118:
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for their improvement towards selected properties such as cellulase for ionic liquid resistance, protease with increased detergent tolerance, glucose oxidase for analytical application, phytase with increased thermostability and monooxygenase with improved catalytic efficiency using alternative electron donors. SeSaM is patent protected by US770374 B2 in over 13 countries and is one of the platform technologies of
59:. These nucleotides are then replaced by standard nucleotides, allowing for a broad distribution of nucleic acid mutations spread over the gene sequence with a preference to transversions and with a unique focus on consecutive point mutations, both difficult to generate by other mutagenesis techniques. The technique was developed by Professor Ulrich Schwaneberg at
164:
These generated so-called fwd template and rev templates are now amplified in a PCR reaction with a pre-defined mixture of phosphorothioate and standard nucleotides to ensure an even distribution of inserted mutations over the full length of the gene. PCR products of Step 1 are cleaved specifically
188:
SeSaM is used to directly optimize proteins on amino acid level, but also to preliminarily identify amino acid positions to test in saturation mutagenesis for the ideal amino acid exchange. SeSaM has been successfully applied in numerous directed evolution campaigns of different classes of enzymes
148:
The SeSaM-method consists of four PCR-based steps which can be executed within two to three days. Major parts include the incorporation of phosphorothioate nucleotides, the chemical fragmentation at these positions, the introduction of universal or degenerate bases and their replacement by natural
160:
Initially, universal “SeSaM”-sequences are inserted by PCR with gene-specific primers binding in front of and behind the gene of interest. The gene of interest with its flanking regions is amplified to introduce these SeSaM_fwd and SeSaM_rev sequences and to generate template for consecutive PCR
139:
polymerase in Step III of the SeSaM-TV-II method and the addition of an alternative degenerate nucleotide for efficient substitution of thymine and cytosine bases and increased mutation frequency in SeSaM-P/R, generated libraries were further improved with regard to transversion number and the
131:
and the use of optimized DNA polymerases, further increasing the ratio of introduced transversions. This modified SeSaM-TV+ method in addition allows for and favors the introduction of two consecutive nucleotide exchanges, broadening strongly the spectrum of amino acids that may be substituted.
113:
are strongly prevalent. By non-specific introduction of universal bases at every position in the gene sequence, SeSaM overcomes the polymerase bias favoring transitory substitutions at specific positions but opens the complete gene sequence to a diverse array of amino acid exchanges.
179:
By replacement of the universal/degenerate bases in the gene sequence by random standard nucleotides in SeSaM Step 4, a diverse array of full-length gene sequences with substitution mutations is generated, including a high load of transversions and subsequent substitution mutations.
383:
Wong, T.S.; Roccatano, D.; Loakes, D.; Tee, K.L.; Schenk, A.; Hauer, B.; Schwaneberg, U. (2008). "Transversion-enriched sequence saturation mutagenesis (SeSaM-Tv+): A random mutagenesis method with consecutive nucleotide exchanges that complements the bias of error-prone PCR".
121:
Comparison of the amino acid substitution pattern obtainable using standard epPCR methods (single nucleotide substitutions with transition bias) and the
Sequence saturation mutagenesis method (introducing consecutive nucleotide substitutions with an increased ratio of
126:
During the development of the SeSaM-method, several modifications were introduced that allowed for the introduction of several mutations simultaneously. Another advancement of the method was achieved by introduction of degenerate bases instead of universal
80:
and thus encounter limitations which mainly result from the circumstance that only single, but very rarely consecutive, nucleic acid substitutions are performed and that these substitutions occur usually at specific, favored positions only. In addition,
175:
Subsequently, in Step 3 a PCR is performed recombining the single stranded DNA fragments with the corresponding full-length reverse template, generating the full-length double stranded gene including universal or degenerate bases in its sequence.
75:
SeSaM has been developed in order to overcome several of the major limitations encountered when working with standard mutagenesis methods based on simple error-prone PCR (epPCR) techniques. These epPCR techniques rely on the use of
519:
Ruff, A.J.; Marienhagen, J.; Verma, R.; Roccatano, D.; Genieser, H.-G.; Niemann, R.; Shivange, A.V.; Schwaneberg, U. (2012). "dRTP and dPTP a complementary nucleotide couple for the
Sequence Saturation Mutagenesis (SeSaM) method".
590:
Pottkämper, J.; Barthen, P.; Ilmberger, N.; Schwaneberg, U.; Schenk, A.; Schulte, M.; Ignatiev, N.; Streit, W. (2009). "Applying metagenomics for the identification of bacterial cellulases that are stable in ionic liquids".
618:
Li, Z.; Roccatano, D.; Lorenz, M.; Schwaneberg, U. (2012). "Directed evolution of subtilisin E into a highly active and guanidinium chloride- and sodium dodecylsulfate-tolerant protease".
