Staijen I E, Marcionelli R, Witholt B
Institut für Biotechnologie, Swiss Federal Institute of Technology (ETH), ETH Hönggerberg, HPT, 8093 Zürich, Switzerland.
J Bacteriol. 1999 Mar;181(5):1610-6. doi: 10.1128/JB.181.5.1610-1616.1999.
The alk genes are located on the OCT plasmid of Pseudomonas oleovorans and encode an inducible pathway for the utilization of n-alkanes as carbon and energy sources. We have investigated the influence of alternative carbon sources on the induction of this pathway in P. oleovorans and Escherichia coli alk+ recombinants. In doing so, we confirmed earlier reports that induction of alkane hydroxylase activity in pseudomonads is subject to carbon catabolite repression. Specifically, synthesis of the monooxygenase component AlkB is repressed at the transcriptional level. The alk genes have been cloned into plasmid pGEc47, which has a copy number of about 5 to 10 per cell in both E. coli and pseudomonads. Pseudomonas putida GPo12 is a P. oleovorans derivative cured of the OCT plasmid. Upon introduction of pGEc47 in this strain, carbon catabolite repression of alkane hydroxylase activity was reduced significantly. In cultures of recombinant E. coli HB101 and W3110 carrying pGEc47, induction of AlkB and transcription of the alkB gene were no longer subject to carbon catabolite repression. This suggests that carbon catabolite repression of alkane degradation is regulated differently in Pseudomonas and in E. coli strains. These results also indicate that PalkBFGHJKL, the Palk promoter, might be useful in attaining high expression levels of heterologous genes in E. coli grown on inexpensive carbon sources which normally trigger carbon catabolite repression of native expression systems in this host.
alk基因位于食油假单胞菌的OCT质粒上，编码一条以正构烷烃作为碳源和能源的诱导途径。我们研究了替代碳源对食油假单胞菌和大肠杆菌alk⁺重组体中该途径诱导的影响。在此过程中，我们证实了早期的报道，即假单胞菌中烷烃羟化酶活性的诱导受到碳分解代谢物阻遏的影响。具体而言，单加氧酶组分AlkB的合成在转录水平受到抑制。alk基因已被克隆到质粒pGEc47中，该质粒在大肠杆菌和假单胞菌中的每个细胞拷贝数约为5至10个。恶臭假单胞菌GPo12是一种不含OCT质粒的食油假单胞菌衍生物。将pGEc47引入该菌株后，烷烃羟化酶活性的碳分解代谢物阻遏显著降低。在携带pGEc47的重组大肠杆菌HB101和W3110培养物中，AlkB的诱导和alkB基因的转录不再受碳分解代谢物阻遏的影响。这表明假单胞菌和大肠杆菌菌株中烷烃降解的碳分解代谢物阻遏调控方式不同。这些结果还表明，PalkBFGHJKL（Palk启动子）可能有助于在以通常会触发该宿主中天然表达系统碳分解代谢物阻遏的廉价碳源生长的大肠杆菌中实现异源基因的高表达水平。
