Menzella Hugo G, Ceccarelli Eduardo A, Gramajo Hugo C
Microbiology, IBR, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, (S2002LRK) Rosario, Argentina.
Biotechnol Bioeng. 2003 Jun 30;82(7):809-17. doi: 10.1002/bit.10630.
An important characteristic of promoters used in recombinant protein production in Escherichi coli is their inducibility in a simple and cost-effective manner. The IPTG inducible promoters lac, tac, and trc are powerful and widely used for basic research. However, the use of IPTG in large-scale production is undesirable due to its high cost and toxicity. The promoters mentioned above can also be induced by the addition of lactose, which has the double role of inducer and carbon and energy source. Nevertheless, the use of this sugar in industrial processes has several drawbacks, which result in low volumetric yields and difficulties in process control. We have genetically engineered a BL21 strain to allow the efficient use of lactose as inducer in fed-batch cultures. Two modifications were introduced, the exchange of the wild-type lac operator by a constitutive one (lacO(c)) and the replacement of the gal alleles to recover the Gal(+) phenotype. The constitutive expression of the lac operon overcame the negative effects that the Lac nongenetic heterogeneity of wild-type E. coli introduces when lactose is used as inducer. The gal(+) genotype allowed the complete use of the lactose as carbon and energy source. The relevance of these two modifications in the efficient utilization of lactose as inducer was demonstrated in fed-batch cultures of the novel recombinant strain MP101 harboring expression vectors containing the calf prochymosin gene or the pccB gene, which encodes for the carboxyltransferase component of a propionyl-CoA carboxylase complex from Streptomyces coelicolor. Similar levels of recombinant protein production (up to 16 g/L) were obtained by using either lactose or IPTG as inducers, which confirmed the success of the genetics modifications introduced.
用于大肠杆菌中重组蛋白生产的启动子的一个重要特性是以简单且经济高效的方式实现诱导性。IPTG诱导型启动子lac、tac和trc功能强大，广泛用于基础研究。然而，由于IPTG成本高且具有毒性，在大规模生产中使用它并不理想。上述启动子也可通过添加乳糖来诱导，乳糖具有诱导剂以及碳源和能源的双重作用。尽管如此，在工业生产过程中使用这种糖存在几个缺点，这会导致体积产量低以及过程控制困难。我们对BL21菌株进行了基因工程改造，使其能够在补料分批培养中高效利用乳糖作为诱导剂。引入了两项改造，将野生型lac操纵子替换为组成型操纵子（lacO(c)），并替换gal等位基因以恢复Gal(+)表型。lac操纵子的组成型表达克服了野生型大肠杆菌在使用乳糖作为诱导剂时Lac非遗传异质性带来的负面影响。gal(+)基因型使乳糖能够完全用作碳源和能源。在含有表达载体的新型重组菌株MP101的补料分批培养中，证明了这两项改造在高效利用乳糖作为诱导剂方面的相关性，该表达载体包含小牛凝乳酶原基因或pccB基因，pccB基因编码来自天蓝色链霉菌的丙酰辅酶A羧化酶复合物的羧基转移酶组分。使用乳糖或IPTG作为诱导剂可获得相似水平的重组蛋白产量（高达16 g/L），这证实了所引入的基因改造的成功。
