination of (R)-β-phenylalanine. In addition, the amination rates
of cinnamic acid yielding optically pure (S)-α- and (R)-β-phenylalanine were doubled.
The applicability of the mutant enzyme for kinetic resolution and asymmetric amination
could be shown by biocatalysis on a preparative scale.
Microscale methods to rapidly
evaluate bioprocess options for
increasing bioconversion yields:
application to the ω-transaminase
synthesis of chiral amines.
Halim, M.; Rios-Solis, L.; Micheletti, M.;
Ward, J.; Lye, G. Microscale Methods
to Rapidly Evaluate Bioprocess Options
for Increasing Bioconversion Yields:
Application to the ω-Transaminase
Synthesis of Chiral Amines. Bioprocess
Biosyst. Eng. 2013, 1–11.
Abstract: This work aims to establish
microscale methods to rapidly explore
bioprocess options that might be used
to enhance bioconversion reaction
yields: either by shifting unfavourable
reaction equilibria or by overcoming
substrate and/or product inhibition.
As a typical and industrially relevant
example of the problems faced we have
examined the asymmetric synthesis of
(2S,3R)-2-amino-1,3,4-butanetriol from
L-erythrulose using the ω-transaminase
from Chromobacterium violaceum
DSM30191 (CV2025 ω-TAm) and
methylbenzylamine as the amino donor.
The first process option involves the
use of alternative amino donors. The
second couples the CV2025 ω-TAm with
alcohol dehydrogenase and glucose
dehydrogenase for removal of the
acetophenone (AP) by-product by in situ
conversion to (R)-1-phenylethanol. The
final approaches involve physical in-situ
product removal methods. Reduced
pressure conditions, attained using a
96-well vacuum manifold were used to
selectively increase evaporation of the
volatile AP while polymeric resins were
also utilised for selective adsorption of AP
from the bioconversion medium. For the
particular reaction studied here the most
promising bioprocess options were use
of an alternative amino donor, such as
isopropylamine, which enabled a 2.8-fold
increase in reaction yield, or use of a
second enzyme system which achieved a
3.3-fold increase in yield.
Synthesis of 9-Oxononanoic Acid, a Precursor for Biopolymers
Otte, K. B.; Kirtz, M.; Nestl, B. M.; Hauer, B. Synthesis of 9-Oxononanoic Acid, a
Precursor for Biopolymers. ChemSusChem 2013, 6, 2149–2156.
Abstract: Polymers based on renewable resources have become increasingly
important. The natural functionalization of fats and oils enables an easy access
to interesting monomeric building blocks, which in turn transform the derivative
biopolymers into high-performance materials. Unfortunately, interesting building
blocks of medium-chain length are difficult to obtain by traditional chemical
means. Herein, a biotechnological pathway is established that could provide an
environmentally suitable and sustainable alternative. A multiple enzyme two-step
one-pot process efficiently catalyzed by a coupled 9S-lipoxygenase (St-LOX1,
Solanum tuberosum) and 9/13-hydroperoxide lyase (Cm-9/13HPL, Cucumis melo)
cascade reaction is proposed as a potential route for the conversion of linoleic
acid into 9-oxononanoic acid, which is a precursor for biopolymers. Lipoxygenase
catalyzes the insertion of oxygen into linoleic acid through a radical mechanism to
give 9S-hydroperoxy-octadecadienoic acid (9S-HPODE) as a cascade intermediate,
which is subsequently cleaved by the action of Cm-9/13HPL. This one-pot
process afforded a yield of 73 % combined with high selectivity. The best reaction
performance was achieved when lipoxygenase and hydroperoxide lyase were
applied in a successive rather than a simultaneous manner. Green leaf volatiles,
which are desired flavor and fragrance products, are formed as by-products in this
reaction cascade. Furthermore, we have investigated the enantioselectivity of 9/13-
HPLs, which exhibited a strong preference for 9S-HPODE over 9R-HPODE.
Structure and activity of NADPH-
dependent reductase Q1EQE0 from
Streptomyces kanamyceticus,
which catalyses the R-selective
reduction of an imine substrate.
Rodríguez-Mata, M.; Frank, A.;
Wells, E.; Leipold, F.; Turner, N. J.;
Hart, S.; Turkenburg, J. P.; Grogan,
G. Structure and Activity of NADPH-
Dependent Reductase Q1EQE0 from
Streptomyces Kanamyceticus, Which
Catalyses the R-Selective Reduction
of an Imine Substrate. ChemBioChem
2013, 14, 1372–1379.
