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semi rational mutagenesis

Yogan Khatri, Ilona K Jóźwik, Michael Ringle, Irina Alexandra Ionescu, Martin Litzenburger, Michael Christopher Hutter, Andy-Mark W H Thunnissen, Rita Bernhardt
The production of regio- and stereoselectively hydroxylated steroids is of high pharmaceutical interest and can be achieved by cytochrome P450-based biocatalysts. CYP260A1 from Sorangium cellulosum strain So ce56 catalyzes hydroxylation of C19 or C21 steroids at the very unique 1-position. However, the conversion of progesterone (PROG) by CYP260A1 is very unselective. In order to improve its selectivity we applied a semi-rational protein engineering approach, resulting in two different, highly regio- and stereoselective mutants by replacing a single serine residue (S276) of the substrate recognition site 5 with an asparagine or isoleucine...
March 6, 2018: ACS Chemical Biology
Gaia Pasqualetto, Andrea Brancale, Mark T Young
P2X receptors are trimeric eukaryotic ATP-gated cation channels. Extracellular ATP-their physiological ligand-is released as a neurotransmitter and in conditions of cell damage such as inflammation, and substantial evidence implicates P2X receptors in diseases including neuropathic pain, cancer, and arthritis. In 2009, the first P2X crystal structure, Danio rerio P2X4 in the apo - state, was published, and this was followed in 2012 by the ATP-bound structure. These structures transformed our understanding of the conformational changes induced by ATP binding and the mechanism of ligand specificity, and enabled homology modeling of mammalian P2X receptors for ligand docking and rational design of receptor modulators...
2018: Frontiers in Pharmacology
Simon d'Oelsnitz, Andrew Ellington
Continuous directed evolution is the 'while loop' of synthetic biology, autonomous cycles of mutation, selection, and self-replication that can lead to the rapid development of industrially relevant organisms, pathways, or molecules. Although this engineering strategy requires particular mutagenesis methods and well-defined selections, recent advances have facilitated its implementation. Control over selection pressure has been augmented by novel cost-effective continuous culturing devices with open source designs...
February 11, 2018: Current Opinion in Biotechnology
Ge Qu, Jing Zhao, Ping Zheng, Jibin Sun, Zhoutong Sun
Screening is the bottleneck of directed evolution. In order to address this problem, a series of novel semi-rational designed strategies have been developed based on combinatorial active-site saturation test and iterative saturation mutagenesis, including single code saturation mutagenesis, double code saturation mutagenesis and triple code saturation mutagenesis. By creation of "small and smart" high qualified mutant libraries and combinatorial mutagenesis of specific sites, these new strategies have been successfully applied in multiparameter optimization, e...
January 25, 2018: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Eunok Jung, Beom Gi Park, Hee-Wang Yoo, Joonwon Kim, Kwon-Young Choi, Byung-Gee Kim
CYP153A35 from Gordonia alkanivorans was recently characterized as fatty acid ω-hydroxylase. To enhance the catalytic activity of CYP153A35 toward palmitic acid, site-directed saturation mutagenesis was attempted using a semi-rational approach that combined structure-based computational analysis and subsequent saturation mutagenesis. Using colorimetric high-throughput screening (HTS) method based on O-demethylation activity of P450, CYP153A35 D131S and D131F mutants were selected. The best mutant, D131S, having a single mutation on BC-loop, showed 13- and 17-fold improvement in total turnover number (TTN) and catalytic efficiency (k cat/K M) toward palmitic acid compared to wild-type, respectively...
January 2018: Applied Microbiology and Biotechnology
Zhongyi Cheng, Lukasz Peplowski, Wenjing Cui, Yuanyuan Xia, Zhongmei Liu, Jialei Zhang, Michihiko Kobayashi, Zhemin Zhou
Optically pure compounds are important in the synthesis of fine chemicals. Using directed evolution of enzymes to obtain biocatalysts that can selectively produce high-value chiral chemicals is often time-, money- and resource-intensive; traditional semi-rational designs based on structural data and docking experiments are still limited due to the lack of accurate selection of hot-spot residues. In this study, through ligand-protein collision counts based on steered molecular dynamics simulation, we accurately identified four residues related to improving nitrile hydratase stereoselectivity towards rac-mandelonitrile (MAN)...
October 28, 2017: Biotechnology and Bioengineering
Stefan Scheiblbrandner, Erik Breslmayr, Florian Csarman, Regina Paukner, Johannes Führer, Peter L Herzog, Sergey V Shleev, Evgeny M Osipov, Tamara V Tikhonova, Vladimir O Popov, Dietmar Haltrich, Roland Ludwig, Roman Kittl
Fungal high redox potential laccases are proposed as cathodic biocatalysts in implantable enzymatic fuel cells to generate high cell voltages. Their application is limited mainly through their acidic pH optimum and chloride inhibition. This work investigates evolutionary and engineering strategies to increase the pH optimum of a chloride-tolerant, high redox potential laccase from the ascomycete Botrytis aclada. The laccase was subjected to two rounds of directed evolution and the clones screened for increased stability and activity at pH 6...
