Enzyme expression in bacteria and yeasts

There are various processes for the production of enzymes. One possibility is the direct use of microbial isolates for enzyme production such as white rot fungi for the production of ligninolytic enzymes or chitinolytic bacteria for the production of chitinases. Many of these microorganisms already form the desired enzymes in sufficient quantity under the correct cultivation conditions and without genetic modifications, which can be used directly or after purification in industrial processes.


However, microorganisms often produce not only the desired enzyme, but also a cocktail of different enzymes. In addition, many microbial isolates are not accessible for genetic engineering methods. As a result, modifications of the enzyme to optimize activity, specificity or purification (e.g. by altering the active center or by attaching affinity tags) or to the organism itself that improve enzyme production (e.g. by overexpression of folding aids) can hardly be carried out. In such cases, the desired enzyme can be produced heterologously in established, microbial production strains such as Escherichia coli.


Fraunhofer IGB deals both with the identification of microbial isolates that produce desired enzymes or enzyme mixtures and with the generation of microbial production strains for heterologous enzyme production. Prokaryotic expression systems such as Escherichia coli or Bacillus subtilis and eukaryotic systems such as the yeast Kluyveromyces lactis and the methylotrophic yeast Komagataella pastoris (formerly Pichia pastoris) are used for heterologous enzyme production.

Advantages of heterologous expression

  • Gene expression can be significantly increased by using strong promoters or by introducing several gene copies coding for the desired enzyme.
  • The desired enzyme can be specifically expressed by inducible promoters, thus decoupling the growth and production phases.
  • Targeted mutations are possible to improve the activity or specificity of the enzyme.
  • Many production strains are optimized for enzyme production, e.g. for the production of sequences particularly rich in GC. Protease-deficient strains are also available, which minimizes yield losses due to degradation of the enzyme product during cultivation.
  • Intracellular enzymes can be secreted into the culture medium by linking them to a signal sequence. This means that cell disruption is no longer necessary and the enzyme product is purified for the first time during cultivation.


Range of services

  • Recombinant production of perhydrolases, lipases, peroxidases, phospholipases, dismutases, alcohol dehydrogenases, ligninolytic enzymes, chitinases
  • Identification and characterization of suitable enzyme producers
  • Development and optimization of stable prokaryotic or eukaryotic expression systems for industrial enzymes
  • Process development and optimization for enzyme production up to pilot scale (10 m3)

Objectives and strategies

Our goal is to establish stable microbial expression systems for selected enzymes and to develop optimized processes for their large-scale production. Several 1-liter parallel fermenters and a 42-liter bioreactors with methanol probe for the cultivation of K. pastoris are available at Fraunhofer IGB in Stuttgart to investigate enzyme production on a laboratory scale. The multifunctional plant at Fraunhofer CBP at the Leuna chemical site enables scale-up of optimized processes up to the pilot scale of 10 m3. The use of methanol as an inductor for protein expression is a problem in the fermentation of K. pastoris. Methanol is toxic and highly flammable, which is why certain safety requirements must be met for large-scale fermentation. These requirements were explicitly considered in the design of the multifunctional plant at Fraunhofer CBP in order to be able to carry out fermentations by K. pastoris on a technical scale.

Results and outlook

Enzymes already produced at the institute and their possible fields of application.
Enzymes already produced at the institute and their possible fields of application.
Lipase spray-dried.
Lipase spray-dried.

Several enzymes are already expressed heterologously at Fraunhofer IGB in E. coli or the yeasts K. pastoris and K. lactis. Examples can be found in the adjacent table. Current work focuses on process optimization for the efficient production of some of the enzymes mentioned above.

The expression of a lipase in K. pastoris could already be successfully carried out at Fraunhofer CBP up to the 1 m3 scale and several kilograms of spray-dried or lyophilized enzyme could be produced. The lipase produced is currently being investigated for the chemo-enzymatic epoxidation of vegetable oils and is to be used on a larger scale for the production of biobased epoxides. Some of the enzymes are produced in different E. coli systems. Different strains were compared for growth and enzyme expression and the strain with the highest expression was selected. By varying parameters such as culture medium, carbon source, expression temperature and duration as well as induction time, the yield of active enzyme could be further increased. Similar optimizations are being carried out for other enzymes. Furthermore, the scale-up of these results to fermentations is carried out first on a laboratory scale and later on a pilot scale up to 10 m3 (Fraunhofer CBP Leuna).


Werner N, Hirth T, Rupp S, Zibek S. 2015. Expression of a codon-optimized Carica papaya papain sequence in the methylotrophic yeast Pichia pastoris. in: Journal of Microbial and Biochemical Technology. https://doi:10.4172/1948-5948.1000231


Blaschke L, Wagner W, Werkmeister C, Wild, M, Gehring A, Rupp S, Zibek S. 2017. Development of a simplified purification method for a novel formaldehyde dismutase variant from Pseudomonas putida J3. in Journal of Biotechnology, Vol. 241, pp. 69-75. https://doi.org/10.1016/j.jbiotec.2016.11.007