A Directed Evolution System for Lysine Deacetylases

2020 ◽  
pp. 319-337
Author(s):  
Martin Spinck ◽  
Maria Ecke ◽  
Damian Schiller ◽  
Heinz Neumann
Keyword(s):  
Directed Evolution ◽  
Evolution System ◽  
Lysine Deacetylases

scholarly journals Directed Evolution of Retrovirus Envelope Protein Cytoplasmic Tails Guided by Functional Incorporation into Lentivirus Particles

2005 ◽  
Vol 79 (2) ◽  
pp. 834-840 ◽  
Author(s):  
Christoph A. Merten ◽  
Jörn Stitz ◽  
Gundula Braun ◽  
Eric M. Poeschla ◽  
Klaus Cichutek ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Cleavage Site ◽  
Leukemia Virus ◽  
Cytoplasmic Tail ◽  
Hiv Protease ◽  
Evolution System ◽  
Peptide Cleavage ◽  
C Terminus ◽  
Hiv 1 ◽  
Vector Particles

ABSTRACT In contrast to most gammaretrovirus envelope proteins (Env), the Gibbon ape leukemia virus (GaLV) Env protein does not mediate the infectivity of human immunodeficiency virus type 1 (HIV-1) particles. We made use of this observation to set up a directed evolution system by creating a library of GaLV Env variants diversified at three critical amino acids, all located around the R-peptide cleavage site within the cytoplasmic tail. This library was screened for variants that were able to functionally pseudotype HIV-1 vector particles. All selected Env variants mediated the infectivity of HIV-1 vector particles and encoded novel cytoplasmic tail motifs. They were efficiently incorporated into HIV particles, and the R peptide was processed by the HIV protease. Interestingly, in some of the selected variants, the R-peptide cleavage site had shifted closer to the C terminus. These data demonstrate a valuable approach for the engineering of chimeric viruses and vector particles.

scholarly journals In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6

2008 ◽  
Vol 75 (2) ◽  
pp. 337-344 ◽  
Author(s):  
Daniel J. Koch ◽  
Mike M. Chen ◽  
Jan B. van Beilen ◽  
Frances H. Arnold
Keyword(s):  
Directed Evolution ◽  
Chain Length ◽  
Carbon Sources ◽  
Evolution System ◽  
Alkane Hydroxylase ◽  
Medium Chain ◽  
Medium Chain Length ◽  
Selection For ◽  
Alkane Hydroxylases

ABSTRACT Enzymes of the AlkB and CYP153 families catalyze the first step in the catabolism of medium-chain-length alkanes, selective oxidation of the alkane to the 1-alkanol, and enable their host organisms to utilize alkanes as carbon sources. Small, gaseous alkanes, however, are converted to alkanols by evolutionarily unrelated methane monooxygenases. Propane and butane can be oxidized by CYP enzymes engineered in the laboratory, but these produce predominantly the 2-alkanols. Here we report the in vivo-directed evolution of two medium-chain-length terminal alkane hydroxylases, the integral membrane di-iron enzyme AlkB from Pseudomonas putida GPo1 and the class II-type soluble CYP153A6 from Mycobacterium sp. strain HXN-1500, to enhance their activity on small alkanes. We established a P. putida evolution system that enables selection for terminal alkane hydroxylase activity and used it to select propane- and butane-oxidizing enzymes based on enhanced growth complementation of an adapted P. putida GPo12(pGEc47ΔB) strain. The resulting enzymes exhibited higher rates of 1-butanol production from butane and maintained their preference for terminal hydroxylation. This in vivo evolution system could be useful for directed evolution of enzymes that function efficiently to hydroxylate small alkanes in engineered hosts.

scholarly journals A novel C-terminal degron identified in bacterial aldehyde decarbonylases using directed evolution

2020 ◽  
Author(s):  
yilan liu ◽  
jinjin chen ◽  
Anna N. Khusnutdinova ◽  
Kevin Correia ◽  
Patrick Diep ◽  
...  
Keyword(s):  
Directed Evolution ◽  
High Stability ◽  
Degradation Mechanism ◽  
Bioinformatic Analysis ◽  
Evolution System ◽  
Marine Cyanobacterium ◽  
E Coli ◽  
Renewable Feedstock ◽  
Promising Solution

Abstract Background: Aldehyde decarbonylases (ADs), which convert acyl aldehydes into alkanes, supply promising solution for producing alkanes from renewable feedstock. However the instability of ADs impedes their further application. Therefore, the current study aimed to investigate the degradation mechanism of ADs and engineer it towards high stability.Results: Here, we describe the discovery of a degradation tag (degron) in the AD from marine cyanobacterium Prochlorococcus marinus using error-prone PCR based directed evolution system. Bioinformatic analysis revealed that this C-terminal degron is common in bacterial ADs and identified a conserved C-terminal motif, RMSAYGLAAA, representing the AD degron (ADcon). Furthermore, we demonstrated that the ATP-dependent proteases ClpAP and Lon are involved in the degradation of AD-tagged proteins in E. coli, thereby limiting alkane production. Deletion or modification of the degron motif increased alkane production in vivo. Conclusion: This work revealed the presence of a novel degron in bacterial ADs responsible for its instability. The in vivo experiments proved eliminating or modifying the degron could stabilize AD, thereby producing higher titers of alkanes.

scholarly journals A novel C-terminal degron identified in bacterial aldehyde decarbonylases using directed evolution

2020 ◽  
Author(s):  
yilan liu ◽  
jinjin chen ◽  
Anna N. Khusnutdinova ◽  
Kevin Correia ◽  
Patrick Diep ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Degradation Mechanism ◽  
Bioinformatic Analysis ◽  
Evolution System ◽  
E Coli ◽  
In Vivo Experiments ◽  
Renewable Feedstock ◽  
The Family ◽  
Promising Solution

Abstract Background: Aldehyde decarbonylase (AD), which converts acyl aldehydes into alkanes, supplies promising solution for producing alkanes from renewable feedstock. However the instability of AD impeded its further application. Therefore, the current study aimed to investigate the degradation mechanism of AD and engineer it towards high stability. Results: Here, we describe the discovery of a degradation tag (degron) in the AD from marine cyanobacterium Prochlorococcus marinus via error-prone PCR based directed evolution system. Bioinformatic analysis revealed this C-terminal degron is common in the family of bacterial ADs and identified a conserved C-terminal motif, RMSAYGLAAA, representing the AD degron (ADcon). Furthermore, we demonstrated that the ATP-dependent proteases ClpAP and Lon are involved in the degradation of AD-tagged proteins in E. coli , thereby limiting alkane production. Deletion or modification of the degron motif increased alkane production in vivo . Conclusions: This work revealed the presence of a novel degron in bacterial ADs responsible for its instability. The in vivo experiments proved eliminating or modifying the degron could stabilize AD, thereby producing higher titers of alkanes.

Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

2019 ◽  
Author(s):  
Huifang Xu ◽  
Weinan Liang ◽  
Linlin Ning ◽  
Yuanyuan Jiang ◽  
Wenxia Yang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Fatty Acid ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Screening Method ◽  
Random Mutagenesis ◽  
Direct Production ◽  
Consumption Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient <i>Escherichia coli</i> host strain and report an HTS approach based on colorimetric detection of H<sub>2</sub>O<sub>2</sub>-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H<sub>2</sub>O<sub>2</sub> as co-substrate.

Sign in / Sign up

Export Citation Format

Share Document