Directed Evolution of an Enantioselective Bacillus subtilis Lipase

Funke, Susanne Aileen; Eipper, Andreas

doi:10.1080/10242420301787

Cited by 3 publications

(3 citation statements)

References 15 publications

(18 reference statements)

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“…and ) (Wong et al, ). The latter might explain why in many mutagenesis studies (Funke et al, ; Reetz, ; Zha et al, ) little improvement can be achieved after several rounds of epPCR under similar mutagenic conditions.…”

Section: Discussionmentioning

confidence: 99%

Lessons from diversity of directed evolution experiments by an analysis of 3,000 mutations

Zhao

Kardashliev

Ruff

et al. 2014

Biotech & Bioengineering

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Diversity generation by random mutagenesis is often the first key step in directed evolution experiments and screening of 1,000-2,000 clones is in most directed evolution campaigns sufficient to identify improved variants. For experimentalists important questions such as how many positions are mutated in the targeted gene and what amino acid substitutions can be expected after screening of 1,000-2,000 clones are surprisingly not answered by a statistical analysis of mutant libraries. Therefore three random mutagenesis experiments (epPCR with a low- and a high-mutation frequency and a transversion-enriched sequence saturation mutagenesis method named SeSaM-Tv P/P) were performed on the lipase BSLA and in total 3,000 mutations were analyzed to determine the diversity in random mutagenesis libraries employed in directed evolution experiments. The active fraction of the population ranged from 15% (epPCR-high), to 52% (SeSaM-Tv P/P), and 55% (epPCR-low) which correlates well with the average number of amino acid substitutions per protein (4.1, 1.6 and 1.1). In the epPCR libraries transitions were the predominant mutations (>72%), and >82% of all mutations occurred at A- or T-nts. Consecutive nucleotide (nt) mutations were obtained only with a low fraction (2.8%) under highly error-prone conditions. SeSaM-Tv P/P was enriched in transversions (43%; >1.7-fold more than epPCR libraries), and consecutive nt mutations (30.5%; 11-fold more than epPCR-high). A high fraction of wild-type BSLA protein (33%) was found in the epPCR-low mutant library compared to 2% in epPCR-high and 13% in SeSaM-Tv P/P. An average of 1.8-1.9 amino acid substitutions per residue was obtained with epPCR-low and -high compared to 2.1 via SeSaM-Tv P/P. The chemical composition of the amino acid substitutions differed, however, significantly from the two epPCR methods to SeSaM-Tv P/P.

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Section: Discussionmentioning

confidence: 99%

Lessons from diversity of directed evolution experiments by an analysis of 3,000 mutations

Zhao

Kardashliev

Ruff

et al. 2014

Biotech & Bioengineering

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“…The bsla gene which encodes for the lipase A from Bacillus subtilis is an ideal gene to advance random mutagenesis methods due to its relatively low size of only 181 amino acids (19 kDa) (Funke et al, ; van Pouderoyen et al, ). Systematic studies for the improvement of BSLA toward resistance of organic solvents and ILs revealed up to 104 beneficial amino acid positions and their respective beneficial substitutions (up to 69% chemically different amino acid substitutions (Frauenkron‐Machedjou et al, ; Zhao et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Casting epPCR (cepPCR): A simple random mutagenesis method to generate high quality mutant libraries

Yang

Ruff

Arlt

et al. 2017

Biotech & Bioengineering

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During the last decade, directed evolution has become a standard protein engineering strategy to reengineer proteins for industrial applications under high stress conditions (e.g., high temperature, extreme pH, ionic liquids, or organic solvents). The most commonly employed method for diversity generation to improve biocatalysts for these properties is random mutagenesis by error-prone polymerase chain reaction (epPCR). However, recent reports show that epPCR often fails to produce >70% of beneficial positions/amino acid exchanges which improve enzyme properties such as organic solvent or ionic liquid resistance. In this report, bsla (543 bp, small lipase gene from Bacillus subtilis) was divided into three fragments (147, 192, 204 bp). Each fragment was subjected to an epPCR with a high mutation load (22, 31, and 33 mutations per kb) in order to increase the number of identified beneficial positions while maintaining a fraction of active population which can efficiently be screened in agar plate or microtiter plate format. The use of this "casting epPCR" process termed as (cepPCR), doubles the number of identified beneficial positions (from 14% to 29%), when compared to standard epPCR for the BSLA enzyme model. A further increase to 39% of beneficial positions is obtainable through combination of cepPCR with the transversion biased sequence saturation mutagenesis (SeSaM) method. Furthermore, sequencing of up to 600 mutations per fragment provided valuable insights into the correlation of total throughput and number of identified beneficial positions as well as how an efficient balance of screening efforts to obtainable results can be achieved in directed evolution campaigns. Biotechnol. Bioeng. 2017;114: 1921-1927. © 2017 Wiley Periodicals, Inc.

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“…Als Matrizen dienten Plasmide mit den kodierenden Sequenzen der betreffenden Enzyme, die freundlicherweise von der Arbeitsgruppe von Prof. Jaeger vom Institut für Molekulare Enzymtechnologie (IMET) in Jülich zur Verfügung gestellt wurden. Im Falle der Bacillus subtilis Lipase LipA handelte es sich dabei um das Plasmid pET22lipA [233] (2.2.4). Die zur Amplifikation verwendeten Oligonukleotide waren BS-LipA-Sfi-up und BS-LipA-Sfi-lo (2.4).…”

Section: Ergebnisse Und Diskussionunclassified

Neue Zugänge zu enantioselektiven lipolytischen Enzymen durch fluoreszenzbasierte Durchmusterung kombinatorischer Bibliotheken

Becker¹

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Directed Evolution of an Enantioselective Bacillus subtilis Lipase

Cited by 3 publications

References 15 publications

Lessons from diversity of directed evolution experiments by an analysis of 3,000 mutations

Lessons from diversity of directed evolution experiments by an analysis of 3,000 mutations

Casting epPCR (cepPCR): A simple random mutagenesis method to generate high quality mutant libraries

Neue Zugänge zu enantioselektiven lipolytischen Enzymen durch fluoreszenzbasierte Durchmusterung kombinatorischer Bibliotheken

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