Promoters are the most important tools to control and regulate the gene expression in synthetic biology and metabolic engineering. The expression of target genes in Escherichia coli is usually controlled by the high-strength inducible promoter with the result that the abnormally high transcription of these genes creates excessive metabolic load on the host, which decreases product formation. The constitutive expression systems are capable of avoiding these defects. In this study, to enrich the application of constitutive promoters in metabolic engineering, four promoters from the glycolytic pathway of E. coli were cloned and characterized using the enhanced green fluorescent protein as reporter. Among these promoters, Pgap Awas determined as the strongest one, the strength of which was about 8.92% of that of the widely used inducible promoter PT7. This promoter was used to control the expression of heterologous xylose reductase in E. coli for xylitol synthesis so as to verify its function in pathway engineering. The maximum xylitol titer(40.6 g·L-1) produced by engineered E. coli under the control of the constitutive promoter Pgap Awas obviously higher than that under the control of the inducible promoter PT7,indicating the feasibility and superiority of promoter Pgap Ain the metabolic engineering of E. coli.
Glycyrrhetic acid 3-O-mono-β-D-glucuronide （GAMG）, the major functional ingredient in licorice, has widespread applications in food, pharmacy and cosmetics industry. The production of GAMG through Penicillium purpurogenum Li-3 cultivation was for the first time performed through both batch and fed-batch processes in bioreactors. In batch process, under optimal conditions （pH 5.0, temperature 32℃, agitation speed 100 r. rain 1）, 3.55 g. L^-1 GAMG was obtained in a 2.5 L fermentor. To further enhance GAMG production, a fine fed-batch process was developed by using pH and DO as feedback parameters. Starting from 48 h, 100 m190 g-L 1 substrate Glycyrrhizin （GL） was fed each time when pH increased to above 5.0 and DO was increased to above 80%. This strategy can significantly enhance GAMG production： the achieved GL conversion was 95.34% with GAMG yield of 95.15%, and GAMG concentration was 16.62 g. L^-1 which was 5 times higher than that of batch. Then, a two-step separation strat- egy was established to separate GAMG from fermentation broth by crude extraction of 15 ml column packed with D10I resin followed by fine purification with preparative C18 chromatography. The obtained GAMG purity was 95.79%. This study provides a new insight into the industrial bioprocess of high-level GAMG production.
