Presenting - Special Edition - your link to Carbon Chemistry - CarbaZymes
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e-Newsletter No3. September 2017
your link to Carbon Chemistry
= your link to a Green Future!
Presenting
Special Edition
This project has received funding from the European Union’s
Horizon 2020 Research and Innovation Programme under
Grant Agreement No 635595
WELCOME FROM THE COORDINATOR
It was a great opportunity for all consortium partners to meet again at the Eötvös Lóránd
University´s modern facilities in Budapest for the 2 nd CarbaZymes one-day progress meet-
ing. Our meeting was held as a satellite event on the occasion of the 13 th BioTrans Sympo-
sium (July 09 –13, 2017), Europe’s major conference in applied biocatalysis, which started
the evening of the same day!
The CarbaZymes consortium held a constructive progress and coordination meeting, and
the partners had the chance to gather once more in person for in-depth discussions. This
time it took place in the beautiful surroundings of Budapest, which greeted us with unusu-
ally hot summer temperatures, in line with our hot science: indeed, I am proud to an-
nounce that the CarbaZymes consortium now has filed for two technology patents so far, arising from our innova-
tive research and significant discoveries!
Following our internal meeting, the CarbaZymes consortium continued with a strong presence at the BioTrans con-
ference, where most partners participated with lectures and posters, SME partner PROZOMIX was one of the main
conference sponsors and SME partner Bio-Prodict held a company stand: All partners disseminated non-
confidential results of the project, and Bio-Prodict presented their top technology throughout the conference:
Hence the CarbaZymes project and its outcome was prominently made known to more than 520 top level scien-
tists present, in a gathering of Europe’s expertise in the field of biocatalysis and biotransformation.
Moreover, on this occasion our upcoming CarbaZymes conference – “Novel Enzymes 2018” — planned to be held
in October 2018 at the coordinator´s facilities of TU Darmstadt, Germany (together with the H2020 project
ROBOX), was officially announced by distributing informative flyers to all BioTrans participants.
The CarbaZymes presence was strong at BioTrans 2017 and now we aspire to see you together with just as many
participants in our final showcase meeting in Darmstadt next year!
2
ABOUT CARBAZYMES We have been busy researching…
Carbon-carbon bond forming reactions are key
The CarbaZymes project is a 48-month col-
processes in industrial organic synthesis, and rou-
laborative project funded by the European
tinely done by applying inorganic chemical and of-
Commission under the Horizon 2020 Pro- ten toxic catalysts while at the same time these
gramme with grant agreement number processes produce large amounts of toxic waste.
635595 and a total budget of 8,2M€. During its However, milder, efficient biocatalytic routes to
course, the project will implement the bio- such syntheses remain insufficiently explored due
catalytic synthesis of Active Pharmaceutical to the lack of suitable enzymes. The scientists be-
Ingredients (APIs) and bulk chemicals by using hind CarbaZymes address this major industrial and
a broad platform of unique environmental challenge by researching and devel-
C-C bond forming enzymes. oping a broad toolbox of carbon-carbon bond form-
ing biocatalysts tailored to industrial needs, which
will lead to novel safer and more efficient indus-
trial processes, to benefit the industry the consum-
ers and the environment simultaneously.
The CarbaZymes consortium has already identified
and developed a broad panel of improved enzymes
for carbon-carbon bond formation in a supra-
disciplinary manner, based on their successfully es-
tablished, advanced high-throughput genome min-
ing technology and bioinformatics tools.
Using these novel biocatalysts, innovative carboligation cascade processes could be realised and are now be-
ing tested using microreactor technology for bioprocess characterization and advanced reaction engineer-
ing. This will enable the development of scalable syntheses of the targeted high value API and bulk chemical
compounds.
Overall, CarbaZymes aims to promote innovation in the field of carbon-carbon bond formation at large scale,
by developing natural enzyme-catalyzed routes to important specialty and bulk chemical precursors. The
range of novel and improved biocatalysts generated from this research will also provide opportunities for the
synthesis of many additional valuable products, thus making a profound impact on reducing emissions, en-
ergy consumption and toxic waste. We are on the way to achieving this!
