Partner und Internationale Organisationen
(Englisch)
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Genencor International (NL); 2x University of Nottingham, (UK); Ruhr Universität Bochum (D); CNRS, Marseille (F); Utrecht University (NL); University of Basel (CH)
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Abstract
(Englisch)
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The overall objective of this project was to develop the Gram-negative bacterium Pseudomonas into a general secretory host for the production of industrial enzymes such as extracellular lipase.
Our contribution towards this goal was the design of economical and optimally controllable expression systems for exo-proteins based on two types of signals: oxygen limitation (i.e. an inexpensive inducer) and cell density-dependent regulators (i.e., naturally produced signal molecules).
In a first expression system which we developed, a gene of interest can be cloned into a broad-host-range vector under the control of an ANR-dependent, oxygen-controlled promoter. Under oxygen limitation, the transcriptional regulator ANR can activate this promoter leading to high-level expression of the desired protein. In P. aeruginosa, this promoter is stronger than the induced tac promoter. The P. aeruginosa lipAH genes, which encode extracellular lipase and lipase foldase, respectively, were cloned under this ANR-dependent, oxygen-controlled promoter. Extracellular lipase was overproduced 30-fold compared to the production by an unengineered P. aeruginosa strain. To further improve lipase production, we constructed a cascade induction system, by placing the T7 RNA polymerase gene under an ANR-dependent promoter. T7 RNA polymerase formed at high cell density efficiently transcribed the lipAH genes driven by the T7 promoter. Using this second expression system, lipase production could be increased 3000-fold over the level observed in wildtype P. aeruginosa. In Escherichia coli and Salmonella typhimurium, an anaerobically inducible expression system has been used previously to overproduce proteins of industrial interest, such as antigens for vaccine purposes. Our cascade induction system is an economic expression system for biotechnological production in fluorescent pseudomonads.
In the second part of the project we have analysed in detail the regulation of the hydrogen cyanide (HCN) biosynthetic genes in P. aeruginosa. The hcn promoter is controlled by oxygen limitation and cell-density and could eventually be used to develop new regulatory cassettes for controlled expression of exoproteins. Two different transcriptional start sites were attributed to this double transcriptional control which involves the transcriptional regulator ANR and two members (LasR and RhlR) of the quorum sensing machinery of P. aeruginosa. However, hcn expression was found to be also under posttranscriptional control mediated by the global activator GacA and its sensor protein GacS. This two-component regulatory system controls the formation of various exoproteins and secondary metabolites in Gram-negative bacteria. In P. aeruginosa, GacA positively controls the quorum-sensing machinery and hence the production of HCN, pyocyanin, and lipase. GacA regulation involves a specific ribosome binding site (RBS), which may represent a target site for RsmA, a small RNA binding protein and repressor of secondary metabolism. According to our model, GacA and RsmA belong to a common regulatory cascade controlling secondary metabolites and exoenzymes in Gram-negative bacteria. A detailed understanding of this general posttranscriptional control is therefore of academic and industrial interest and will be investigated further, hopefully during a future EU project for which an application has been filed (PsEUdomonas for value) on May 31,1999.
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