Contribution of single amino acid and codon substitutions to the production and secretion of a lipase by Bacillus subtilis

Microbial Cell Factories

Research

Christina Nutschel¹  Anuseema Bhadauriya¹  Holger Gohlke²  Andreas Knapp³  Kristina Volkenborn³  Karl-Erich Jaeger⁴  Pia Skoczinski⁵  Alexander Fulton⁶ 

[1] Institute for Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University Düsseldorf, 40225, Düsseldorf, Germany;Institute for Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University Düsseldorf, 40225, Düsseldorf, Germany;John von Neumann Institute for Computing (NIC), Jülich Supercomputing Centre (JSC) & Institute for Complex Systems - Structural Biochemistry (ICS6), Forschungszentrum Jülich GmbH, 52425, Jülich, Germany;Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, 40225, Düsseldorf, Germany;Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, 40225, Düsseldorf, Germany;Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany;Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, 40225, Düsseldorf, Germany;Macromolecular Chemistry and New Polymeric Materials, Zernike Institute of Advanced Materials, University of Groningen, Nijenborgh 4, 9747AG, Groningen, The Netherlands;Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, 40225, Düsseldorf, Germany;Novozymes A/S, Krogshoejvej 36, 2880, Bagsvaerd, Denmark;

关键词: Bacillus subtilis;    Lipase;    Protein production;    Secretion;    Optimization;   

DOI  :  10.1186/s12934-017-0772-z

 received in 2017-05-23, accepted in 2017-09-13,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundBacillus subtilis produces and secretes proteins in amounts of up to 20 g/l under optimal conditions. However, protein production can be challenging if transcription and cotranslational secretion are negatively affected, or the target protein is degraded by extracellular proteases. This study aims at elucidating the influence of a target protein on its own production by a systematic mutational analysis of the homologous B. subtilis model protein lipase A (LipA). We have covered the full natural diversity of single amino acid substitutions at 155 positions of LipA by site saturation mutagenesis excluding only highly conserved residues and qualitatively and quantitatively screened about 30,000 clones for extracellular LipA production. Identified variants with beneficial effects on production were sequenced and analyzed regarding B. subtilis growth behavior, extracellular lipase activity and amount as well as changes in lipase transcript levels.ResultsIn total, 26 LipA variants were identified showing an up to twofold increase in either amount or activity of extracellular lipase. These variants harbor single amino acid or codon substitutions that did not substantially affect B. subtilis growth. Subsequent exemplary combination of beneficial single amino acid substitutions revealed an additive effect solely at the level of extracellular lipase amount; however, lipase amount and activity could not be increased simultaneously.ConclusionsSingle amino acid and codon substitutions can affect LipA secretion and production by B. subtilis. Several codon-related effects were observed that either enhance lipA transcription or promote a more efficient folding of LipA. Single amino acid substitutions could improve LipA production by increasing its secretion or stability in the culture supernatant. Our findings indicate that optimization of the expression system is not sufficient for efficient protein production in B. subtilis. The sequence of the target protein should also be considered as an optimization target for successful protein production. Our results further suggest that variants with improved properties might be identified much faster and easier if mutagenesis is prioritized towards elements that contribute to enzymatic activity or structural integrity.

【 授权许可】

CC BY   
© The Author(s) 2017

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