Protein Engineering Research Group of Prof. Schwaneberg
at Jacobs University Bremen
Research
Organization & Research Areas Introduction to the groupWe are experts in biocatalyst engineering with a focus on laboratory evolution of enzymes and related rational methods. Enzymes were designed by nature to be regulated and operated in aqueous solution at a physiological pH, ambient temperature and certain substrate concentrations. However, enzyme properties are often far away from application demands. Laboratory evolution mimics natural evolution and selection in a test tube and allows us to tailor enzymes for our needs in applications and to discover fundamental design principles of proteins. The research of my group can be divided into three parts: Actual challenges in directed protein evolutionThe diversity challengeCurrent random mutagenesis methods suffer from three fundamental problems:
The Figure below shows the consequence of these three fundamental problems for one of the most commonly used diversity generating methods error-prone PCR (epPCR) employing Taq-Polymerase with supplemented MnCl2 and balanced dNTP concentrations. As example Subtilisin from Bacillus lentus has been chosen (from: Schenk, A., Wong, T. S., Roccatano, D., Hauer, B. and Schwaneberg, U. (2006). SeSaM (Sequence Saturation Mutagenesis): Eine Methode zur Saettigungsmutagenese eines Genes, BIOspektrum, 3, 277-279.) 
Statistical analysis of amino acid substitution patterns of Subtilisin from Bacillus lentus by using epPCR.
The left structure show the occurrence of mutagenic hot spots (blue) and barely mutated regions as consequences of mutational bias. The right picture shows the generated diversity. Y-axis shows the original amino acid species and X-axis shows the substitution pattern. The substitution pattern for 20 amino acid species is indicated from red (lowest probability) to blue (highest probability). Amino acid substitutions that do not occur are colored in white. The values on the Y-axis represent the fraction of amino acid substitutions with different amino acid side-chains.
Combined with the redundant organization of the genetic code the following performance can be achieved on the amino acid level with current 19 mutagenesis methods (Wong, T. S., Zhurina, D. and Schwaneberg, U. (2006). The diversity challenge in directed protein evolution, Comb. Chem. High Throughput Screen., 9, 271-289):
Values of a genetically unbiased method are shown in brackets. Statistical analysis has been performed with MAP (Mutagenesis Assistant Program; Wong, T. S., Roccatano, D., Zacharias, M. and Schwaneberg, U. (2006). A statistical analysis of current random mutagenesis methods for directed protein evolution, J. Mol. Biol. 355, 858-871. (cover page)). MAP is publicly available at: http://map.iu-bremen.de. A detailed statistical analyses proves with MAP proves that transition and transversion indicators fail as benchmark for diversity generating methods. Based on this MAP (Mutagenesis Assistant Program) analysis three novel indicators based on amino acid substitution patterns have been defined:
The protein structure indicator describes the influence of structure/function-disrupting amino acid substitutions and comprises two parts, percentage of stop codons and percentage of Gly/Pro substitutions. The amino acid diversity indicator can be regarded as a valuable benchmark for the power of a random mutagenesis method to generate diversity on the protein level. It is a measure for the redundancy of the genetic code and the bias of random mutagenesis methods. The amino acid diversity indicator should be complemented by a codon diversity coefficient that measures the distribution of amino acid substitutions over a whole protein sequence. The chemical diversity indicator describes to which extent each amino acid species has been generated. This analysis impressively shows that we require innovative random mutagenesis methods that enable us to explore sequence space efficiently. Our solution to these challenges is SeSaM: Sequence Saturation Mutagenesis. MAP: Mutagenesis Assistant ProgramMAP is available to you here.More details under: Wong, T. S., Roccatano, D., Zacharias, M. and Schwaneberg, U. (2006). A statistical analysis of current random mutagenesis methods for directed protein evolution, J. Mol. Biol. 355, 858-871. (cover page) MAP assists in developing directed evolution strategies by investigating the consequences of mutational bias on the protein level. Main results with MAP for four enzymes from different enzyme classes and of different origin (BM3, GOx, AEs, ADH) reveals:
Unbiased method with "perfect" nucleotide substitutions and ideal Ts/Tv bias indicator is shown in brackets. Such a method fails to generate diverse amino acid substitution patterns SeSaM: Sequence Saturation Method
The Figure below shows 19 mutagenesis methods classified in three categories by the method used for generating diversity at the gene level (from: Wong, T. S., Zhurina, D. and Schwaneberg, U. (2006). The diversity challenge in directed protein evolution, Comb. Chem. High Throughput Screen., 9, 271-289). The standard method used in directed protein evolution is error-prone PCR (epPCR) likely due to its robustness and simplicity in use. 
