Controlling the metabolic flux through the carotenoid pathway using directed mRNA processing and stabilization.

A synthetic operon containing the crtI and crtY genes, encoding the phytoene desaturase and the lycopene cyclase, respectively, was placed under the control of the araBAD promoter. DNA cassettes encoding mRNA secondary structures were placed at the 5' and 3' ends of the genes and a putative RNase E site was placed between the genes. This construct was transformed into Escherichia coli cells harboring the genes for phytoene production. By varying the mRNA secondary structures, we were able to modulate the flux through the carotenoid pathway, resulting in a 300-fold variation in the production of beta-carotene relative to lycopene. In addition, intermediates in the pathway from phytoene to beta-carotene production that are not observed in cells expressing the recombinant operon were observed when the engineered operons were used, indicating that changes in levels of the enzymes affected the formation of intermediates. These results indicate that it is possible to coordinately regulate the genes encoding the enzymes of a metabolic pathway and balance the production of the intermediates.

Effects of transcription induction homogeneity and transcript stability on expression of two genes in a constructed operon.

A synthetic operon was constructed using the reporter genes gfp and lacZ and the arabinose-inducible araBAD promoter. DNA cassettes encoding mRNA secondary structures were placed at the 3' and 5' ends of the genes and a putative RNase E site was placed between the genes. These mRNA control elements have been shown to affect transcript processing and decay, resulting in altered protein levels. These constructs were transformed into cells harboring the native arabinose-inducible araE gene encoding the arabinose transport protein and engineered cells harboring a constitutively expressed araE. In the strains with arabinose-dependent transport the linear response in the production of both reporter proteins to inducer concentration occurred over a narrow range of arabinose concentrations. In the strains with constitutive transport the linear range of gene expression occurred over a much larger arabinose concentration range than in strains with the arabinose-inducible transport. Strains with the arabinose-inducible transport harboring different operon constructs produced the two reporter proteins at very different levels at low arabinose concentrations; as inducer concentrations increased, differences in relative expression levels decreased. In contrast, strains with constitutive transport harboring different operon constructs produced the reporter proteins at very different levels across the entire range of inducer concentrations, pointing to the importance of optimizing gene expression control at various levels to control the production of heterologous proteins.

Coordinated, differential expression of two genes through directed mRNA cleavage and stabilization by secondary structures.

Metabolic engineering and multisubunit protein production necessitate the expression of multiple genes at coordinated levels. In bacteria, genes for multisubunit proteins or metabolic pathways are often expressed in operons under the control of a single promoter; expression of the genes is coordinated by varying transcript stability and the rate of translation initiation. We have developed a system to place multiple genes under the control of a single promoter and produce proteins encoded in that novel operon in different ratios over a range of inducer concentrations. RNase E sites identified in the Rhodobacter capsulatus puf operon and Escherichia coli pap operon were separately placed between the coding regions of two reporter genes, and novel secondary structures were engineered into the 5' and 3' ends of the coding regions. The introduced RNase E site directed cleavage between the coding regions to produce two secondary transcripts, each containing a single coding region. The secondary transcripts were protected from exonuclease cleavage by engineered 3' secondary structures, and one of the secondary transcripts was protected from RNase E cleavage by secondary structures at the 5' end. The relative expression levels of two reporter genes could be varied up to fourfold, depending on inducer concentration, by controlling RNase cleavage of the primary and secondary transcripts. Coupled with the ability to vary translation initiation by changing the ribosome binding site, this technology should allow one to create new operons and coordinate, yet separately control, the expression levels of genes expressed in that operon.