3
CarbaZymes Research is Editor‘s Choice!
The research of Professor Clapes and his team,
published under the title 2-Keto-3- Deoxy-l-
Rhamnonate Aldolase (YfaU) as Catalyst in Aldol
Additions of Pyruvate to Amino Aldehyde Deriva-
tives has been featured as „Editor´s Choice“ in
the Chemicals & Chemistry newsletter.
The newsletter reports on the latest research,
product and marketing trends in the chemical industry. Its contents are read more
than 100,000 times per year and are invaluable to academics, researchers, biotech-
nology and healthcare companies, as well as governments and consulting firms. Car-
baZymes is expanding its appeal!
PUBLICATION’S ABSTRACT
4-Hydroxy-2-keto acid derivatives are versatile building
blocks for the synthesis of amino acids, hydroxy carboxylic
acids and chiral aldehydes. Pyruvate aldolases are privi-
leged catalysts for a straightforward access to this class of
keto acid compounds. In this work, a Class II pyruvate al-
dolase from Escherichia coli K-12, 2-keto-3-deoxy-l-
rhamnonate aldolase (YfaU), was evaluated for the syn-
thesis of amino acid derivatives of proline, pipecolic acid,
and pyrrolizidine-3-carboxylic acid. The aldol addition of
pyruvate to N-protected amino aldehydes was the key en-
zymatic aldol addition step followed by catalytic intramolecular reductive amination. The corresponding N-Cbz-
amino-4-hydroxy-2-keto acid (Cbz=benzyloxycarbonyl) precursors were obtained in 51–95% isolated yields and en-
antioselectivity ratios from 26:74 to 95:5, with chiral α-substituted N-Cbz-amino aldehydes. (S)-N-Cbz-amino alde-
hydes gave aldol adducts with preferentially (R)-configuration at the newly formed stereocenter, whereas the con-
trary is true for (R)-N-Cbz-amino aldehydes. Addition reactions to achiral amino aldehydes rendered racemic aldol
adducts. Molecular models of the pre-reaction ternary complexes YfaU-pyruvate enolate-acceptor aldehyde were
constructed to explain the observed stereochemical outcome of the reactions. Catalytic reductive amination of the
aldol adducts yielded 4-hydroxy-2-pipecolic acid, and unprecedented C-5 substituted 4-hydroxyproline and pyrrol-
izidine-3-carboxylic acid derivatives.
4
BIOTRANS PLENARY LECTURE: PROF. PERE CLAPÉS
ENGINEERED CARBOLIGASES WITH UNPRECEDENTED SUBSTRATE TOLERANCE
Stereoselective carbon–carbon bond formation is a pivotal process in the asymmetric
construction of the skeletal frameworks of complex chiral molecules from simple start-
ing materials. Catalytic C─C coupling through aldol addition is particularly valuable in
asymmetric synthesis because of its potential for stereodivergent product generation,
by which multiple stereoisomeric products can be derived from common synthetic
building blocks.[1]
Using enzyme catalysis (i.e. carboligases), molecular complexity can be rapidly built up
under mild conditions, without a need for protecting sensitive or reactive functional
groups, with high chemical efficiency and often with uncompromised stereochemical fidelity. [2] In this sense, suita-
bly engineered known and novel carboligases are promising candidates to fuel future developments in the field.
In this communication, engineered D-fructose-6-phosphate
aldolase (FSA), L-serine hydroxymethyl transferase (SHMT),
with glycine aldolase activity, and 2-keto-3-deoxy-L-
rhamnonate aldolase (YfaU), will be discussed for diverse syn-
thetic applications (Scheme 1).[3] Wild-type and different vari-
ants of these carboligases will be presented that allow to
modulate its selectivity against different substrates.
Scheme 1. Aldol addition catalyzed by YfaU, FSA, and SHMT
[1] R. Mahrwald, Modern Aldol Reactions, Vol. 2: Metal Catalysis,
Wiley-VCH, Weinheim, 2004.
