GEOPHYSICS. Layered deformation in the Taiwan orogen.

The underthrusting of continental crust during mountain building is an issue of debate for orogens at convergent continental margins. We report three-dimensional seismic anisotropic tomography of Taiwan that shows a nearly 90° rotation of anisotropic fabrics across a 10- to 20-kilometer depth, consistent with the presence of two layers of deformation. The upper crust is dominated by collision-related compressional deformation, whereas the lower crust of Taiwan, mostly the crust of the subducted Eurasian plate, is dominated by convergence-parallel shear deformation. We interpret this lower crustal shearing as driven by the continuous sinking of the Eurasian mantle lithosphere when the surface of the subducted plate is coupled with the orogen. The two-layer deformation clearly defines the role of subduction in the formation of the Taiwan mountain belt.

Effect of pH on high-temperature production of bacterial penicillin acylase in Escherichia coli.

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.

Novel strategy for efficient screening and construction of host/vector systems to overproduce penicillin acylase in Escherichia coli.

A novel and simple method of using penicillin for screening of mutant strains with a high penicillin acylase (PAC) activity was developed. Random mutagenesis was conducted using a PAC-producing strain resistant to 6-aminopenicillanic acid (6-APA) as the parent strain and mutants were screened with penicillin at a high concentration. Results suggest that mutants with a high minimum inhibitory concentration for penicillin (MIC(penG)) usually overproduce PAC. Both volumetric and specific PAC activities of a mutant, MD7, were significantly higher than those of the parent strain, HBPAC101 harboring pCLL2902. The mutation(s) resulting in the enhanced expression was mapped on the host chromosome rather than the plasmid. In addition, the mutant strain of MDDeltaP7, derived by elimination of the harbored plasmid in MD7, was demonstrated to be efficient in production of PAC by using the expression plasmids for which expression of the pac gene is limited by translation. An extremely high specific PAC activity of more than 350 U/L/OD(600) was reached upon cultivation of MDDeltaP7 harboring pTrcKnPAC2902 in a bioreactor. As such, the strategy is effective in terms of constructing PAC overproducers and improving the process yield for production of PAC.

Effect of SecB chaperone on production of periplasmic penicillin acylase in Escherichia coli.

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.

Optimization of the host/vector system and culture conditions for production of penicillin acylase in Escherichia coli.

Culture performance for the production of penicillin acylase (PAC) in a bioreactor was investigated using HB101 or ATCC11105 as the host and pCLL2902, pCLL3201 or pTrcKnPAC2902 as the expression plasmid. We observed that the production of PAC by HB101 harboring pCLL3201 was, similar to ATCC11105, induced by phenyl acetic acid (PAA) and catabolicaily repressed by glucose, whereas the production of PAC by HB101 harboring pCLL2902 did not require PAA for induction and was not repressed by glucose. PAC activity of HB101 harboring pCLL2902 was significantly higher than that of HB101 harboring pCLL3201. There was no significant effect of host or carbon source on the production of PAC using pCLL2902. The production of PAC by HB101 harboring pTrcKnPAC2902, in which the pac gene expression was controlled by the trc promoter system, was about the same as that by HB101 harboring pCLL2902, when the culture was appropriately induced with isopropyl beta-d-thiogalactopyranoside (IPTG). Therefore, the use of both pCLL2902 and pTrcKnPAC2902 could be expected to be feasible for industrial applications. However, optimization of IPTG induction for HB101 harboring pTrcKnPAC2902 might be required, since formation of inclusion bodies tends to limit the production of PAC in some cases.