Identifying the Source of Strain-to-Strain Variability in Isoprenoid Production Capacity of E. coli Using a Systems Biology Approach

Isoprenoids are a large class of natural compounds with diverse industrial applications ranging from pharmaceuticals to biofuels. Extraction of isoprenoids from their native hosts is often costly and inefficient since many high-value isoprenoids are produced in low quantities. Therefore heterologous production in microorganisms presents a desirable alternative. In spite of their remarkable chemical diversity, all isoprenoids are derived from the same five-carbon precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This enables metabolic engineers to access a myriad of valuable compounds through overproduction of these basic building blocks. Of the two different metabolic routes leading to isoprenoid precursors, the methylerythritol phosphate (MEP) pathway used by E. coli and many other bacteria has a higher theoretical
maximum yield than the mevalonate (MVA) pathway1. However, despite intensive metabolic
engineering efforts, isoprenoid product yields via the E. coli MEP pathway remain far below the theoretical maximum. The factors influencing isoprenoid biosynthesis in E. coli are currently poorly understood. Unpredictable results from MEP pathway engineering â?? including inexplicable production thresholds and reduced product formation when all pathway enzymes are highly over- expressed â?? suggest complex regulatory mechanisms 2, 3. While it is known that different E. coli strains vary in their ability to produce isoprenoids 4, 5, most previous studies have focussed on E. coli strains from the K-12 lineage. We have systematically tested E. coli laboratory strains from different phylogenetic groups for their ability to produce lycopene, a model compound for evaluating isoprenoid production in microorganisms. We observed differences in lycopene production of up to an order of magnitude between strains. Of particular significance for metabolic engineering
applications, we also show that different wild-type E. coli strains respond differently to MEP pathway engineering. Overexpression of the rate-limiting enzyme, 1-deoxy-D-xylulose-5-phosphate synthase (dxs), releases the primary main bottleneck in isoprenoid biosynthesis6, 7; however, we have
observed significant differences in the "engineerability" of even genetically closely related strains through dxs overexpression. In particular, an isolate of the widely used laboratory strain E. coli MG1655 produced 5-fold less lycopene than its parental wild-type strain WG1, suggesting a loss of isoprenoid production capacity during the genetic modifications made to obtain this laboratory strain. In order to understand these phenotypic differences, we are currently conducting systems biology analyses using genomics and proteomics. Full genome sequences were obtained for several strains of interest for which genomes were not publicly available. Comparative genomics using these sequences was identified mutations in candidate genes that might be related to the observed phenotypic differences. In the WG1 family (including K-12 strains), no single or simple set of gene mutations can explain the phenotypic differences and proteomics across the lycopene-production period was used to identify the effect of more subtle differences. We are currently evaluating target genes through complementation and expect this to deepen our understanding of MEP pathway flux regulation and suggest new targets for isoprenoid engineering in E. coli.
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