October 20, 2017: Scientific Reports
Claudia Huber, Andreas Marx
Fidelity and selectivity of DNA polymerases are critical determinants for the biology of life, as well as important tools for biotechnological applications. DNA polymerases catalyze the formation of DNA strands by adding deoxynucleotides to a primer, which is complementarily bound to a template. To ensure the integrity of the genome, DNA polymerases select the correct nucleotide and further extend the nascent DNA strand. Thus, DNA polymerase fidelity is pivotal for ensuring that cells can replicate their genome with minimal error...
2017: PloS One
Zhiguo Wang, Shuo Zhou, Shuangling Zhang, Sa Zhang, Fangmeng Zhu, Xiaolu Jin, Zhenming Chen, Xiaoling Xu
A novel aldo-keto reductase Tm1743 characterized from Thermotoga maritima was explored as an effective biocatalyst in chiral alcohol production. Natural Tm1743 catalyzes asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (EOPB) at high efficiency, but the production of, ethyl (S)-2-hydroxy-4-phenylbutyrate ((S)-EHPB), which is less desirable, is preferred with an enantiomeric excess (ee) value of 76.5%. Thus, altering the enantioselectivity of Tm1743 to obtain the more valuable product (R)-EHPB for angiotensin drug synthesis is highly desired...
June 21, 2017: Scientific Reports
Kaori Yasuda, Hiroshi Sugimoto, Keiko Hayashi, Teisuke Takita, Kiyoshi Yasukawa, Miho Ohta, Masaki Kamakura, Shinichi Ikushiro, Yoshitsugu Shiro, Toshiyuki Sakaki
Cytochrome P450 enzymes belonging to the CYP105 family are predominantly found in bacteria belonging to the phylum Actinobacteria and the order Actinomycetales. In this review, we focused on the protein engineering of P450s belonging to the CYP105 family for industrial use. Two Arg substitutions to Ala of CYP105A1 enhanced its vitamin D3 25- and 1α-hydroxylation activities by 400 and 100-fold, respectively. The coupling efficiency between product formation and NADPH oxidation was largely improved by the R84A mutation...
June 2, 2017: Biochimica et Biophysica Acta
Kaiyuan Tian, Kee Tai, Bryan Jian Wei Chua, Zhi Li
Engineering a methanol tolerant lipase is of great importance in biodiesel production. Here, the first semi-rational method for directed enzyme evolution to enhance methanol tolerance by targeting high B-factor residues for iterative saturation mutagenesis (ISM) is reported. The best double mutant, TLL-S105C/D27R, retained 71% of its original activity after incubation in methanol, showing 30% greater methanol tolerance than TLL. TLL-S105C/D27R also displayed 27% higher activity over TLL. Structure modelling suggested that the increased stability of TLL-S105C/D27R was caused by the formation of a new hydrogen bond which stabilized the protein structure...
May 19, 2017: Bioresource Technology
Juan Pablo Acevedo, Manfred T Reetz, Juan A Asenjo, Loreto P Parra
Enzymes active at low temperature are of great interest for industrial bioprocesses due to their high efficiency at a low energy cost. One of the particularities of naturally evolved cold-active enzymes is their increased enzymatic activity at low temperature, however the low thermostability presented in this type of enzymes is still a major drawback for their application in biocatalysis. Directed evolution of cold-adapted enzymes to a more thermostable version, appears as an attractive strategy to fulfill the stability and activity requirements for the industry...
May 2017: Enzyme and Microbial Technology
Tea Pavkov-Keller, Kerstin Steiner, Mario Faber, Martin Tengg, Helmut Schwab, Mandana Gruber-Khadjawi, Karl Gruber
Friedel-Crafts alkylation of aromatic systems is a classic reaction in organic chemistry, for which regiospecific mono-alkylation, however, is generally difficult to achieve. In nature, methyltransferases catalyze the addition of methyl groups to a wide range of biomolecules thereby modulating the physico-chemical properties of these compounds. Specifically, S-adenosyl-L-methionine dependent C-methyltransferases possess a high potential to serve as biocatalysts in environmentally benign organic syntheses. Here, we report on the high resolution crystal structure of CouO, a C-methyltransferase from Streptomyces rishiriensis involved in the biosynthesis of the antibiotic coumermycin A1...
