Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon <i>Sulfolobus acidocaldarius</i>

<jats:p>Multiple gene knockout systems developed in the thermoacidophilic crenarchaeon <jats:italic>Sulfolobus acidocaldarius</jats:italic> are powerful genetic tools. However, plasmid construction typically requires several steps. Alternatively, PCR tailing for high-throughput gene disruption was also developed in <jats:italic>S. acidocaldarius</jats:italic>, but repeated gene knockout based on PCR tailing has been limited due to lack of a genetic marker system. In this study, we demonstrated efficient homologous recombination frequency (2.8 × 10<jats:sup>4</jats:sup> ± 6.9 × 10<jats:sup>3</jats:sup> colonies/<jats:italic>μ</jats:italic>g DNA) by optimizing the transformation conditions. This optimized protocol allowed to develop reliable gene knockout via double crossover using short homologous arms and to establish the multiple gene knockout system with one-step PCR (MONSTER). In the MONSTER, a multiple gene knockout cassette was simply and rapidly constructed by one-step PCR without plasmid construction, and the PCR product can be immediately used for target gene deletion. As an example of the applications of this strategy, we successfully made a DNA photolyase- (<jats:italic>phr-</jats:italic>) and arginine decarboxylase- (<jats:italic>argD-</jats:italic>) deficient strain of <jats:italic>S. acidocaldarius</jats:italic>. In addition, an agmatine selection system consisting of an agmatine-auxotrophic strain and <jats:italic>argD</jats:italic> marker was also established. The MONSTER provides an alternative strategy that enables the very simple construction of multiple gene knockout cassettes for genetic studies in <jats:italic>S. acidocaldarius</jats:italic>.</jats:p>