title: Efficient gene targeting in Penicillium chrysogenum using novel Agrobacterium-mediated transformation approaches.
abstract: The industrial production of β-lactam antibiotics by Penicillium chrysogenum has increased tremendously over the last decades, however, further optimization via classical strain and process improvement has reached its limits. The availability of the genome sequence provides new opportunities for directed strain improvement, but this requires the establishment of an efficient gene targeting (GT) system. Recently, mutations affecting the non-homologous end joining (NHEJ) pathway were shown to increase GT efficiencies following PEG-mediated DNA transfer in P. chrysogenum from 1% to 50%. Apart from direct DNA transfer many fungi can efficiently be transformed using the T-DNA transfer system of the soil bacterium Agrobacterium tumefaciens, however, for P. chrysogenum no robust system for Agrobacterium-mediated transformation was available. We obtained efficient AMT of P. chrysogenum spores with the nourseothricin acetyltransferase gene as selection marker, and using this system we investigated if AMT in a NHEJ mutant background could further enhance GT efficiencies. In general, AMT resulted in higher GT efficiencies than direct DNA transfer, although the final frequencies depended on the Agrobacterium strain and plasmid backbone used. Providing overlapping and complementing fragments on two different plasmid backbones via the same Agrobacterium host was shown to be most effective. This so-called split-marker or bi-partite method resulted in highly efficient GT (>97%) almost exclusively without additional ectopic T-DNA insertions. As this method provides for an efficient GT method independent of protoplasts, it can be applied to other fungi for which no protoplasts can be generated or for which protoplast transformation leads to varying results.
authors.affiliation: Add2X Biosciences B.V., Sylviusweg 72, 2333 BE Leiden, The Netherlands. Electronic address: paulo.deboer@tno.nl.
authors.forename: Paulo
authors.initials: P
authors.lastname: de Boer
authors.forename: Jurian
authors.initials: J
authors.lastname: Bronkhof
authors.forename: Karolina
authors.initials: K
authors.lastname: Dukiќ
authors.forename: Richard
authors.initials: R
authors.lastname: Kerkman
authors.forename: Hesselien
authors.initials: H
authors.lastname: Touw
authors.forename: Marco
authors.initials: M
authors.lastname: van den Berg
authors.forename: Remko
authors.initials: R
authors.lastname: Offringa
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D005827:Genetics, Microbial / Q000379:methods*; D010408:Penicillium chrysogenum / Q000235:genetics*; D012641:Selection, Genetic; D014170:Transformation, Genetic
keywords: Agrobacterium-mediated transformation; Bi-partite method; Gene targeting; Non-homologous end joining; Penicillium chrysogenum