- Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations
The demands on a valuable expression system are, in a nutshell, to receive high product yields, to provide a stable and tightly regulatable expression and to ensure high plasmid stability. The properties of such a system are determined by the combination of a specific organism with the desired gene, whereas the plasmid features play a crucial role, too. The effects of two or more determinants can not easily be estimated by addition of singular effects, because combined features can cancel each other out. Therefore, several expression systems have to be tested and the conditions have to be adjusted.
Owing to Escherichia coli being a model organism for genetic studies, a multitude of well-established regulatable promoters are available. A distinction is drawn between positively and negatively controlled regulatory mechanisms. For many promoters, especially those involved in carbohydrate catabolism, both possibilities are implemented, which is true for the well characterized lac-operon for instance. In other cases, such as the L-arabinose operon or the L-rhamnose operon, the expression is positively regulated. These systems are often characterized by a slower response with very low basal transcriptional activity, which can be a great advantage for the production of proteins that are detrimental to the host cell. The L-rhamnose system has successfully been used to express a variety of genes [1-3]. This system often provides better results compared to other vectors, especially if the expression of a gene usually leads to a large moiety of insoluble protein.
L-rhamnose is taken up by the RhaT transport system, converted to L-rhamnulose by an isomerase (RhaA) and then phosphorylated by a kinase (RhaB). Subsequently, the resulting rhamnulose-1-phosphate is hydrolyzed by an aldolase (RhaD) into dihydroxyacetone phosphate, which is metabolized in glycolysis, and L-lactaldehyde. The latter can be oxidized into lactate under aerobic conditions and be reduced into L-1,2-propanediol under unaerobic conditions. The genes rhaBAD are organized in one operon which is controlled by the rhaPBAD promoter. This promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which is located in opposite direction of rhaBAD. If L-rhamnose is available, RhaR binds to the rhaPRS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose in turn binds to the rhaPBAD and the rhaPT promoter and activates the transcription of the structural genes. However, for the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaPBAD promoter has to be cloned upstream of the gene that is to be expressed. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex , which is a key regulator of catabolite repression.
In addition to transcriptional regulation, the degradation of messenger RNA (mRNA) as well as translation initiation appear to be important factors in controlling the level of gene expression. Most bacterial mRNAs show a high turnover rate which allow to rapidly adjust gene expression to the specific needs of the cells. RNase E is the principal endonuclease involved in mRNA decay in E. coli. The action of RNase E is favored by an accessible 5' terminus carrying a monophosphate residue . Therefore, sequence independent thermodynamically stable 5'stem-loop structures protect mRNA from endonucleolytic attack by RNase E as seen in ompA or T7 gene 10 mRNA which show unusual long half-lifes [6,7]. Translation initiation is greatly enhanced in E. coli and other bacteria by the Shine-Dalgarno sequence (SD) in mRNA, located 5–9 base pairs upstream of the initiation codon . The canonical sequence (5'-AAGGAGG-3') is complementary to a sequence close to the 3' end of the 16 S rRNA. Numerous studies suggest, that mRNA translation is less efficient when the SD sequence has a lower degree of complementarity to the 16 S rRNA or a different distance to the start codon .
Since a read-through by the RNA polymerase can lead to severe instability of the expression system, it is recommended to insert a transcription terminator downstream of the desired gene. The vectors used in this study contain a Rho-independent terminator from the rrnB operon, which forms a stem-loop structure. Besides the transcription termination, the stability of a vector can be affected by miscellanous parameters.
The multimerization of plasmids reduces the copy number per cell and leads to segregational instability, a phenomenon known as dimer catastrophe . Multimers can be resolved to monomers by site-specific recombination via the Xer-cer System of ColE1 . Additionally, the promoter Pcer within cer directs the synthesis of the 90 nt transcript Rcd (regulator of cell division), whose overexpression strongly inhibits the growth of cells on solid media, whereas in broth culture the growth is slowed down but not stopped [12,13]. Thus Rcd might be part of a checkpoint which causes a delay in cell division until multimers are converted into monomers, and Rcd seems to play a role in plasmid maintenance, which is functionally independent of dimer resolution . Dimer-containing cells grow more slowly than their monomer-containing counterparts, and the appearance of Rcd correlates with the inhibition of division of multimer-containing cells, perhaps in order to provide the opportunity to resolve the multimers . Due to the fact that an antisense target on the E. coli chromosome could not be found, it has been suggested, that Rcd might interact directly with a protein target .
Another parameter that can be of importance to the expression output is the number of plasmids contained by the cells. In ColE1-type plasmids an efficient regulation mechanism has been evolved, that helps to maintain a constant copy number by counteracting occasional deviations from the steady state level. This inhibitor-target mechanism is based on the negative control of the frequency of replication initiation events, mediated by the interaction of two RNA molecules, RNAI and RNAII and Rop (repressor of primer), a protein consisting of 63 amino acids. In this system RNAII is a preprimer whose processing into a primer by RNase H is inhibited by the hybridization with RNAI. Additionally, the interaction of target and inhibitor is enforced by Rop, and in absence of Rop the copy number is quintupled [17,18].
Beside the possibility to ensure plasmid maintenance by an improved stability, this task can also be performed by killing or inhibiting all cells which have lost their plasmids. Beyond the application of antibiotics combined with resistance markers on the plasmids, this function can be fulfilled by so called addiction modules, which induce programmed cell death in case of loss. This genetic system consists of two components, a stable toxin and an unstable antitoxin. Such systems were mainly found in low-copy plasmids of E. coli, where cured cells were killed because the unstable antidote is degraded faster than the toxin and leads to the postsegregational killing effect (reviewed in . One example amongst others is the ccd addiction system (couples cell division; [20,21]) of the Escherichia coli F plasmid, which codes for a stable toxin (CcdB) and a less stable antidote (CcdA). CcdB inhibits GyrA, a subunit of the heterotetrameric DNA gyrase consisting of GyrA and GyrB, and thereby causes gyrase-dependent killing of the cells . This inactivation can be prevented and reversed in the presence of CcdA protein. The products of treating the inactive GyrA-CcdB complex with CcdA are free GyrA and a CcdB-CcdA complex . Moreover, the formation of the complex might prevent CcdA from being degraded by Lon protease in an ATP-dependent manner . Though Ccd is one of the best understood addiction systems, some key mechanisms of the regulation remain unclear, presumedly because the CcdA-CcdB interaction and its stoichiometry is unexpectedly complex . Eventually, upon plasmid loss, CcdB outlives CcdA and kills the cell by poisoning GyrA.
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