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 efficient ethanol production by ethanologenic recombinants of Escherichia coli B such as LY01 which contain chromosomally integrated Zymomonas mobilis genes (pdc,adhB) encoding the ethanol pathway. The basis for this requirement 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 increasing the level of pyruvate decarboxylase activity. This was accomplished by adding a multicopy plasmid containing pdc alone (but not adhB alone) to strain LY01, and by adding multicopy plasmids which express pdc and adhB from strong promoters. New strong promoters were isolated from random fragments of Z. mobilis DNA and characterized but were not used to construct integrated biocatalysts. These promoters contained regions resembling recognition sites for 3 different E. coli sigma factors: sigma(70), sigma(38), and sigma(28). The most effective plasmid-based promoters for fermentation were recognized by multiple sigma factors, expressed both pdc and adhB at high levels, and produced ethanol efficiently while allowing up to 80% reduction in complex nutrients as compared to LY01. The ability to utilize multiple sigma factors may be advantageous to maintain the high levels of PDC and ADH needed for efficient ethanol production throughout batch fermentation. From this work, we propose that the activation of biosynthetic genes in nutrient-poor media creates a biosynthetic burden that reduces the expression of chromosomal pdc and adhB by competing for transcriptional and translational machinery. This reduced expression can be viewed as analogous to the effect of plasmids (plasmid burden) on the expression of native chromosomal genes.

Transcriptional regulation of the human asparagine synthetase gene by carbohydrate availability.

Transcription of the asparagine synthetase (AS) gene is induced by amino acid deprivation. The present data illustrate that this gene is also under transcriptional control by carbohydrate availability. Incubation of human HepG2 hepatoma cells in glucose-free medium resulted in an increased AS mRNA content, reaching a maximum of about 14-fold over control cells after approx. 12 h. Extracellular glucose caused the repression of the content of AS mRNA in a concentration-dependent manner, with a k1/2 (concentration causing a half-maximal repression) of 1 mM. Fructose, galactose, mannose, 2-deoxyglucose and xylitol were found to maintain the mRNA content of both AS and the glucose-regulated protein GRP78 in a state of repression, whereas 3-O-methylglucose did not. Incubation in either histidine-free or glucose-free medium also resulted in adaptive regulation of the AS gene in BNL-CL.2 mouse hepatocytes, rat C6 glioma cells and human MOLT4 lymphocytes, in addition to HepG2 cells. In contrast, the steady-state mRNA content of GRP78 was unaffected by amino acid availability. Transient transfection assays using a reporter gene construct documented that glucose deprivation increases AS gene transcription via elements within the proximal 3 kbp of the AS promoter. These results illustrate that human AS gene transcription is induced following glucose limitation of the cells.