The effect of SecB chaperone on production of periplasmic penicillin acylase (PAC) in Escherichia coli was investigated. It appears that formation of PAC required the function of SecB chaperone and the amount of SecB required was at a basal level. The secB mutant was defective in production of PAC, and the impairment could be complemented by extrachromosomally supplementing SecB in trans. The function of SecB might be primarily stabilizing the cytoplasmic PAC precursors. Overproduction of SecB chaperone usually resulted in an increase in the amount of PAC precursors without enhancing PAC activity. In addition, most of the PAC precursors were located in the periplasm, suggesting that formation of active PAC was likely limited by periplasmic processing steps.

No abstract

High-temperature-oriented production of bacterial penicillin acylase (PAC), which is usually expressed at low temperatures (less than 30 degrees C), was demonstrated in this study via heterologous expression of the Providencia rettgeri (P. rettgeri) pac gene in Escherichia coli (E. coli). While it is possible to produce PAC at a temperature as high as 37 degrees C, the environmental condition (specifically, culture pH) critically affected culture performance. Production of PAC at 37 degrees C was feasible only when culture pH was close to neutral (i.e., 6.5-7.5). Outside this pH range, cell physiology for the host/vector system was seriously affected, resulting in poor culture performance. In acidic culture environments, temperature significantly affected the pac expression level and specific PAC activity decreased with an increase in culture temperature. In basic culture environments, cell growth was seriously inhibited though the pac expression level was minimally affected by temperature. Such unusual types of pH and temperature effects on pac expression were never reported for bacterial PACs. The results suggest that culture pH should be precisely controlled for the current host/vector systems being applied on the overproduction of P. rettgeri PAC in E. coli at high temperatures.

The production of recombinant penicillin acylase (PAC) in Escherichia coli was optimized in this study. The effect of using a selection of host/vector systems as well as varying culture conditions on the production of PAC was investigated. The production of PAC based on the use of the native pac promoter was inef®cient and could be signi®cantly improved by using the strong trc promoter for regulation of pac expression. A mutant strain MDDP7 was shown to be a suitable host for the production of PAC since the ef®ciency of both pac translation and posttranslational processing for MDDP7 was signi®cantly higher than that for the parent strain HBPAC101. However, the accumulation of inclusion bodies tended to limit the production of PAC as pac transcriptional and translational ef®ciency was increased. It has been demonstrated that, in addition to the increase in pac transcriptional and translational ef®ciency, the protein synthesis¯ux throughout pac expression steps should be balanced for enhancing the production of PAC in E. coli. With the optimization of the host/vector system and culture conditions, culture performance for the production of recombinant PAC was greatly improved. Process bottlenecks limiting the production of PAC were also discussed.