[2] P. Clapés, in Biocatalysis in Organic Synthesis Vol. 2 (Eds.: K. Faber, W.-D. Fessner, N. J. Turner), Georg Thieme Verlag KG, Stuttgart
(Germany), 2015, pp. 31.
[3] a) A. Szekrenyi, X. Garrabou, T. Parella, J. Joglar, J. Bujons, P. Clapés, Nat. Chem. 2015, 7, 724; b) D. Güclü, A. Szekrenyi, X.
Garrabou, M. Kickstein, S. Junker, P. Clapés, W.-D. Fessner, ACS Catal. 2016, 6, 1848. c) K. Hernandez, I. Zelen, G. Petrillo, I. Usón, C.
M. Wandtke, J. Bujons, J. Joglar, T. Parella, P. Clapés, Angew. Chem. Int. Ed. 2015, 54, 3013; d) [5] K. Hernandez, J. Bujons, J. Joglar,
S. J. Charnock, P. Domínguez de María, W. D. Fessner, P. Clapés, ACS Catal. 2017, 7, 1707.
ABOUT THE AUTHOR:
Prof. Pere Clapés of the Catalonia Institute for Advanced Chemistry (IQAC–CSIC) is a chemist
specializing in biocatalysis and biotransformation in organic asymmetric synthesis of bioac-
tive molecules. He has published over 100 scientific papers and patents in the field of bio-
catalysis. Currently he is head of the Biotransformation and Bioactive Molecules Group were
he conducts research on the exploitation of biocatalysts in asymmetric carbon-carbon bond
formation as a means to rapidly create chiral structures of high complexity to produce novel
compounds for bioactivity testing. The research includes cascade chemoenzymatic synthesis,
analysis, purification, computational models and biological evaluation of products. Professor
Clapés product research is focused on cancer, particularly in metastasis, and the prevention
of the metabolic syndrome, with particular focus on the control of blood sugar.
5
BIOTRANS LECTURE: DR ANNA SZEKRENYI
FLUOROGENIC KINETIC ASSAY FOR HIGH-THROUGHPUT DISCOVERY OF STEREOSELECTIVE KETOREDUCTASES
RELEVANT TO PHARMACEUTICAL SYNTHESIS
Enantiomerically pure chiral alcohols, especially those derived from acetophe-
none type precursors, are frequent and important building blocks for the produc-
tion of pharmaceutical intermediates. Particularly, NAD(P)H dependent ketore-
ductases (KREDs) are a well established and reliable source of chiral alcohols with
high enantiomeric purity on industrial scale.
An assay concept was developed that allows the screening of novel enzymes with selected properties, either from
natural sources or by optimization of known candidates by directed evolution in vitro. This assay further allows a
systematic modular variation of the substrate structure to facilitate a “substrate morphing” (or “substrate walk-
ing”) approach.
We established a highly sensitive, reliable and simple assay method for the rapid determination of activity meas-
urements and enantioselectivity typing of unknown KREDs. The assay principle is based on the generation of
brightly fluorescent acetophenone derivatives from enantiomerically pure carbinols. Because of its simplicity in
microtiter plate format the assay qualifies for the discovery of novel KREDs of yet unknown specificity among this
vast enzyme superfamily. The suitability of this approach is illustrated by a quick exemplary screening of a large
collection of hundreds of short-chain
dehydrogenase/reductase (SDR) en-
zymes arrayed from metagenomic ap-
proach.
We believe that this assay format
should match well the pharmaceutical
industry’s demand for acetophenone- Figure 1. Fluorogenic kinetic assay applied for quick and reliable screening of a
type substrates and the continuing in- large collection of KREDs.
terest in new enzymes with broad sub-
strate promiscuity for the synthesis of chiral, non-racemic carbinols.
ABOUT THE AUTHOR:
Dr Anna Szekrenyi is a postdoctoral researcher currently based at the Technische Universität
Darmstadt. She works in Professor Fessner´s group developing and testing novel C–C bond
biocatalysts and processess.