Abstract: NADPH-dependent
oxidoreductase Q1EQE0 from
Streptomyces kanamyceticus
catalyzes the asymmetric reduction
of the prochiral monocyclic imine
2-methyl-1-pyrroline to the chiral
amine (R)-2-methylpyrrolidine with
>99% ee, and is thus of interest
as a potential biocatalyst for the
production of optically active
amines. The structures of Q1EQE0
in native form, and in complex with
the nicotinamide cofactor NADPH
have been solved and refined to a
resolution of 2.7 Å. Q1EQE0 functions
as a dimer in which the monomer
consists of an N-terminal Rossman-
fold motif attached to a helical
C-terminal domain through a helix of
28 amino acids. The dimer is formed
through reciprocal domain sharing
in which the C-terminal domains are
swapped, with a substrate-binding
cleft formed between the N-terminal
subunit of monomer A and the
C-terminal subunit of monomer B. The
structure is related to those of known
β-hydroxyacid dehydrogenases,
except that the essential lysine, which
serves as an acid/base in the (de)
protonation of the nascent alcohol
in those enzymes, is replaced by
an aspartate residue, Asp187 in
Q1EQE0. Mutation of Asp187 to either
asparagine or alanine resulted in an
inactive enzyme.
Regulation of Pichia pastoris promoters and its consequences for protein
production.
Vogl, T.; Glieder, A. Regulation of Pichia Pastoris Promoters and Its Consequences
for Protein Production. N. Biotechnol. 2013, 30, 385–404.
Abstract: The methylotrophic yeast Pichia pastoris is a widely used host for
heterologous protein production. Along with favorable properties such as growth
to high cell density and high capacities for protein secretion, P. pastoris provides
a strong, methanol inducible promoter of the alcohol oxidase 1 (AOX1) gene.
The regulation of this promoter has been extensively studied in recent years by
characterizing cis-acting sequence elements and trans-acting factors, revealing
insights into underlying molecular mechanisms. However, new alternative promoters
have also been identified and characterized by means of their transcriptional
regulation and feasibility for protein production using P. pastoris. Besides the
often applied GAP promoter, these include a variety of constitutive promoters
from housekeeping genes (e.g. TEF1, PGK1, TPI1) and inducible promoters from
particular biochemical pathways (e.g. PHO89, THI11, AOD). In addition to these
promoter sequence/function based studies, transcriptional regulation has also
been investigated by characterizing transcription factors (TFs) and their modes
of controlling bioprocess relevant traits. TFs involved in such diverse cellular
processes such as the unfolded protein response (UPR) (Hac1p), iron uptake
(Fep1p) and oxidative stress response (Yap1p) have been studied. Understanding
of these natural transcriptional regulatory networks is a helpful basis for synthetic
biology and metabolic engineering approaches that enable the design of tailor-made
production strains.
Asymmetric Reduction of Cyclic Imines Catalyzed by a Whole-
Cell Biocatalyst Containing an (S)-Imine Reductase
Leipold, F.; Hussain, S.; Ghislieri, D.; Turner, N. J. Asymmetric
Reduction of Cyclic Imines Catalyzed by a Whole-Cell Biocatalyst
Containing an (S)-Imine Reductase. ChemCatChem 2013, 5, 3505–
3508.
Abstract:
Biocatalytic imine reduction: A whole-cell
recombinant E. coli system, producing an
(S)-selective imine reductase (IRED) from
Streptomyces sp. GF3546, is developed. This
biocatalyst is used for the enantioselective
reduction of a broad range of substrates such as
dihydroisoquinolines and dihydro-β-carbolines as
well as iminium ions.

5. In the Press In the Press
08 http://bionexgen-fp7.eu The BIONEXGEN newsletter edition 5 09
Priming ammonia lyases and aminomutases for industrial and therapeutic
applications
Heberling, M. M.; Wu, B.; Bartsch, S.; Janssen, D. B. Priming Ammonia Lyases and
Aminomutases for Industrial and Therapeutic Applications. Curr. Opin. Chem. Biol.
2013, 17, 250–260.
Abstract: Ammonia lyases (AL) and aminomutases (AM) are emerging in green
synthetic routes to chiral amines and an AL is being explored as an enzyme
therapeutic for treating phenylketonuria and cancer. Although the restricted
substrate range of the wild-type enzymes limits their widespread application, the
non-reliance on external cofactors and direct functionalization of an olefinic bond
make ammonia lyases attractive biocatalysts for use in the synthesis of natural
and non-natural amino acids, including β-amino acids. The approach of combining
structure-guided enzyme engineering with efficient mutant library screening has
extended the synthetic scope of these enzymes in recent years and has resolved
important mechanistic issues for AMs and ALs, including those containing the MIO
(4-methylideneimidazole-5-one) internal cofactor.
