English (United Kingdom)

https://curated-unify.zendy.io/wp-json/zendy-region/v1/featured_content/oa?rat=en

https://curated-unify.zendy.io/wp-json/zendy-region/v1/highlighted_journal/

Global: Zendy Plus annual

Presents the access of premium content as premium feature

Premium Content

Presents the keyphrase highlighting as premium feature

Keyphrase Highlighting

Presents the summarisation as premium feature

Summarisation

Insights

Presents the pdf analysis as premium feature

PDF Analysis

Presents the zaia usage as premium feature

ZAIA

Global: Zendy Tools annual

Global: Zendy Free Plan

Global: Zendy Tools monthly

Global: Zendy Plus monthly

Background  The microbial transglutaminase (MTG) is inactive when only the mature sequence is expressed in  Pichia pastoris.  Although co-expression of MTG and its N-terminal pro-peptide can obtain the active MTG, the enzyme activity was still low. One of the basic steps for strain improvement is to ensure a sufficient level of transcription of the heterologous gene, based on promoter strength and gene copy number. To date, high-copy-number recombinants of  P. pastoris  are achievable only by cloning of gene concatemers, so methods for rapid and reliable multicopy strains are therefore desirable.    Results  The coexpression strains harboring different copies  mtg  were obtained successfully by stepwise increasing Zeocin concentration based on the rDNA sequence of  P. pastoris . The genome of coexpression strains with the highest enzyme activity was analyzed by real-time fluorescence quantitative PCR, and three copies of  mtg  gene ( mtg- 3c) was calculated according to the standard curve of  gap  and  mtg  genes ( gap  is regarded as the single-copy reference gene). The maximum enzyme activity of  mtg- 3c was up to 1.41 U/mL after being inducted for 72 h in 1 L flask under optimal culture conditions, and two protein bands were observed at the expected molecular weights (40 kDa and 5 kDa) by Western blot. Furthermore, among the strains detected, compared with  mtg -2c,  mtg -6c or  mtg -8c,  mtg -3c is the highest expression level and enzyme activity, implying that  mtg -3c is the most suitable for co-expression pro-peptide and MTG.    Conclusions  This study provides an effective strategy for improving the expression level of active MTG by directional increasing of  mtg  copies in  P. pastoris.Electronic supplementary material  The online version of this article (10.1186/s12896-019-0542-6) contains supplementary material, which is available to authorized users.

Improved the expression level of active transglutaminase by directional increasing copy of mtg gene in Pichia pastoris