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“Virtual
Molecular
Biology”
and
its
role
in
Industrial
Biotransformation
Santanu Datta PhD
Anand Anandkumar PhD
Cellworks Research India Pvt Ltd.
There seems to be a gradual paradigm shift in experimental molecular biology. From
individual gene cloning and expression, the science has now moved to ‘pathway
engineering’ and beyond. The point of inflexion was brought to limelight when Craig
Venter and his group synthesized the first functioning synthetic bacteria. However,
what is not always appreciated is that the back end of such endeavours is supported by
virtual molecular biology. This branch of science which is still in its infancy is driven
by large scale genome information. Till date about 188 individual organisms and over
2000 species have been completely sequenced. Following the annotation of the genes,
large scale pathways networks are being stitched. At present most of these networks
are static, akin to road maps of a city. Just as road maps of a city in absence of data on
traffic flow, handicaps a city planner entrusted in building new roads to ease traffic
flow, a static pathway of an organism is of limited use to when one intends to
‘engineer’ recombinant bugs that can generate products of high value to Industries
ranging from Pharmaceutical to Nutrition to Cosmetics to Agro.
To bring life to these static networks one needs to add on kinetic parameters of
enzymes its substrates and ligands. However the values of these parameters like Km,
Vmax and Kd are minimally available. The problem then is how does one generate a
kinetic model in absence of these values. There are two distinct ways that can initiate
this process. One is the popular FBA ( flux balance analysis) technique and the other
process championed by Cellworks is of reverse engineering. The former assumes that
for a given carbon source the flux through various pathways are balanced such that
the cellular biomass is maximized. In contrast the technique of reverse engineering is
to intelligently plug in values of kinetic parameters such that the platform produces
alignment with experimental data. Cellworks is pioneering the creation of Dynamic
maps of various Organisms, that allow for these Organisms to be ‘engineered’ to
partake in various functions of interest to the Industrial Biotransformation segement.
Engineering Whole cell Hosts that drive Biotransformation:
The kinetic platform developed for the bacteria like E.coli and Pseudomonas putida
can be leveraged in Industrial Biotechnology (IBT) specifically in the area of green
chemistry. Environmental awareness in general populace has resulted in tougher
regulations leading to drastic changes in industrial practice. This has resulted in
pharmaceutical industry slowly moving from traditional synthetic chemistry to
environmentally friendly processes of biotransformation. Though biotransformation
have been traditionally used for ages to make alcoholic beverages, its use to generate
metabolites and chiral compounds of pharmaceutical value is in its infancy. With over
70% of drugs being chiral and the FDA guidelines preventing future use of racemic
compounds, there is a major push to use biotransformation to generate these
molecules. Enzymes by its inherent nature are not only safer but superior to metal
catalyst used for chiral synthesis. However one of the major roadblocks in this area is
that many metabolite and chiral synthons require stochiometric amounts of cofactors
like NADH, NADPH for its synthesis. Any large scale synthesis of specific
metabolite thus require large scale production of these expensive molecules. Hence
invitro based biotransformation techniques have a scalability limitation.
However these molecules (NADPH, NADH) are part of the energy currency of a cell.
If a bacterium is driven to produce these metabolites it is starved of energy causing an
energy imbalance that may lead to death, stasis or stunted growth. The kinetic
platform of E.coli gives for the first time an understanding of the energy flow (flux of
NAD, NADH, NADP, NADPH, ATP and ADP ) such that one can redistribute and
optimize the energy flow in a way that cellular biomass increases as also the
production of metabolites. This is somewhat similar to how there are many time zones
in countries of large land mass. Time zones are in effect to redistribute electricity
when necessary (lighting and heating) for optimising work efficiency. In the same
way we would like to optimize the gene network in E.coli (by knocking out some ,
overexpressing others and bringing in new pathways from other organisms) such that
the metabolite production is simultaneously maximised while keeping the cell
healthy. We should remember that a normal bacterium has not evolved to do this job.
It is true that this is not only a challenging task but also it will not be a “one size fits
all” approach. Each metabolite will have its own optimum circuit. This is where
virtual molecular biology will be executed. From a possible of thousands of
combinations, in-silico simulations will predict a small set of optimum rewiring
possibilities of gene network. These will then be experimentally constructed to
produce the desired remodelled bacteria. We are at the cusp of such a dawn.
Dr. Anand Anandkumar serves as the Managing Director and Chairman of the BOD
of the India operations of Cellworks.
Anandkumar received his MS and PhD in Biomedical & Electrical Engineering from
George Washington University, Washington DC. He is a veteran of the
Semiconductor Industry with more than 18 years of experience, specializing in
Electronic Design Automation (EDA) Software products, IC design and in setting up
and running global operations in Emerging markets India and China. Before joining
Cellworks, Anandkumar was the founding Managing Director of the India operations
of Magma Design Automation, where he was responsible for setting up and driving
many aspects of the Operations including RD, Product Engineering, Business
Development and links to Government/Academia. The India operation of Magma
which Anandkumar founded in 2003 is Magma's largest subsidiary with roughly 25%
of the worldwide headcount.
Dr. Santanu Datta, Head of Science, BugWorks
Before joining Cellworks in 2009 he was the Principal Research Scientist in
AstraZeneca India, where he spent more than 2 decades working in Infectious
diseases, with a focus on Malaria and MTB. His current interest is in anti-infective
drug discovery and recombininat DNA technology and its application in Industrial
Biotechnology. He has published widely and has over ten international patents.
Dr. Santanu, received his PhD from Calcutta University in Biophysics in 1981 and
was trained as a molecular biologist during his post doctoral stint in Baylor College of
Medicine USA and Karolinska Institute Sweden.