The division Molecular Bioeconomy engineers proteins and enzymes to develop improved catalyst for a sustainable bioeconomy and application of the catalyst in chemical and pharmaceutical synthesis. Key technologies we developed are the ligase independent cloning method PLICing, the mutagenesis methods SeSaM - Sequence Saturation Mutagenesis and OmniChange - simultaneous saturation of up to five codons, the recombination method PTRec as well as expression tools like the promotor toolbox and P-Link - generating multicomponent fusion proteins with variable linker length.
The developed strategies in the division Molecular Bioeconomy support the core expertise of the institute and the divisions in the rational and evolutive design of proteins. We aim to develop diversity generation methods in directed enzyme evolution, powerful enough to obtain sufficient numbers of variants to cover the theoretical diversity of the generated library. In order to redefine the library generation step no longer as a bottleneck in directed evolution.
Recently, the efficient protein engineering strategy named KnowVolution 1 , was developed and validated in six protein engineering campaigns. KnowVolution yielded e.g. a glucose oxidase 2 for diabetes analytics; a phytase for animal feeding3, and proteases4 for laundry applications. In three cases evolved enzymes were commercialized by industrial partners and one further enzyme is in the upscaling process. The KnowVolution (Knowledge gaining directed evolution) Strategy is significant conceptional advancement in protein engineering, which speeds up directed evolution, gains a molecular understanding, and reduces significantly screening efforts and time consumption (Figure 1).Bio VI
KnowVolution is divided in four phases: I. Identification of potential beneficial positions, II. Determination of beneficial substitutions at positions from I., III. Structural analysis to determine whether substitutions might interact or not, i. e. cooperative, and IV. Recombination of beneficial positions/substitutions depending on analysis on III to maximize property improvements.
The KnowVolution strategy enables us to understand the structure function relationship, and allows on molecular level to investigate the interactions between the catalyst and the substrate under synthesis conditions.
In the division Molecular Bioeconomy, we are using the developed key technologies and strategies to foster our current application areas. Core enzyme classes are the Monooxygenases and Phytases.
Additional enzyme classes that we successfully evolved comprise Dehydrogenases, for example Alcohol Dehydrogenases or Carbonyl reductase, Oxidases like Glucoseoxidase for medical analytics, lipases for the polymer synthesis or degradation of polymers, proteases for laundry applications, Glycosyltransferases for the synthesis of nucleotide sugars as pharmaceuticals, Cutinases for the establishment of secretion signal library platform, and plastic degradation, Polymerases for recombination methods, and dioxygenases in the biosynthesis of Saffron.
Highlights in Monoxygenase research comprise a whole cell ultra-high throughput screening platform for P450 monooxygenases based on flow cytometry, improved BM3 properties, for example solvent resistance, inverted selectivity, broadened substrate profiles, for example monosubstituted benzenes from the EU OXYGREEN project, and novel concepts in whole cell hydroxylation like the double-oxidation of short chain alkanes and cycloalkanes with direct co-factor regeneration as well as an innovative solution to improve substrate uptake in whole cells for efficient oxygenation of aromatics.
The chemoselective introduction of oxygen functionalities is hardly possible by conventional organic chemistry or strong oxidizing argents. Therefore, the identification and improvement of biocatalysts that can oxidize industrial important compounds by using molecular oxygen under mild conditions with high selectivity is of high synthetic value. Notably, the monooxygenase P450 BM3 from Bacillus megaterium has gained great interest within the last decades due to its extraordinary nature to catalyze the CH-activating oxidative hydroxylation of organic compounds. P450 BM3 has been intensively studied and redesigned as catalyst for the synthesis of organic building blocks. In the EU funded project ROBOX, the engineering of robust P450 BM3 variants with improved hydroxylation activity and coupling efficiency towards aromatic and olefin building blocks is tackled. Furthermore we are investigating the whole cell cascade reaction to generate valuable compounds by coupling the enzyme classes we are evolving for example the coexpression of Monooxygenases and Alcohol dehydrogenases for the synthesis of short chain or cyclic alkanes.
Further core topics are described in the following paragraphs, including the substrate diffusion barrier over the membrane in biotransformation and the phosphate stewardship.
Microbial biotransformations represent a promising way to satisfy the growing demand for scalable production of plant metabolite-based pharmaceuticals. Chemoselective hydroxylations are chemical dream reactions, catalyzed by oxygenases, which are found in many secondary metabolic pathways of plants. Monooxygenases are for instance attractive catalysts to convert, aromatics and short chain fatty acids to highly valuable compounds/intermediates. Industrial applications of monooxygenases, for example cytosolic expressed P450 BM3, have so far been restricted to more cost-effective whole-cell systems that enable efficient NAD(P)H recycling and have in general a higher process stability with lower downstream processing costs than cell-free systems. However, a general limitation for several compound classes is the limited substrate uptake and low efflux of products out of bacterial production systems such as E. coli. The latter often limits productivities and overall reaction rates. We developed an innovative and general solution to improve substrate uptake and product efflux through an engineered passive diffusion channel, which is expressed in the outer E. coli membrane and harbors inside a ~1.3 nm pore which enables translocation of even single stranded DNA. We are presenting a novel class of whole cell catalysts, whose successful application in the conversion of aromatic substrates by P450 BM3 suggests that the E. coli system could also be applicable to further cytosolic expressed catalysts.
Our methods for diversity generation are used to tailor enzymes for an integrated and sustainable bioeconomy. Core enzymes are phytases for the recovery of valuable compounds from agro waste to supports a sustainable bioeconomy. Targets are the phosphorous cycle, supplementation to animal feeds, and production of plant metabolites.
Phosphate stewardship and ultimately recycling is one of the great challenges of humankind. Against this backdrop we propose a new value chain to recover phosphate from plant waste material and to convert it to polyphosphates of industrial value. The approach is based on naturally occurring enzymes that free the phosphate bound in an organic form, so mainly phytate in oil seeds, and microbes that effectively collect the soluble phosphate to store it as polyphosphate.
In the division MolBio a broad interdisciplinary team is assembled, ranging from molecular biologists and biotechnologists to chemists and process engineers. Competences to efficiently communicate with bioeconomists and thereby support value chain assessment as well as development of novel value chains and production processes are of high relevance for the division MolBio. The division MolBio is located in the Sammelbau Biologie II at the RWTH Aachen University.
1 Cheng, F., Zhu, L., Schwaneberg, U. Chem Comm. 2015, doi: 10.1039/C5CC01594D, review.
2 Arango Gutierreza, E., Meier,T., Duefel, H., Mundhada, H., Bocola, M., Schwaneberg, U. Biosensors and Bioelectronics, 2013, 50, 84-90.
3 Shivange, A.V., Serwe, A., Dennig, A., Roccatano, D., Haefner, S., Schwaneberg, U. Appl. Microbiol. Biotechnol., 2012, 95, 405-418.
4 Martinez, R., Jakob, F., Tu, R., Siegert, P., Maurer, K.H., Schwaneberg U. Biotechnol. Bioeng. 2013, 110, 711-720.