Metabolic engineering of Escherichia coli for the production of coenzyme Q10

Coenzyme Q10 is required for respiratory electron transport and protects biological membranes against oxidative damage. As coenzyme Q10 supplements are used to treat or to alleviate symptoms associated with an increasing number of health conditions, there is growing interest in the development of bioprocesses for its production. The biosynthesis of coenzyme Q10 involves the condensation of an isoprenoid, decaprenyl diphosphate, with an aromatic compound, para-hydroxybenzoate, followed by a series of modifications of the aromatic moiety of the molecule via the ubiquinone pathway. Escherichia coli naturally produces coenzyme Q8, but replacement of its octaprenyl diphosphate synthase by a decaprenyl diphosphate synthase is sufficient to eliminate the production of coenzyme Q8 and favor the synthesis of coenzyme Q10. A rational genetic engineering approach was used to create a strain of E. coli capable of producing high levels of coenzyme Q10. First, the endogenous octaprenyl diphosphate synthase gene was deleted and functionally replaced by a decaprenyl diphosphate synthase-encoding gene derived from Sphingomonas baekryungensis. Additionally, this strain was engineered to produce elevated levels of para-hydroxybenzoate by over-expressing genes encoding enzymes of the E. coli shikimate pathway. The production of isoprenoid was increased by introducing a heterologous mevalonate pathway. Decaprenyl diphosphate and para-hydroxybenzoate were further directed toward the ubiquinone pathway by overexpressing a para-hydroxybenzoate prenyltransferase. The resulting recombinant strain was capable of producing elevated levels of coenzyme Q10. In order to further enhance production of this antioxidant, an investigation into the interplay between coenzyme Q10 biosynthesis and primary metabolism was conducted. This investigation revealed a link between sorbitol catabolism and coenzyme Q10 production in the engineered strain. Moreover, abrogating carbon flux to acetate by selected gene knock-outs also enhanced coenzyme Q10 accumulation. However, the engineered strains developed through this research were found to be highly unstable, leading to high variability in coenzyme Q10 production. This instability was hypothesized to result from the burden exerted by the engineered aromatic and foreign mevalonate pathways on primary metabolism. As a result, further optimization of these engineered strains of E. coli will be required in order to develop a suitable platform for coenzyme Q10 production

Functional characterization of a gain-of-function mutant of AtMKK9 in Arabidopsis thaliana

The relatively small number of MAPKKs encoded in the Arabidopsis genome suggests that this particular class of kinases acts as a point of convergence within the plant's 'integration of external stimuli and their transduction to elicit biological responses. In an effort to gain information about the function of the MAPKK, AtMKK9 , in Arabidopsis, I have characterized several aspects of the phenotype of DEX:CA-MKK9-FLAG transgenic plants, which express an inducible constitutively active version of AtMKK9, CA-MKK9. I have found that CA-MKK9 expression can control the production of ethylene by activating a downstream MAPK, AtMPK6, which is known to promote the stabilization of ethylene biosynthesis enzymes. CA-MKK9 induction was correlated with an increase in AtMPK6 activity in planta, and was rapidly followed by production of a burst of ethylene in the induced plant tissues. I hypothesized that CA-MKK9 directly activates AtMPK6, and demonstrated that a recombinant version of CA-MKK9 was capable of phosphorylating AtMPK6 in vitro. In addition, the production of the hormone was abolished when C A - MKK9was expressed in a mpk6 knock-out background, thus proving that AtMPK6 is required for CA-MKK9 -mediated ethylene biosynthesis. I have also confirmed preliminary data from our laboratory suggesting that CA-MKK9 plays a role in oxidative programmed cell death. The necrotic lesions induced by CA-MKK9 were still observed in the mpk6 background, indicating that programmed cell death was triggered by CA-MKK9 activity independently of AtMPK6 activity and of ethylene overproduction. In addition, in order to investigate short-term transcriptional events triggered by CA-MKK9, I attempted to capture the transcriptional profile of DEX:CA-MKK9-FLAG plants using two-channel oligonucleotide microarrays. The CA-MKK9 - affected genes included a number of genes involved in the octadecanoid pathway, and their promoters were enriched in ABRE-like elements. However, my attempts to validate the microarray results using additional biological replicates and quantitative real-time PCR revealed that the majority of these early-response microarray results were apparently false positives, indicating that the microarray experiment was probably inappropriately constructed for capturing early transcriptional responses to CA-MKK9 using the dexamethasone-inducible system.Science, Faculty ofBotany, Department ofGraduat