Underpinning technology developments for successful IB processes

Professor Anton Glieder of the Austrian Centre for Industrial Biotechnology (ACIB) discussed the key technologies for enzyme engineering and chemical production by IB with a specific focus on protein expression. Download Prof Gliederís presentation here. Enzyme production is a large and growing market of which the industrial enzyme market was valued at Ä3.3bn (BCC Res Jan 2011: Enzymes in industrial applications: global markets) across a multitude of sectors to include household, beverages, food and feed.

Most industrial enzymes are produced by recombinant GRAS organisms. Biocatalysis for pharma have a high catalyst cost that yield a high value product(s).  Catalyst examples include transaminases, keto reductases, P450s, BVMOs, esterases, aldolases of which most are intracellular enzymes, often produced by E. Coli.  One of the key bottlenecks in industrial biotechnology is microbial protein production at a low cost, high quantity and quality.  The typical production hosts include Aspergillus, Trichoderma, C1, Bacillus and Pichia.  Also of interest is any host which provides active enzymes, examples of which are:

    • E. coli, Pseudomonas, yeasts, extremophiles, insect cells
    • cheap catalysts and protein materials
    • correctly folded and active enzymes
    • balanced biosynthetic pathways

As part of the FP7 projects, ACIB have been working to improve the expression host / vector system and to improve upon or adapt bioreactor cultivation methodologies to increase microbial protein production. Methodologies that have been investigated include increasing the complexity of the expression systems by adding duel or multi-gene expressions, and improving the host strains. ACIB are well positioned to capitalise on the multiplicative effects of improvements and realise that to deliver the necessary innovations at each step of the bioprocess would require a vision beyond a typical three year project.  He adds that EU projects are essential in delivering such innovations and allow for a perfect mix of consortia and network of projects.
Professor Bernhard Hauer, University of Stuttgart discussed the advancements in biopolymer synthesis.  Presentation is available to download here. The importance and need for enzymes was highlighted by drawing upon a societal challenge that affects all of us; how do you fit 7 billion people onto 1 planet that has a sustainable capacity of 2 billion. The solution in part is enzymes, they are innate and they are within us and around us.  Enzymes therefore are essential for both plant and animal life on the planet.
The group are interested in products to include aroma compounds, fatty acids (including derivatives) and pharmaceuticals (including precursors) examples of which are provided below:

Some of the strategies adopted by the group to identify novel biocatalytic activity are highlighted below:

Professor Hauer provided the following examples of functionalized fatty acids - intermediates for polymers and fragrances currently under investigation:

The techniques adopted include substrate characterization, modeling of mutants, site-directed mutagenesis of hotspots, construction of production strain and scale up (collaboration with DTU). Overall the Institute of Technical Biochemistry at the University of Stuttgart are focused on the provision of novel biocatalysts to further expand the spectrum of accessible biocatalytic reactions along with the genetic selection of material-specific peptides and their use as building blocks for controlled preparation of new biomaterials for various applications in biotechnology.
Professor Dick Janssen (University of Groningen) discussed the role of enzymology in developing enzymes for synthesis.
The role of enzymes for synthesis is widely recognized as an opportunity in the preparation of a wide range of chemical intermediates and products.  This is true not only for academic syntheses, but also for industrial-scale applications.  For numerous intermediates and products the synthetic routes based on enzyme synthesis have turned out to be competitive (and in some cases enhanced) compared with the classic chemical as well as chemocatalytic synthetic approaches.  Thus, enzymatic synthesis is becoming increasingly recognized by chemists in both academia and industry as an attractive alternative compared to conventional preparations utilising metal catalysis and organocatalysis.
Enzyme kinetics is the study of the chemical reactions that are catalysed by enzymes.  In enzyme kinetics, the reaction rate is measured and the effects of varying the conditions of the reaction are investigated.  Studying an enzyme's kinetics in this way can reveal the catalytic mechanism of the enzyme, its function, how its activity is controlled, and what might inhibit the enzyme.  The kinetic mechanism can be investigated further by varying parameters and properties to include:

  • catalytic rates, substrate affinity, selectivity
  • pH and inhibitor effects
  • product inhibition
  • enantioselectivity (e.g. kinetic resolution, asymmetric conversion)
  • synthesis vs. hydrolysis properties (e.g. for coupling reactions)

The Biotransformation and Biocatalysis group at the University of Groningen utilise a number of developed techniques to investigate enzyme kinetics.  As a group they are well positioned to obtain insights into enzymology of the microbial transformation of synthetic compounds, and the development of improved biocatalysts for such transformations.  The group studies catalytic mechanisms, kinetic properties and structure-function relationships in enzymes that can be used for region- and enantioselective synthesis reactions for preparing fine chemicals.  The group’s competencies extend further to include obtaining new enzymes by enrichment, genome mining, and high throughput screening as well as their improvement by structure-inspired directed evolution.