enGenes: New research study demonstrates enGenes X-press glycosyltransferase yield advantages

Recombinant protein specialist manufacturer enGenes Biotech GmbH (enGenes) has welcomed a new scientific publication showing successful application of its proprietary enGenes-X-press technology in significantly improving recombinant protein yields of difficult-to-express plant glycosyltransferases.

It demonstrates five-fold increases in yields of glycosyltransferase when using the enGenes-X-press strain compared to a state-of-the-art strain BL21(DE3), with a doubling in enzyme quality.

Peer-reviewed study

The study ‘Decoupling of recombinant protein production from Escherichia coli cell growth enhances functional expression of plant Leloir glycosyltransferases’ has been published in the prestigious journal Biotechnology and Bioengineering (DOI:10.1002/bit.26934).

The study has been leaded by Prof. Bernd Nidetzky, Head of the Institute of Biotechnology and Biochemical Engineering at Technical University Graz and CSO of ACIB GmbH, with contributions from enGenes CEO Dr. Juergen Mairhofer, and three other TU Graz and ACIB GmbH researchers, Martin Lemmerer, Alexander Lepak and Karin Longus and enGenes long-time collaborator Assoc. Prof. Dr. Rainer Hahn, affiliated to the University of Natural Resources and Life Sciences Vienna (BOKU).

The study is the result of collaborative research funded by the Austrian Research Promotion Agency, the FFG.

Releasing Leloir glycosyltransferase applications

The research team focused on sugar nucleotide‐dependent (Leloir) glycosyltransferases from plants as important catalysts for the glycosylation of small molecules and natural products whose application for biocatalytic synthesis is limited by their low protein expression of under 10 mg per liter of culture in standard microbial hosts.

The research focused on two representative glycosyltransferases, sucrose synthase from soybean and UGT71A15 from apple. They found that a synthetic biology‐based strategy of decoupling the enzyme expression from cell growth, as achievable with enGenes proprietary X-press platform technology, was effective in enhancing their individual production yield of correctly folded, biologically active proteins by up to five times, with a doubling in combined yield.

12-fold active enzyme increase

The glycosyltransferase genes were encoded on conventional pET‐based expression plasmids that allowed T7 RNA polymerase‐driven inducible expression by isopropyl‐β‐D‐galactoside. Laboratory batch and scaled‐up (20 L) fed‐batch bioreactor cultivations demonstrated up to 12‐fold improvements in overall yield of active enzyme as the result of production under growth‐decoupled conditions.

In batch culture, sucrose synthase and UGT71A15 were obtained, respectively, at 115 U/g and 2.30 U/g cell dry weight, corresponding to ∼5% and ∼1% of total intracellular protein.

Fed‐batch production gave sucrose synthase in a yield of 2,300 U/L of culture (830 mg protein/L).

Recombinant glycosyltransferase production

“Analyzing the isolated glycosyltransferase, we were able to show that improvement in the enzyme production was due to 5.3‐fold enhancement in yield and 2.3‐fold increase in quality of the soluble sucrose synthase,” commented first author Dr. Martin Lemmerer.

“Enzyme preparation from decoupled production comprised an increased portion (61% compared with 26%) of the active sucrose synthase homo‐tetramer. In summary, therefore, we show that expression in growth‐arrested E. coli is promising for recombinant production of plant Leloir glycosyltransferases,” the article abstract concludes.

Dr. Mairhofer commented: “This is a significant milestone for enGenes X-press, demonstrating that our arguments for its advantages are not just marketing ones but can be demonstrated in rigorous peer-reviewed academic research.”

“It also shows that the technology can achieve remarkable results in the hands of others in gaining significant yield improvements compared to state-of-the-art expression host strains,” he added.

About enGenes Biotech

enGenes Biotech GmbH (enGenes) is a contract research, development and manufacturing company that provides leading edge technologies and production services focused on recombinant proteins in bacteria. The company’s mission is to provide cost-effective and scalable production of recombinant proteins at a fraction of current cost, allied to a vision of developing a world-class portfolio of cutting-edge protein production technologies, relevant to a broad spectrum of application fields.

enGenes has developed advanced technologies to drive more cost-effective recombinant protein production processes., including its proprietary enGenes-X-press™ E. coli platform that achieves outstanding yields of soluble and active recombinant protein. enGenes-X-press has been successful applied for the manufacturing of enzymes and biopharmaceutical products that failed to give economically feasible yields in a conventional expression host.

enGenes Biotech offers development and manufacturing services tailored around the needs of pharmaceutical and industrial biotech companies. The services include expression strain and vector development, fermentation process development and optimization, downstream process development, production of purified protein, technology transfer and scale-up support with technology out-licensing and co-development opportunities.

About ACIB GmbH

The Austrian Centre of Industrial Biotechnology (ACIB GmbH) is an international Research Centre for Industrial Biotechnology. ACIB scientists harness natural mechanisms to replace traditional industrial methods with new, more economic and ecological technologies.

Currently, more than 250 ACIB researchers are engaged in some 170 research projects at sites in Austria (Vienna, Graz, Innsbruck, Tulln), in Germany (Hamburg, Heidelberg, Bielefeld), Italy (Pavia),Spain (Barcelona), Australia, New Zealand and Taiwan as part of an international network of more than 150 international universities and industry partners, including BASF, DSM, Sandoz, Boehringer Ingelheim RCV, Jungbunzlauer and VTU Technology.

ACIB is co-owned by the Universities of Innsbruck and Graz, Graz University of Technology, the University of Natural Resources and Life Sciences, Vienna and Joanneum Research.

The ACIB Competence Center is sponsored by BMVIT, BMWFW and the provinces of Styria, Tyrol, Lower Austria and Vienna as part of the COMET (Austrian Competence Centres for Excellent Technologies) program managed by FFG, the Austrian Research Agency.

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