Various recombinant proteins can be produced by the yeast Saccharomyces cerevisiae cell factories; therefore, efficient recombinant protein production techniques are desirable. In this study, to establish an efficient recombinant protein production technique in S. cerevisiae, the secretory production of recombinant protein endoglucanase II (TrEG) was tested. We developed 2 novel methods for TrEG production via clustered regularly interspaced short palindromic repeat (CRISPR) -δ-integration as well as multiple promoter shuffling, which involved the pre-breakdown of the δ-sequence by the CRISPR system and subsequent δ-integration as well as the conjugation of TrEG with various promoters and subsequent δ-integration, respectively. Moreover, simultaneous use of the CRISPR-δ-integration and multiple promoter shuffling methods was also examined. The CRISPR-δ-integration method was effective for improvement of the integrated TrEG copy number and its activity, and the multiple promoter shuffling method was also beneficial for enhancing the transcriptional level of TrEG and its activity. Furthermore, simultaneous use of CRISPR-δ-integration and multiple promoter shuffling methods was the most useful. The carboxymethyl cellulase activity of the TrEG expressing transformant YPH499/24CP constructed by the method reached 559 U/L, and it was 17.3-fold higher than that of the transformant constructed by the conventional YEp type vector. Overall, the simultaneous use of CRISPR-δ-integration and multiple promoter shuffling can be useful and easily applied for recombinant protein production.

• PMID: 30554640
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Journal Article

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Sasaki Y, Mitsui R, Yamada R, Ogino H

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