20 July 2020

Protein engineering with reduced risk

Using the REDUCE method, the “protein engineers" at BRAIN AG can optimize a protein in a targeted manner while it is already located in the production host. Enzyme developers can use the method to avoid undesirable developments and save time.

REDUCE – the acronym stands for the Rational Enzyme Design Using CRISPR Editing technology. The acronym is intended to bring to mind "reduction", even though the technology is more of a gain for the company itself. With the technology developed in-house and already registered as a trademark in some countries, BRAIN AG has expanded its biotechnology portfolio and can now precisely modify proteins without having to change the microorganism (host organism) used for production after the proteins have been optimized. This drastically reduces the effort involved in typical protein engineering experiments. Enzyme developers and producers alike appreciate this advantage.

Avoid undesirable developments in enzyme optimization

The selection of a suitable production host, i.e. the microorganism cell that will later produce the modified enzyme, plays a major role in the optimization and production of enzymes. The cost-effective production of a specialized enzyme depends largely on the host organism and its function as an expression system and on the success of protein engineering.

In general, protein engineering experiments in the lab are performed with plasmid-based expression systems. When it comes to production the host usually has to be changed by isolating the previously modified gene and integrating it into the genome of the actual production host. However, this two-stage process can be disadvantageous for enzyme production because host organisms generally differ from each other, e.g. in terms of their transcriptional and translational control elements, their gene structure or in the way proteins are secreted or folded. In particular, posttranslational modifications, the processing of molecules and the incorporation of cofactors can dramatically change the enzyme properties in an unwelcome direction. Therefore, experts strongly recommend performing protein engineering experiments directly in the selected production organism in order to avoid or minimize such undesirable developments and the possibly associated negative effects on enzyme optimization.

Amylases from yeast cells

In the course of their R&D activities for enzyme optimization, scientists at BRAIN have developed a technique that allows protein engineering directly in the genome of the selected production organism. To this end, they have cleverly combined "Rational Enzyme Design" (rational prediction of amino acid exchanges) and CRISPR Editing (targeted cutting of DNA) the result is "Rational Enzyme Design Using CRISPR Editing", REDUCE.

The scientists used two alpha-amylases as their “guinea pigs”. These enzymes are used, for example, in the production of bioethanol when starch-containing raw materials are fermented. In order to optimize the amylases, the protein engineers at BRAIN, together with their bioinformatics colleagues, designed an amylase library. In their production organism, they were able to show that the CRISPR/Cas9 gene scissors are suitable for the targeted genetic manipulation of chromosomal gene copies in order to modify the desired protein. Incidentally, the host organism was Pichia pastoris, one of the biotechnologists’ favorite yeasts.

Using the REDUCE method, the BRAIN team was able to set specific mutations in the form of exact base exchanges in the DNA of the host organism. During the subsequent enzyme screening, several hits with improved properties were identified. In a next step, the optimized amylases were biochemically characterized so that they can now be offered to enzyme companies as development candidates.


REDUCE can drastically reduce the risk of erroneous developments in classical protein engineering experiments. The technology replaces plasmid-based screening to identify hit mutants prior to integration into the designated production strain. There is no need to validate the beneficial mutations when they are later produced in the genomic context of the finally selected production strain. In this way, REDUCE helps BRAIN to achieve rapid enzyme development results for its customers. This all adds up to protein engineering with reduced risk – also for our customers.

Note: REDUCE has already been registered as a trademark of BRAIN AG in the USA, Singapore and the EU. A patent application has also been submitted for the technology. The PCT and EP applications are published under WO 2019/224105 A1 and EP 3572512 A1 respectively.

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