Synthesis of ω-hydroxy dodecanoic acid based on an engineered CYP153A
fusion construct
Honda Malca, S.; Scheps, D.; Kuhnel, L.; Venegas-Venegas, E.; Seifert, A.; Nestl,
B. M.; Hauer, B. Bacterial CYP153A Monooxygenases for the Synthesis of Omega-
Hydroxylated Fatty Acids. Chem. Commun. 2012, 48, 5115–5117.
Abstract: A bacterial P450 monooxygenase-based whole cell biocatalyst using
Escherichia coli has been applied in the production of ω-hydroxy dodecanoic acid from
dodecanoic acid (C12-FA) or the corresponding methyl ester. We have constructed
and purified a chimeric protein where the fusion of the monooxygenase CYP153A
from Marinobacter aquaeloei to the reductase domain of P450 BM3 from Bacillus
megaterium ensures optimal protein expression and efficient electron coupling. The
chimera was demonstrated to be functional and three times more efficient than other
sets of redox components evaluated. The established fusion protein (CYP153AM. aq.-
CPR) was used for the hydroxylation of C12-FA in in vivo studies. These experiments
yielded 1.2 g l–1
ω-hydroxy dodecanoic from 10 g l–1
C12-FA with high regioselectivity
(> 95%) for the terminal position. As a second strategy, we utilized C12-FA methyl
ester as substrate in a two-phase system (5:1 aqueous/organic phase) configuration
to overcome low substrate solubility and product toxicity by continuous extraction.
The biocatalytic system was further improved with the coexpression of an additional
outer membrane transport system (AlkL) to increase the substrate transfer into the
cell, resulting in the production of 4 g l–1
ω-hydroxy dodecanoic acid. We further
summarized the most important aspects of the whole-cell process and thereupon
discuss the limits of the applied oxygenation reactions referring to hydrogen peroxide,
acetate and P450 concentrations that impact the efficiency of the production host
negatively
Substrate promiscuity of
cytochrome P450 RhF
O’Reilly, E.; Corbett, M.; Hussain, S.;
Kelly, P. P.; Richardson, D.; Flitsch, S.
L.; Turner, N. J. Substrate Promiscuity
of Cytochrome P450 RhF. Catal. Sci.
Technol. 2013, 3, 1490–1492.
Abstract: Cytochrome P450
RhF displays a high degree of
substrate promiscuity, mediating a
range of O-dealkylations, aromatic
hydroxylations, epoxidations and
asymmetric sulfoxidations. The self-
sufficient nature of this CYP coupled
with its ability to catalyse the oxidation
of a wide range of functional groups
highlights this enzyme as an excellent
starting template for directed evolution
and promising alternate to P450 BM3.
Synthesis of ω-hydroxy dodecanoic acid based on an engineered CYP153A
fusion construct.
Scheps, D.; Honda Malca, S.; Richter, S. M.; Marisch, K.; Nestl, B. M.; Hauer,
B. Synthesis of ω-Hydroxy Dodecanoic Acid Based on an Engineered CYP153A
Fusion Construct. Microb. Biotechnol. 2013, 6, 694–707.
Abstract: A bacterial P450 monooxygenase-based whole cell biocatalyst using
Escherichia coli has been applied in the production of ω-hydroxy dodecanoic
acid from dodecanoic acid (C12-FA) or the corresponding methyl ester. We have
constructed and purified a chimeric protein where the fusion of the monooxygenase
CYP153A from Marinobacter aquaeloei to the reductase domain of P450 BM3
from Bacillus megaterium ensures optimal protein expression and efficient electron
coupling. The chimera was demonstrated to be functional and three times more
efficient than other sets of redox components evaluated. The established fusion
protein (CYP153AM. aq. -CPR) was used for the hydroxylation of C12-FA in in vivo
studies. These experiments yielded 1.2 g l-1 ω-hydroxy dodecanoic from 10 g l-1
C12-FA with high regioselectivity (> 95%) for the terminal position. As a second
strategy, we utilized C12-FA methyl ester as substrate in a two-phase system
(5:1 aqueous/organic phase) configuration to overcome low substrate solubility
and product toxicity by continuous extraction. The biocatalytic system was further
improved with the coexpression of an additional outer membrane transport
system (AlkL) to increase the substrate transfer into the cell, resulting in the
production of 4 g l-1 ω-hydroxy dodecanoic acid. We further summarized the most
important aspects of the whole-cell process and thereupon discuss the limits of the
applied oxygenation reactions referring to hydrogen peroxide, acetate and P450
concentrations that impact the efficiency of the production host negatively.
