Development of chimeric laccases by directed evolution.
DNA recombination methods are useful tools to generate diversity in directed evolution protein engineering studies. We have designed an array of chimeric laccases with high-redox potential by in vitro and in vivo DNA recombination of two fungal laccases (from Pycnoporus cinnabarinus and PM1 basidiomycete), which were previously tailored by laboratory evolution for functional expression in Saccharomyces cerevisiae. The laccase fusion genes (including the evolved α-factor prepro-leaders for secretion in yeast) were subjected to a round of family shuffling to construct chimeric libraries and the best laccase hybrids were identified in dual high-throughput screening (HTS) assays. Using this approach, we identified chimeras with up to six crossover events in the whole sequence, and we obtained active hybrid laccases with combined characteristics in terms of pH activity and thermostability.
Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Isabel
I
Pardo
Ana Isabel
AI
Vicente
Diana M
DM
Mate
Miguel
M
Alcalde
Susana
S
Camarero
D001487:Basidiomycota / Q000201:enzymology / Q000235:genetics; D052160:Benzothiazoles / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D042845:Laccase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008957:Models, Genetic; D008958:Models, Molecular; D015202:Protein Engineering; D011748:Pyrogallol / Q000031:analogs & derivatives / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013451:Sulfonic Acids / Q000378:metabolism
D052160:Benzothiazoles; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D013451:Sulfonic Acids; D011748:Pyrogallol; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; C010120:pyrogallol 1,3-dimethyl ether; D042845:Laccase