2017: PloS One
Jian-Miao Xu, Feng Cheng, Fang-Tian Fu, Hai-Feng Hu, Yu-Guo Zheng
L-2-aminobutyric acid (L-ABA) as a precursor for the anticonvulsant and the antituberculotic is a key intermediate in the chemical and pharmaceutical industries. Recently, leucine dehydrogenase (LeuDH) with NAD(+) regeneration was developed for L-ABA production on a large scale. Previously, the L-ABA yield was improved by optimizing conversion conditions, including cofactor regeneration and enzyme immobilization but not protein engineering on LeuDH due to lacking an applicable high-throughput screening (HTS) method...
July 2017: Applied Biochemistry and Biotechnology
Jorick Franceus, Tom Verhaeghe, Tom Desmet
Statistical analysis of a protein multiple sequence alignment can reveal groups of positions that undergo interdependent mutations throughout evolution. At these so-called correlated positions, only certain combinations of amino acids appear to be viable for maintaining proper folding, stability, catalytic activity or specificity. Therefore, it is often speculated that they could be interesting guides for semi-rational protein engineering purposes. Because they are a fingerprint from protein evolution, their analysis may provide valuable insight into a protein's structure or function and furthermore, they may also be suitable target positions for mutagenesis...
May 2017: Journal of Industrial Microbiology & Biotechnology
Matthew Deaner, Hal S Alper
Control of gene expression is crucial to optimize metabolic pathways and synthetic gene networks. Promoters and terminators are stretches of DNA upstream and downstream (respectively) of genes that control both the rate at which the gene is transcribed and the rate at which mRNA is degraded. As a result, both of these elements control net protein expression from a synthetic construct. Thus, it is highly important to discover and engineer promoters and terminators with desired characteristics. This chapter highlights various approaches taken to catalogue these important synthetic elements...
2018: Advances in Biochemical Engineering/biotechnology
Sandra Notonier, Łukasz Gricman, Jürgen Pleiss, Bernhard Hauer
The regioselective terminal hydroxylation of alkanes and fatty acids is of great interest in a variety of industrial applications, such as in cosmetics, in fine chemicals, and in the fragrance industry. The chemically challenging activation and oxidation of non-activated C-H bonds can be achieved with cytochrome P450 enzymes. -CPRBM3 is an artificial fusion construct consisting of the heme domain from Marinobacter aquaeolei and the reductase domain of CYP102A1 from Bacillus megaterium. It has the ability to hydroxylate medium- and long-chain fatty acids selectively at their terminal positions...
August 17, 2016: Chembiochem: a European Journal of Chemical Biology
Xiaoyue Wang, Guanglu Wang, Xinli Li, Jing Fu, Tao Chen, Zhiwen Wang, Xueming Zhao
Adenylosuccinate synthetase (EC. encoded by purA in Bacillus subtilis, catalyzing the first step of the conversion of IMP to AMP, plays an important role in flux distribution in the purine biosynthetic pathway. In this study, we described the use of site saturation mutagenesis to obtain a desired enzyme activity of adenylosuccinate synthetase and its application in flux regulation. Based on sequence alignment and structural modeling, a library of enzyme variants was created by a semi-rational evolution strategy in position Thr238 and Pro242...
August 10, 2016: Journal of Biotechnology
Yan Duan, Lina Ba, Jianwei Gao, Xianxing Gao, Dunming Zhu, René M de Jong, Daniel Mink, Iwona Kaluzna, Zhanglin Lin
ω-Hydroxy oleic acid is an important intermediate for the synthesis of certain polyesters and polyamides. In this study, a functional CYP153A/putidaredoxin (Pdx)/putidaredoxin reductase (Pdr) hybrid system was engineered for improved ω-hydroxylation activity towards oleic acid. By the combination of site-directed saturation mutagenesis (SDSM) and iterative saturation mutagenesis (ISM), a best mutant (Variant II) was obtained with mutations at two sites (S120 and P165) at the Pdx interaction interface with CYP153A, and one site (S453) in the substrate binding pocket...
October 2016: Applied Microbiology and Biotechnology
Kai Wu, Hualei Wang, Lifeng Chen, Haiyang Fan, Zhiqiang Zhao, Dongzhi Wei
Enantiopure styrene oxide (SO) and its derivatives are important building blocks for chiral synthesis. In this study, we developed an attractive "1-pot, 2-step" chemoenzymatic approach for producing enantiopure SO with 100 % theoretical yield. This approach involved asymmetric reduction of α-chloroacetophenone by an alcohol dehydrogenase (ADH; step 1), followed by base-induced ring closure (epoxidation) of enantiopure 2-chloro-1-phenylethanol produced by the ADH (step 2). By-product formation during epoxidation was suppressed to <1 % by adding methyl tert-butyl ether (MTBE) as the second phase...
October 2016: Applied Microbiology and Biotechnology
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