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首页> 美国卫生研究院文献>Microbial Biotechnology >A multi‐functional genetic manipulation system and its use in high‐level expression of a β‐mannanase mutant with high specific activity in Pichia pastoris
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A multi‐functional genetic manipulation system and its use in high‐level expression of a β‐mannanase mutant with high specific activity in Pichia pastoris

机译：一种多功能遗传操作系统及其在Pichia巴氏菌高特异性活性的高水平表达中的应用

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摘要

To further extend the practical application of a thermostable and acidic resistance β‐mannanase (ManAK) in animal feed additives, an effective strategy that combined directed evolution and metabolic engineering was developed. Four positive mutants (P191M, P194E, S199G and S268Q) with enhanced specific activity (25.5%–60.9%) were obtained. The S199G mutant exhibited 56.7% enhancement of specific activity at 37°C and good thermostability, and this was selected for high‐level expression in P. pastoris X33. A multi‐functional and scarless genetic manipulation system was proposed and functionally verified (gene deletion, substitution/insertion and point mutation). This was then subjected to Rox1p (an oxygen related transcription regulator) deletion and Vitreoscilla haemoglobin (VHb) co‐expression for high enzyme productivity in P. pastoris X33VIIManAKS199G. An excellent strain, named X33VIIManAKS199G ∆rox1::VHb, was achieved by combining these two factors, and then the maximum enzymatic activity was further increased to 3753 U ml‐1, which was nearly twice as much as the maximum production of ManAK in P. pastoris. This work provides a systematic and effective method to improve the enzymatic yield of β‐mannanase, promotes the application of ManAK in feed additives, and also demonstrated that a scarless genetic manipulation tool is useful in P. pastoris.
机译：为了进一步延长动物饲料添加剂中热稳定和酸性抗性β-甘露糖酶（MANAK）的实际应用，开发了有效的转向演化和代谢工程的有效策略。获得具有增强的特异性活性（25.5％-60.9％）的四个正突变体（P191M，P194E，S199G和S268Q）。 S199G突变体在37℃下表现出56.7％的特定活性，良好的耐热性，并选择其中P. Pastoris X33中的高水平表达。提出了一种多功能和无缺骨的遗传操作系统，并在功能验证（基因缺失，取代/插入和点突变）。然后将其进行Rox1p（氧相关转录调节剂）缺失和vhb血红蛋白（VHB）的高酶生产率在P. Pastoris X33Viimanaks199G中进行高酶生产率。通过组合这两个因素实现了一个名为X33VIIMANAKS199GΔROX1:: VHB的优异菌株，然后将最大酶活性进一步增加至3753uml-1，这几乎是MANAK在P中最大产量的两倍。牧师。该作品提供了一种系统和有效的方法来提高β-甘露糖酶的酶促产率，促进Manak在饲料添加剂中的应用，并且还证明了无缺陷的遗传操作工具可用于P. Pastoris。

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