Alkylating enzymes
Wessjohann, L. A.; Keim, J.; Weigel, B.; Dippe, M.
Alkylating Enzymes. Curr. Opin. Chem. Biol. 2013, 17,
229–235.
Abstract: Chemospecific and regiospecific modifications
of natural products by methyl, prenyl, or C-glycosyl
moieties are a challenging and cumbersome task
in organic synthesis. Because of the availability
of an increasing number of stable and selective
transferases and cofactor regeneration processes,
enzyme-assisted strategies turn out to be promising
alternatives to classical synthesis. Two categories of
alkylating enzymes become increasingly relevant for
applications: firstly prenyltransferases and terpene
synthases (including terpene cyclases), which are used
in the production of terpenoids such as artemisinin, or
meroterpenoids like alkylated phenolics and indoles,
and secondly methyltransferases, which modify
flavonoids and alkaloids to yield products with a specific
methylation pattern such as 7-O-methylaromadendrin
and scopolamine.

6. The BIONEXGEN newsletter edition 5
The research leading to the results described in this
newsletter has received funding from the European Union
Seventh Framework Programme ([FP7/2007-2013] [FP7/2007-
2011]) under grant agreement n° 266025.
(1) Bartsch, S.; Wybenga, G. G.;
Jansen, M.; Heberling, M. M.; Wu, B.;
Dijkstra, B. W.; Janssen, D. B. Redesign
of a Phenylalanine Aminomutase into
a Phenylalanine Ammonia Lyase.
ChemCatChem 2013, 5, 1797–1802.
(2) Crismaru, C. G.; Wybenga, G.
G.; Szymanski, W.; Wijma, H. J.; Wu,
B.; Bartsch, S.; de Wildeman, S.;
Poelarends, G. J.; Feringa, B. L.; Dijkstra,
B. W.; et al. Biochemical Properties and
Crystal Structure of a β-Phenylalanine
Aminotransferase from Variovorax
Paradoxus. Appl. Environ. Microbiol. 2013,
79, 185–195.
(3) Díaz-Rodríguez, A.; Borzęcka, W.;
Lavandera, I.; Gotor, V. Stereodivergent
Preparation of Valuable γ- or δ-Hydroxy
Esters and Lactones through One-Pot
Cascade or Tandem Chemoenzymatic
Protocols. ACS Catal. 0, 386–393.
(4) Díaz-Rodríguez, A.; Iglesias-
Fernández, J.; Rovira, C.; Gotor-
Fernández, V. Enantioselective
Preparation of δ-Valerolactones with
Horse Liver Alcohol Dehydrogenase.
ChemCatChem 2013, n/a–n/a.
(5) Díaz-Rodríguez, A.; Lavandera, I.;
Kanbak-Aksu, S.; Sheldon, R. A.; Gotor,
V.; Gotor-Fernández, V. From Diols to
Lactones Under Aerobic Conditions Using
a Laccase/TEMPO Catalytic System in
Aqueous Medium. Adv. Synth. Catal. 2012,
354, 3405–3408.
(6) Gerstorferová, D.; Fliedrová,
B.; Halada, P.; Marhol, P.; Křen, V.;
Weignerová, L. Recombinant α-l-
Rhamnosidase from Aspergillus terreus
in Selective Trimming of Rutin. Process
Biochem. 2012, 47, 828–835.
(7) Gerstorferová, D.; Fliedrová,
B.; Halada, P.; Marhol, P.; Křen, V.;
Weignerová, L. Recombinant α-l-
Rhamnosidase from Aspergillus Terreus
in Selective Trimming of Rutin. Process
Biochem. 2012, 47, 828–835.
(8) Gudiminchi, R. K.; Geier, M.;
Glieder, A.; Camattari, A. Screening for
Cytochrome P450 Expression in Pichia
pastoris Whole Cells by P450-Carbon
Monoxide Complex Determination.
Biotechnol. J. 2013, 8, 146–152.
(9) Halim, M.; Rios-Solis, L.; Micheletti,
M.; Ward, J.; Lye, G. Microscale Methods
to Rapidly Evaluate Bioprocess Options
for Increasing Bioconversion Yields:
Application to the ω-Transaminase
Synthesis of Chiral Amines. Bioprocess
Biosyst. Eng. 2013, 1–11.
