Colarusso Andrea, Lauro Concetta, Calvanese Marzia, Parrilli Ermenegilda, Tutino Maria Luisa
Dipartimento di Scienze Chimiche, Complesso Universitario Monte Sant'Angleo, Via Cintia, 80126 Napoli, Italy.
Microorganisms. 2020 Sep 24;8(10):1466. doi: 10.3390/microorganisms8101466.
Our group has used the marine bacterium TAC125 (TAC125) as a platform for the successful recombinant production of "difficult" proteins, including eukaryotic proteins, at low temperatures. However, there is still room for improvement both in the refinement of TAC125 expression plasmids and in the bacterium's intrinsic ability to accumulate and handle heterologous products. Here, we present an integrated approach of plasmid design and strain engineering finalized to increment the recombinant expression and optimize the inducer uptake in TAC125. To this aim, we developed the IPTG-inducible plasmid pP79 and an engineered TAC125 strain called KrPL . This mutant was designed to express the lactose permease and to produce only a truncated version of the endogenous Lon protease through an integration-deletion strategy. In the wild-type strain, pP79 assured a significantly better production of two reporters in comparison to the most recent expression vector employed in TAC125. Nevertheless, the use of KrPL was crucial to achieving satisfying production levels using reasonable IPTG concentrations, even at 0 °C. Both the wild-type and the mutant recombinant strains are characterized by an average graded response upon IPTG induction and they will find different future applications depending on the desired levels of expression.
我们的团队已将海洋细菌TAC125用作一个平台，用于在低温下成功重组生产包括真核蛋白在内的“难”蛋白。然而，无论是在优化TAC125表达质粒方面，还是在该细菌积累和处理异源产物的内在能力方面，仍有改进空间。在此，我们提出一种质粒设计与菌株工程的综合方法，旨在提高TAC125中的重组表达并优化诱导剂摄取。为此，我们构建了IPTG诱导型质粒pP79和一种名为KrPL的工程化TAC125菌株。该突变体经设计可表达乳糖通透酶，并通过整合-缺失策略仅产生内源性Lon蛋白酶的截短版本。在野生型菌株中，与TAC125中使用的最新表达载体相比，pP79确保了两种报告蛋白的产量显著提高。尽管如此，即使在0°C的条件下，使用KrPL对于在合理的IPTG浓度下实现令人满意的产量水平也至关重要。野生型和突变体重组菌株在IPTG诱导后均具有平均分级反应特征，它们将根据所需的表达水平找到不同的未来应用。
