Background - Prof. Sabine Flitsch`s

PhD Studentship in Industrial Biotechnology funded by CoEBio3
Development of new enzyme activities for applications in synthetic biology: Carboxylic acid
amidases (CAA)
A PhD studentship is available to work under supervision of Professors Sabine Flitsch & Nicholas
Turner at the Manchester Institute of Biotechnology, University of Manchester.
The overall aim of the project is to develop Carboxylic Acid Amidases (CAAs) as new tools for the synthesis of
amides from esters and acids. The formation of amides from diverse amines and carboxylic acids or esters has
been identified as a key target for biocatalysis. A number of enzymes and reaction conditions are available to
catalyse amide bond formation, including use of amidases (‘in reverse’) and esterase such as CalB. Reaction
conditions generally require low water conditions to suppress amine protonation and to shift the equilibrium of
the reaction towards synthesis rather than hydrolysis. The unfavourable equilibria in water will make it
challenging to translate such amide bond formations into whole cell biotransformations as part of toolkits for
synthetic biology and biochemistry. In Nature, amide bond formations are generally driven by activation of
carboxylic acids as phosphate esters anhydrides or as thioesters, often using ATP hydrolysis as the driving force.
However, the amidases studied so far are generally part of large and highly specific multienzyme machineries
and are difficult to manipulate. Hence, there is a need for small, generic, ATP driven carboxylic acid amidases
which catalyse the formation of amide bonds from amines and carboxylic acids, as part of the synthetic biology
and biochemistry toolbox.
The project will have the following objectives: (i) Database survey of suitable amide synthase domain
candidates. A benchmark will be a recently reported amidase with broad specificity; (ii) identification of most
promising candidates; (iii) generation of a number of putative CAA genes (iv) protein expression in E.coli; (v) in
vitro assay of expressed proteins for ATP uptake, acid activation to thioester, amidation; (vi) determination of
substrate range of new CAAs ; (vii) whole cell studies; (viii) make active CAAs available to industrial affiliates in
The PhD student will receive training in a broad range of complementary disciplines including synthetic and
analytical chemistry, molecular biology, microbiology and fermentation, biocatalysis, protein engineering and
high-throughput screening techniques. All of the required expertise and equipment to undertake this project is
available within the Manchester Institute of Biotechnology (MIB) at the University of Manchester which
provides an ideal environment for multi-disciplinary projects of this type. The student will join a highly active
and vibrant research group and will receive additional training in problem-solving and presentational skills
through participation at weekly group meetings. Both supervisors have considerable experience of supervising
CASE students and also of collaborating on projects of this type.
Candidates should have a good first degree at Master’s/BSc level (at least 2i or equivalent) in either chemistry
or biological sciences. Full funding including fees and stipends are available for all UK and EU citizens.
Candidates should send a full CV and the names of 2 referees to Professor Flitsch at
[email protected]