6
BIO-PRODICT SHORT PRESENTATION
ENZYME SELECTION AND OPTIMISATION WITH 3DM: A PROTEIN SUPERFAMILY APPROACH
New enzymes often need to be optimized by introducing
mutations to meet the requirements of a (biotechnological)
process. In many cases multiple mutations are needed to reach
these goals, but finding the right combination of mutations still
is problematic. Furthermore, the selection of an optimal starting
enzyme greatly influences the performance of the final enzyme
while this selection should also take into account the freedom to
operate in the enzyme family.
3DM mitigates many of the burdens that researchers face in
dealing with the growing amounts and complexity of biomedical
data. For each protein family a large amount of information that
is extracted from protein structures, alignments and scientific
literature is available among others. All this information is
integrated and validated, and can be analysed via a number of
different methods and tools. We present an update to 3DM, where we have added novel tools that give researchers the
functionalities they need to design novel enzymes through all the stages of target selection, hotspot detection and
enzyme optimisation:
Panel design: Intelligently construct sequence panels that cover the complete sequence space. This approach can
combine phylogeny and active site motifs.
Patent data integration: Visualize freedom to operate and use patent data to modify your panel accordingly.
Iterative enzyme optimisation: Design smart libraries and iteratively improve the desired properties of your enzyme.
These tools are tightly interconnected and tightly integrated with 3DM superfamily data. Comparisons with random de-
signed libraries show that using the 3DM tools results in high quality libraries reducing not only the number of clones
that need to be screened but also increases the chance of finding an enzyme with the desired properties. Initial results
from partners show that expression levels of enzymes are structurally much higher compared to conventional methods.
This demonstrates the opportunities that integrated knowledge databases offer, combined with the right tools that en-
able researchers to intelligently select proteins, residues and mutations.
Bio-Prodict is a spin-off from the Wageningen University that focuses on
bio-informatics in the field of protein engineering, drug design and DNA
diagnostics. Bio-Prodict has proprietary technology (3DM) for the gen-
eration of protein super-family systems that can be used to guide protein
-engineering experiments. The company applies novel approaches to
data mining, storage and analysis of protein data and develop state-of-the-art analysis and visualization tools. Due to
the many customers all over the world (e.g. universities, biotech companies, pharmaceutical companies included large
size companies as Merck, GSK, Sanofi Pasteur, Sandoz and Crucell), and due to the many collaborations in EU project,
Bio-Prodict has extensive experience in bio-informatics software development and data analysis.
7
BIOTRANS 2017 SPONSOR
CarbaZymes partner Prozomix Limited supported the BioTrans 2017 conference as a
major sponsor. Prozomix is a is a research intensive UK biotechnology company and
metagenomics discovery leader, focusing its proprietary GRASP™ high-throughput ge-
nome-mining technology specifically towards the development of novel/maximum di-
versity panels of biocatalysis enzymes. The large panels of enzymes, that comprise the
very popular and effective Prozomix Biocatalysis Enzyme Toolkit, are developed to
guarantee maximum diversity from known primary sequence space, and are then itera-
tively expanded to evenly sample private metagenome sequence data, using HT-hit optimization software -
ProzOMIGO™. Subsequent to the identification of a hit, biocatalysts of interest can be rapidly purchased to any
scale at known list prices, or produced by the client (or their CMO). As Prozomix both discovers and produces all of
its biocatalysis enzymes, lead-times are short and reliable, and many additional biocatalyst optimisation services
can be seamlessly implemented and are free from IP restrictions.
Prozomix has a reputation for helping customers rapidly find hits
against most challenging processes, especially in their focus areas
of APIs, flavours/fragrances, food and health.
Over the last 3 years they have been very busy developing and
applying beyond state-of-the-art in silico / in vitro tools to de-
velop the largest / most diverse range of Biocatalysis Enzymes
available on the market and are now keen on presenting these
leading-edge biocatalysts to the larger biotransformation com-
munity.
