K-101. Redirection of Homogentisate toward Vitamin E Biosynthesis in Rhodococcus

L. B. Willis, T. G. Sambandan, K. Habermann, P. A. Lessard, I. Jhun, A. Grigorescu, C. Rha, A. J. Sinskey;
Massachusetts Inst. of Technology, Cambridge, MA.

Rhodococcus aetherivorans I24 is a gram-positive soil bacterium that produces carotenoids. In plants and cyanobacteria, vitamin E biosynthesis is initiated by the addition of homogentisate to geranylgeranyl diphosphate (a carotenoid precursor). We hypothesized that if R. aetherivorans I24 produces homogentisate it should be possible to express the appropriate heterologous genes to redirect carbon flow away from carotenoid production toward vitamin E production. Homogentisate is typically found as an intermediate in the catabolism of phenylalanine or tyrosine, and consistent with the presence of homogentisate as a metabolite in this strain, we show that R. aetherivorans I24 can utilize hydroxyphenylacetate as a sole carbon source. We identified a putative homogentisate dioxygenase gene, hmgA, based on sequence homology with known enzyme-encoding genes. We disrupted hmgA via homologous recombination in order to increase the availability of homogentisate. The mutation was confirmed via PCR and Southern blot. The mutant strain lacked homogentisate dioxygenase activity, and the enzyme activity could be restored by complementation with the native gene. The mutant strain lost the ability to utilize hydroxyphenylacetic acid as a sole carbon source and, as predicted, the mutant accumulated homogentisate, which autooxidized to form brown ochronotic pigment that could be seen in the growth medium. Using a transcriptional fusion to gusA, we showed that the promoter upstream of hmgA is active in cells grown on LB medium. We constructed a series of E. coli-Rhodococcus expression vectors in which one or more vitamin E biosynthetic genes from Synechocystis or barley were under the control of the hmgA promoter, and transformed these vectors into the hmgA mutant strain. Expression of a codon-optimized homogentisate phytyl transferase gene, but not the non-codon optimized Synechocystis gene encoding the same amino acid sequence, leads to a visible decrease in carotenoid production and ochronotic pigment accumulation, consistent with metabolic redirection. This provides a basis for vitamin E production in a microbial system.