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.

中文翻译：

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通过定向增加巴斯德毕赤酵母中mtg基因的拷贝来提高活性转谷氨酰胺酶的表达水平。

背景技术当在毕赤酵母中仅表达成熟序列时，微生物转谷氨酰胺酶（MTG）是无活性的。虽然MTG及其N末端前肽的共表达可以获得活性MTG，但是酶活性仍然很低。菌株改良的基本步骤之一是基于启动子强度和基因拷贝数，确保异源基因足够水平的转录。迄今为止，仅通过克隆基因级联体就可以实现巴斯德毕赤酵母的高拷贝数重组体，因此需要用于快速和可靠的多拷贝菌株的方法。结果通过基于巴斯德毕赤酵母的rDNA序列逐步增加Zeocin浓度，成功获得了具有不同拷贝数mtg的共表达菌株。通过实时荧光定量PCR分析具有最高酶活性的共表达菌株的基因组，并根据空缺和mtg基因的标准曲线计算出三个拷贝的mtg基因（mtg-3c）（以gap为单复制参考基因）。mtg-3c在最佳培养条件下于1 L烧瓶中诱导72 h后，最大酶活性高达1.41 U / mL，通过Western观察到两条分子量分别为40 kDa和5 kDa的蛋白带。污点。此外，在检测到的菌株中，与mtg-2c，mtg-6c或mtg-8c相比，mtg-3c的表达水平和酶活性最高，这表明mtg-3c最适合于前肽和前肽的共表达。 MTG。