title: Construction of a Kluyveromyces lactis ku80⁻ host strain for recombinant protein production: extracellular secretion of pectin lyase and a streptavidin-pectin lyase chimera.
abstract: In several organisms used for recombinant protein production, integration of the expression cassette into the genome depends on site-specific recombination. In general, the yeast Kluyveromyces lactis shows low gene-targeting efficiency. In this work, two K. lactis ku80⁻ strains defective in the non-homologous end-joining pathway (NHEJ) were constructed using a split-marker strategy and tested as hosts for heterologous gene expression. The NHEJ pathway mediates random integration of exogenous DNA into the genome, and its function depends on the KU80 gene. KU80-defective mutants were constructed using a split-marker strategy. The vectors pKLAC1/Plg1 and pKLAC1/cStpPlg1 were used to evaluate the recovered mutants as hosts for expression of pectin lyase (PNL) and the fusion protein streptavidin-PNL, respectively. The transformation efficiency of the ku80⁻ mutants was higher than the respective parental strains (HP108 and JA6). In addition, PNL secretion was detected by PNL assay in both of the K. lactis ku80⁻ strains. In HP108ku80⁻/cStpPlg1 and JA6ku80⁻/Plg1 cultures, the PNL extracellular specific activity was 551.48 (±38.66) and 369.04 (±66.33) U/mg protein. This study shows that disruption of the KU80 gene is an effective strategy to increase the efficiency of homologous recombination with pKLAC1 vectors and the production and secretion of recombinant proteins in K. lactis transformants.
authors.affiliation: Laboratório de Fisiologia de Micro-organismos, Departamento de Microbiologia, BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, 36570-000, Brazil.
authors.forename: Lívia T
authors.initials: LT
authors.lastname: Colombo
authors.forename: Júlio César C
authors.initials: JC
authors.lastname: Rosa
authors.forename: Caio R S
authors.initials: CR
authors.lastname: Bragança
authors.forename: Raphael P
authors.initials: RP
authors.lastname: Ignacchiti
authors.forename: Mariana C T
authors.initials: MC
authors.lastname: Alvim
authors.forename: Wendel B
authors.initials: WB
authors.lastname: Silveira
authors.forename: Marisa V
authors.initials: MV
authors.lastname: de Queiroz
authors.forename: Denise M S
authors.initials: DM
authors.lastname: Bazzolli
authors.forename: Flávia M L
authors.initials: FM
authors.lastname: Passos
mesh_terms: D059766:DNA End-Joining Repair / Q000235:genetics; D015870:Gene Expression; D007716:Kluyveromyces / Q000166:cytology / Q000235:genetics*; D011133:Polysaccharide-Lyases / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D019809:Streptavidin / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D019809:Streptavidin; D011133:Polysaccharide-Lyases; C027349:pectin lyase