(10) Heberling, M. M.; Wu, B.; Bartsch,
S.; Janssen, D. B. Priming Ammonia
Lyases and Aminomutases for Industrial
and Therapeutic Applications. Curr. Opin.
Chem. Biol. 2013, 17, 250–260.
(11) Honda Malca, S.; Scheps, D.; Kuhnel,
L.; Venegas-Venegas, E.; Seifert, A.;
Nestl, B. M.; Hauer, B. Bacterial CYP153A
Monooxygenases for the Synthesis of
Omega-Hydroxylated Fatty Acids. Chem.
Commun. 2012, 48, 5115–5117.
(12) Leipold, F.; Hussain, S.; Ghislieri,
D.; Turner, N. J. Asymmetric Reduction
of Cyclic Imines Catalyzed by a Whole-
Cell Biocatalyst Containing an (S)-Imine
Reductase. ChemCatChem 2013, 5,
3505–3508.
(13) O’Reilly, E.; Corbett, M.; Hussain,
S.; Kelly, P. P.; Richardson, D.; Flitsch,
S. L.; Turner, N. J. Substrate Promiscuity
of Cytochrome P450 RhF. Catal. Sci.
Technol. 2013, 3, 1490–1492.
(14) Otte, K. B.; Kirtz, M.; Nestl, B. M.;
Hauer, B. Synthesis of 9-Oxononanoic
Acid, a Precursor for Biopolymers.
ChemSusChem 2013, 6, 2149–2156.
(15) Otte, K. B.; Kittelberger, J.; Kirtz,
M.; Nestl, B. M.; Hauer, B. Whole-Cell
One-Pot Biosynthesis of Azelaic Acid.
ChemCatChem 2013, n/a–n/a.
(16) Rebroš, M.; Pilniková, A.; ŠImčíková,
D.; Weignerová, L.; Stloukal, R.; Křen,
V.; Rosenberg, M. Recombinant α-L-
Rhamnosidase of Aspergillus terreus
Immobilization in Polyvinylalcohol
Hydrogel and Its Application in
Rutin Derhamnosylation. Biocatal.
Biotransformation 2013, 31, 329–334.
(17) Rodríguez-Mata, M.; Frank, A.; Wells,
E.; Leipold, F.; Turner, N. J.; Hart, S.;
Turkenburg, J. P.; Grogan, G. Structure
and Activity of NADPH-Dependent
Reductase Q1EQE0 from Streptomyces
kanamyceticus, Which Catalyses the
R-Selective Reduction of an Imine
Substrate. ChemBioChem 2013, 14,
1372–1379.
(18) Šardzík, R.; Green, A. P.; Laurent,
N.; Both, P.; Fontana, C.; Voglmeir, J.;
Weissenborn, M. J.; Haddoub, R.; Grassi,
P.; Haslam, S. M.; et al. Chemoenzymatic
Synthesis of O-Mannosylpeptides in
Solution and on Solid Phase. J. Am.
Chem. Soc. 2012, 134, 4521–4524.
(19) Scheps, D.; Honda Malca, S.; Richter,
S. M.; Marisch, K.; Nestl, B. M.; Hauer, B.
Synthesis of ω-Hydroxy Dodecanoic Acid
Based on an Engineered CYP153A Fusion
Construct. Microb. Biotechnol. 2013, 6,
694–707.
(20) Sheldon, R. A. Laccase CLEAs in
Textile Waste Water Treatment http://www.
wtin.com/article/?articleID=2642270372
(accessed May 10, 2013).
(21) Vogl, T.; Glieder, A. Regulation
of Pichia Pastoris Promoters and Its
Consequences for Protein Production. N.
Biotechnol. 2013, 30, 385–404.
(22) Wessjohann, L. A.; Keim, J.; Weigel,
B.; Dippe, M. Alkylating Enzymes. Curr.
Opin. Chem. Biol. 2013, 17, 229–235.
(23) Wessjohann, L.; Vogt, T.; Kufka, J.;
Klein, R. Prenyl- Und Methyltransferasen
in Natur Und Synthese. BIOspektrum
2012, 18, 22–25.
(24) Zajkoska, P.; Rebroš, M.; Rosenberg,
M. Biocatalysis with Immobilized
Escherichia coli. Appl. Microbiol.
Biotechnol. 2013, 97, 1441–1455.
Complete list of publications resulting
from research undertaken within the
BIONEXGEN project is listed below: