Dr Alexander Pelzer
is our expert in the field of enzyme technology
“One of our biggest strengths is our team composed of experts from diverse scientific fields who work together closely and creatively allowing us to regularly find new solutions to scientific challenges.”
Knowing where to search is a huge benefit. Targeted habitat selection or even substrate-conditioned microbial habitats (enriched cultures) greatly enhance the likelihood of tapping into biodiversity that contains the desired enzyme activity. We specifically sample habitats where an enzyme activity can be anticipated and also sample promising biological material provided by a collaboration partner.
Our bioinformatics pipeline identifies novel enzymes based on homology to a given benchmark sequence or can select specific sequences covering maximum diversity while limiting screening effort. For the identification of homologous neighbors, the pipeline simultaneously searches within the proprietary BRAIN SeqPool and public sequence databases. Our experts rationally select enzyme candidates utilizing sequence-function relationships and refine this selection via structure-based methods including homology modeling and docking. The set of enzyme candidates is produced in vivo in one or more of our recombinant expression strains. Multi-parameter screening under process-relevant conditions is performed to identify enzyme development candidates suitable for the target application.
For the identification of enzymes that perform like no known model enzyme, we screen within our already cloned metagenomic gene expression libraries, the Activity-Based Expression Libraries (ABEL®) and Large Insert Libraries (LIL®). ABEL® libraries express single genes, while LIL® libraries contain large genes or small operons. The only requirement is an activity assay displaying the desired enzyme activity in a high-throughput format. To discover wild-type (non-GMO) enzyme producers, we select strains from our BioArchive and screen them with specific activity assays for the desired activity.
Basic strains are generated within a short space of time in order to
test the production performance and thus the suitability of an
expression system. Subsequently, the production yield is maximized via
directed and/or random strain development. Direct methods target aspects
including translation initiation, gene design, signal peptides, promoters and gene copy numbers. Hosts are also engineered, for example by introducing protease knock outs.
For random strain development, previously developed strains are treated with physical or chemical mutagens to accumulate mutations leading to optimized production strains.
Strain screening and development is conducted in 96-well plate and shake flask format. Different media are available that utilize different feeding and induction strategies for small-scale expression studies. Development candidates are evaluated in 1 L or 2 L fermentation scale to quantify protein production under relevant cultivation conditions. Strong protein-producing strains are handed over to our colleagues from the Bioprocess Technology Unit for production process development.
In addition to heterologous protein and enzyme production, our experts also optimize wild-type producers by “evolution in the lab” and subsequently screen the generated diversity in high-throughput processes to identify enhanced producers.
Our molecular biologists have all the required tools at their disposal for gene construction and cloning as well as diverse PCR methods for mutagenesis. In addition to this, we use our proprietary nuclease-based method REDUCE® that enables us to directly perform protein engineering experiments in almost every production host. This accelerates enzyme development and reduces the likelihood of failure.
Library quality and mutation distribution can be monitored using in-house NGS capabilities. For gene expression and screening, our microbial expression experts provide several prokaryotic and eukaryotic expression strains.
For small and medium-size libraries, we directly use multi-parameter screenings that analyze multiple application-relevant enzyme characteristics simultaneously. For screening of large mutant libraries, we apply agar plate indicator assays, selection assays or FACS-based screenings. A large number of enzyme assays are already established and available. If collaboration partners want to use proprietary activity tests for screening, we can quickly establish and/or adapt these tests for the use at BRAIN.