Manipulation of carbon assimilation with respect to expression of the pac gene for improving production of penicillin acylase in Escherichia coli

Chou, C. Perry; Tseng, Jen-Hung; Lin, Ming-I; Lin, Hua‐Kuo; Yu, Chih‐Chang

doi:10.1016/s0168-1656(98)00202-8

Cited by 14 publications

(7 citation statements)

References 22 publications

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“…Expression of the pac gene for pCLL2902 is regulated by the native pac promoter. However, the gene expression is constitutive, and no phenylacetic acid (PAA) is required since part of the pac regulatory region is truncated (Chou et al, 1999a). The Eco RI/ Pst I fragment containing the pac gene at 2.9 kb from pCLL2902 was subcloned into pTrcKn99A, a trc ‐expression vector derived from pTrc99A (Amann et al, 1988) by replacing the ampicillin (Ap)‐resistance gene with a kanamycin (Kn)‐resistance gene, to form pTrcKnPAC2902 (Chou et al, 1999b).…”

Section: Methodsmentioning

confidence: 99%

Effect of SecB Chaperone on Production of Periplasmic Penicillin Acylase in Escherichia coli

et al. 1999

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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.

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Section: Methodsmentioning

confidence: 99%

Effect of SecB Chaperone on Production of Periplasmic Penicillin Acylase in Escherichia coli

et al. 1999

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“…Due to the difference in the pac regulatory region (Fig. 2), expression of PAC was induced by PAA for pCLL3201 and was constitutive for pCLL2902 (Chou et al, 1999). The two plasmids gave relatively high PAC expression levels by which investigation on the effects of transcription or translation was possible.…”

Section: Genetically Modulating Transcription And/or Translation Effimentioning

confidence: 99%

“…The correlation was elucidated by expression of the pac gene devoid of this regulatory region using pCLL2902. Not only was the expression no longer subjected to PAA-induction and glucose catabolite repression (Chou et al, 1999), but the specific PAC activity for pCLL2902 was also much higher than that for pCLL3201 (Table II). It was then presumed that the extra 241 bps in the pac regulatory region for pCLL3201 somehow hindered transcription of the pac gene.…”

Section: Effects Of Transcription And/or Translation Efficiencies On mentioning

confidence: 99%

Genetic manipulation to identify limiting steps and develop strategies for high‐level expression of penicillin acylase in Escherichia coli

Chou

Tseng

et al. 1999

Biotechnol. Bioeng.

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We have identified the bottleneck steps limiting expression of penicillin acylase (PAC) through comparison of the expression performance for various PAC-expression vectors constructed by genetically modulating the efficiencies of transcription and/or translation of the pac gene. To our knowledge, this is the first report demonstrating that expression of PAC could be limited by various steps, such as transcription, translation, and post-translational steps (i.e. translocation and periplasmic processing), depending on the host/vector systems. Results also indicate that the structure of the wild-type pac gene might not be optimal for direct use in production of PAC using recombinant DNA technology. To improve the gene expression, transcription was enhanced by manipulating certain DNA bases in the pac regulatory region, whereas translation was enhanced by enlarging the spacing between the ribosome binding site and the ATG initiation codon to increase the initiation efficiency. The information is useful in terms of developing genetic strategies for overproduction of recombinant PAC in Escherichia coli.

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“…pCLL3201 contains the whole pac gene from E. coli ATCC11105, whereas a portion of the pac regulatory region is truncated in pCLL2902. Due to the difference in the pac regulatory region between the two plasmids, production of PAC is PAA-inducible for pCLL3201, whereas constitutive for pCLL2902 (Chou et al, 1999b). In addition, PAC activity expressed from pCLL2902 is significantly higher than that from pCLL3201 due to more efficient transcription of the pac gene (Chou et al, 1999c).…”

Section: Construction Of Penicillin-sensitive But 6-apa-resistant Mutmentioning

confidence: 99%

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

Chou¹,

Yu²,

Lin³

et al. 1999

Biotechnol. Bioeng.

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Manipulation of carbon assimilation with respect to expression of the pac gene for improving production of penicillin acylase in Escherichia coli

Cited by 14 publications

References 22 publications

Effect of SecB Chaperone on Production of Periplasmic Penicillin Acylase in Escherichia coli

Effect of SecB Chaperone on Production of Periplasmic Penicillin Acylase in Escherichia coli

Genetic manipulation to identify limiting steps and develop strategies for high‐level expression of penicillin acylase in Escherichia coli

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

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