Biosynthetic burden and plasmid burden limit expression of chromosomally integrated heterologous genes (pdc, adhB) in Escherichia coli

Previous studies have shown an unexpectedly high nutrient requirement for e fficient ethanol production by ethanologenic recombinants of Escherichia co li B such as LY01 which contain chromosomally integrated Zymomonas mobilis genes (pdc,adhB) encoding the ethanol pathway. The basis for this requireme nt has been identified as a media-dependent effect on the expression of the Z. mobilis genes rather than a nutritional limitation. Ethanol production was substantially increased without additional nutrients simply by increasi ng the level of pyruvate decarboxylase activity. This was accomplished by a dding a multicopy plasmid containing pdc alone (but not adhB alone) to stra in LY01, and by adding multicopy plasmids which express pde and adhB from s trong promoters. New strong promoters were isolated from random fragments o f Z. mobilis DNA and characterized but were not used to construct integrate d biocatalysts. These promoters contained regions resembling recognition si tes for 3 different E. coli sigma factors: sigma(70), sigma(38), and sigma( 28). The most effective plasmid-based promoters for fermentation were recog nized by multiple sigma factors, expressed both pde and adhB at high levels , and produced ethanol efficiently while allowing up to 80% reduction in co mplex nutrients as compared to LY01. The ability to utilize multiple sigma factors may be advantageous to maintain the high levels of PDC and ADH need ed for efficient ethanol production throughout batch fermentation. From thi s work, we propose that the activation of biosynthetic genes in nutrient-po or media creates a biosynthetic burden that reduces the expression of chrom osomal pdc and adhB by competing for transcriptional and translational mach inery. This reduced expression can be viewed as analogous to the effect of plasmids (plasmid burden) on the expression of native chromosomal genes.