661:
Gutierrez, E.A.; Mundhada, H.; Meier, T.; Duefuel, H.; Bocola, M.; Schwaneberg, U. (2013). "Reengineered glucose oxidase for amperometric glucose determination in diabetes analytics".
476:
Mundhada, H.; Marienhagen, J.; Scacioc, A.; Schenk, A.; Roccatano, D.; Schwaneberg, U. (2011). "SeSaM-Tv-II generates a protein sequence space that is unobtainable by epPCR".
547:
Zhao, J.; Kardashliev, T.; Ruff, A.J.; Bocola, M.; Schwaneberg, M. (2014). "Lessons from diversity of directed evolution experiments by an analysis of 3000 mutations".
168:
In Step 2 of SeSaM, the DNA single strands are elongated by one to several universal or degenerate bases (depending on the modification of SeSaM applied) catalyzed by
264:
Wong, T.S.; Roccatano, D.; Zacharias, M.; Schwaneberg, U. (2006). "A statistical analysis of random mutagenesis methods used for directed protein evolution".
165:
at the phosphorothioate bonds, generating a pool of single-stranded DNA fragments of different lengths starting from the universal primer.
24:
698:"Iterative key-residues interrogation of a phytase with thermostability increasing substitutions identified in directed evolution"
427:
d'Abbadie, M.; Hofreiter, M.; Vaisman, A.; Loakes, D.; Gasparutto, D.; Cadet, J.; Woodgate, R.; Pääbo, S.; Holliger, P. (2007).
348:
Wong, T.S.; Tee, K.L.; Hauer, B.; Schwaneberg, U. (2005). "Sequence saturation mutagenesis with tunable mutation frequencies".
156:
Experimental steps for generation of a non-biased random mutagenesis library using the sequence saturation mutagenesis method.
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number of consecutive mutations was raised to 2–4 consecutive mutations with a rate of consecutive mutations of up to 30%.
172:(TdT). This step is the key step to introduce the characteristic consecutive mutations to randomly mutate entire codons.
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Belsare, K.D.; Horn, T.; Ruff, A.J.; Martinez, R.; Magnusson, A.; Holtmann, D.; Schrader, J.; Schwaneberg, U. (2017).
93:. These characteristics of epPCR catalyzed nucleic acid exchanges together with the fact that the genetic code is
245:
FĂĽllen, G.; Youvan D.C. (1994). "Genetic algorithms and recursive ensemble mutagenesis in protein engineering".
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Wong, T.S.; Zhurina, D.; Schwaneberg, U. (2006). "The diversity challenge in directed protein evolution".
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44:
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301:"Sequence Saturation Mutagenesis (SeSaM): a novel method for directed protein evolution"
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sequence, cleaved and the resulting fragments elongated by universal or degenerate
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By several optimizations including the application of an improved
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745:"Directed evolution of P450cin for mediated electron transfer"
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and require specifically designed polymerases with an altered
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Wong, T.S.; Tee, K.L.; Hauer, B.; Schwaneberg, U. (2004).
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with similar physico-chemical properties such as size and
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Shivange, A.V.; Roccatano, D.; Schwaneberg, U. (2016).
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85:of nucleic acids are much less likely than
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749:Protein Engineering Design and Selection
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97:decrease the resulting diversity on the
149:nucleotides inserting point mutations.
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71:Technology, development and advantages
170:terminal deoxynucleotidyl transferase
105:lead to amino acid preservation or
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212:Comb. Chem. High Throughput Screen
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41:saturation mutagenesis techniques
51:nucleotides are inserted in the
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39:. It is one of the most common
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278:10.1016/j.jmb.2005.10.082
362:10.1016/j.ab.2005.03.023
103:Synonymous substitutions
61:Jacobs University Bremen
47:-based reaction steps,
27:method applied for the
23:) is a chemo-enzymatic
762:10.1093/protein/gzw072
632:10.1002/cbic.201100714
490:10.1002/cbic.201100010
398:10.1002/biot.200700193
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107:conservative mutations
65:RWTH Aachen University
702:Appl. Microbiol. Biot
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663:Biosens. Bioelectron
801:Protein engineering
522:J Mol Catal B-Enzym
791:Molecular genetics
317:10.1093/nar/gnh028
191:SeSaM-Biotech GmbH
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29:directed evolution
25:random mutagenesis
561:10.1002/bit.25302
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484:(10): 1595–1601.
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247:Complex Int
95:degenerated
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785:Categories
593:Green Chem
311:(3): e26.
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99:amino acid
43:. In four
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144:Procedure
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335:14872057
286:16325201
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33:proteins
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