Current epPCR and whole cell random mutagenesis methods suffer from three fundamental problems: • Bias of the polymerase or mutagenic agent that results in mutagenic "hot spots" and limits amino acid substitutions, • Significant fraction of stop codons and destabilizing amino acid substitutions (e.g. proline and glycine in helices), • Lack of subsequent mutations due to methodological limitationsSeSaM: Sequence Saturation Method is a four step method (click here for details) that
SeSaM is novel method for creating diversity at the gene level and exploring sequence space with unique control over mutational bias. SeSaM allows to develop novel directed evolution strategies. Main advantages of the SeSaM method over epPCR (standard): mutational bias independent from polymerase, control over mutational bias through universal bases, each nucleotide of a gene targeted (no mutagenic hot spots), tunable mutation frequencies, and increased diversity in mutant libraries. SeSaM has been patented in EU, USA, China, Japan, Switzerland and others through BASF AG. More details about SeSaM >>
Novel high throughput screening systemsOur screening systems in 96-well plate format. All listed screening systems have been used successfully for directed protein evolution (click on the assay system for more details):
pNCA-Assay family: a continuous assay based on p-nitrophenolate formation
For improving properties of enzymes that hydroxylate terminally fatty acids, amids, nitriles, alcohols, amines or alkanes.
4-AAP-Assay: an end-point assay based on 4-aminoantipyrine that reacts with phenolic compounds For improving enzyme activity toward aromatic and O-heterocyclic compounds and identifying novel substrates.. pNTP-Assay: a continuous discoloration screen based on p-nitrothiophenolate consumption For improving enzymes activities toward epoxydation reactions. GODA: Glucose Oxidase Detection Assay: a product based end-point assay for gluconolactone formation. For improving glucose oxidizing enzymes based on product and not hydrogen peroxide formation. Apart from these assay various solid phase screening systems and microtiter plate assays have been established for example for hydrogen peroxide, NAD(P)H, aldehyde and thiol-detection. References to screening systemspNCA-Assay
Screening options in 96-well plate formatWe further have possibilities to screen in 96-well plates with solids (see Figure left), organic solvents (Figure center), and perform screen for evaporated compounds (Figure right).
Applications of our core expertise in directed evolution on biocatalysts
Heme domain of P450 BM-3 from Bacillus megaterium
Our industrially most significant success stories are sealed due to secrecy agreements. However, our model systems for generating knowledge by elucidating structure-function relationships comprise:
The research team combines at the Jacobs University Bremen the expertise in directed evolution of P450 BM-3 (Schwaneberg group, 13 manuscripts, 6 patents on P450 BM-3; 1 on GOx), with the computational expertise in docking (6 manuscripts; Zacharias group) and in solvation effects (12 papers; Dr. Roccatano). Through a collaboration (Prof. Wilmanns; EMBL at Desy Hamburg) we will have access to crystallization expertise and facilities for validating our models. This integrative approach (three joint publications) will generate knowledge to understand principles for modulating electron transfer and organic solvent resistance of P450 BM-3 that are beyond the reach of each individual research group. We are particularly interested in understanding at the molecular level fundamental principles for efficient electron transfer from electrode surfaces or mediators to P450 BM-3. Based on joint research efforts we aim to understand how organic cosolvents affect potentials within oxidoreductases and thereby reduce electron transfer rates and activities without affecting the overall protein structure. For instance, some highlights on directed evolution of P450 BM-3. We improved by directed evolution:
For details look at the references related to monooxygenases and glucose oxidase. References related to monooxygenases and glucose oxidase
Synthetic liposomes (Synthosomes/Nanocompartments)A Synthosome is a hollow sphere consisting of a mechanically stable vesicle with a block copolymer membrane and an engineered transmembrane protein acting as selective gate. Among other functions, the interior contains an enzyme catalyzing a reaction or a charged macromolecule species as a trap for compounds. 
Schematic representation of Synthosome systems designed for applications in biocatalysis (left) and selective product recovery (right). Biocatalysis is performed by enzymes encapsulated in Synthosomes. Selective product recovery is performed by loading Synthosomes with positively charged macromolecules as traps for negatively charged compounds. Synthosomes are mechanically stable vesicles with a block copolymer membrane and an engineered transmembrane protein acting as selective gate. The polymer vesicles are nanometer-sized (50-1000 nm) and functionalized by loading them with enzymes for bioconversions or encapsulating charged macromolecules for selective compound recovery/release. The Synthosome system has the potential become a novel technology platform for biocatalysis and selective product recovery. The Synthosome principle has been patented by Jacobs University Bremen/UniBasel. Progress in Synthosome research comprises employed block copolymers, transmembrane channel engineering, and functionalizations. A critical assessment of the Synthosome for prospective applications in industrial (white) biotechnology in press. For details and first proof of principles look at the references related to Synthosome technology platform. Reference related to Synthosome technology platform
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