Prozomix scientists provided the microplate screening panel of
metagenomic KRED enzymes that were investigated by fluoro-
750 L In-house Pilot Lab
genic assay typing in Dr Szekrenyi’s presentation (page No. 5).
For further information contact: sales@prozomix.com
8
POSTER PRESENTATIONS
During BioTrans, Carbazymes was present with 14 posters cover-
ing all research areas of the Project. This was a great opportunity
for our young researchers to present their great results. In the
following table there is a list of all the posters, their authors and
a link to the pdf version of the poster located in our webpage.
Feel free to download them!
Click to Click to
Title Author Partner Title Author Partner
download download
Development of a one-
Engineered class II pyruvate aldo- pot, two-enzyme cascade Mrs Yen-Chi
Dr Jordi TUDA
lase from E. coli, YfaU, with unpre- CSIC for the synthesis of a Thai
Bujons
cendented nucleophile promiscuity
lovastatin analog
2-Keto-3-deoxy-l-rhamnonate aldo- An improved monomeric
lase (YfaU) as catalyst in aldol addi- Dr Jesús Mrs Sandra
CSIC folding reporter to select UAM
tions of pyruvate to aminoaldehyde Joglar Bosch
thermostable proteins
derivatives
Synthesis of cyclic ß-hydroxy-alfa-
Selection of optimal proc-
amino acid derivatives by 3-methyl- Dr Roser Prof Ana Vrsa-
CSIC ess design in the synthesis UZAG
2-oxobutanoate hydroxymethyl- Marin Valls lović Presečki
of statin intermediate
transferase
D-Fructose-6-phospate-aldolase
Mathematical modelling
catalyzed asymmetric aldol addi-
Mrs Raquel of lactone synthesis cata- Mrs Anera
tions of simple aliphatic ketones CSIC UZAG
Roldan lyzed by aldehyde dehy- Švarc
and aldehydes. Synthesis
drogenase
of pyranose synthons
Mutability-landscape guided engi- Enzymatic cascade reac- Mrs
Mr Lieuwe
neering of a promiscuous enzyme RUG tion for the synthesis of Morana Ces- UZAG
Biewenga
for c-c bond-forming reactions imino sugar precursors nik
Optimization of enzyme
Improving enzymatic C-C-bond Dr Willem Prof Zvjez-
TUBS catalyzed aldol addition of
formation Dijkman dana Findrik UZAG
propanal and formalde-
Blazevic
hyde
Enzyme immobilization in the Dr Katharina Reaction engineering of
chemoenzymatic synthesis of a Gloria TUBS the mend-catalyzed 1,4- Dr Martina
statin intermediate Hugentobler UZAG
addition of a-ketoglutaric Sudar
acid to acrylonitrile
9
UPCOMING
CARBAZYMES EVENTS
Joint H2020 Summer School 2017 - Siena, Italy
Download the Programme here
Biocatalysis as a Key Enabling Technology CarbaZymes/ Robox/Metafluidics
Tuesday 03 – Friday 06 October 2017
Expoquimia: Networking activities - Barcelona, Spain
CarbaZymes representatives will participate When October 3rd 2017
in the networking activities of Expoquimia, a Where Fira de Barcelona, Gran Via venue map
unique international chemicals fair which
Preliminary agenda
takes place every three years in Barcelona.
Three partners from the Project will present 16:20 Dr Roberto Horcajada (IUCT)
breakthrough results and outcomes. 16:40 Dr Pablo Dominguez (SUSMOM)
17:00 Dr Jaume Mir (Biochemize)
CPhI Worldwide - Frankfurt, Germany
Biospain 2018 - to be announced
CarbaZymes Entrepreneurship in the Life Sciences workshop will be
organized at Biospain2018
10Sustainable industrial processes based
on a C-C bond-forming enzyme platform
This project has received funding from the European Union’s
Horizon 2020 Research and Innovation Programme under
Grant Agreement No 635595
For more information:
info@carbazymes.com
www.carbazymes.com
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@carbazymes
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