Recombinant plasmids with genes for the biosynthesis of alkaline phosphatase of Escherichia coli

Boidol, Werner; Simonis, Marianne; Töpert, Michael; Siewert, Gerhard

doi:10.1007/bf00334150

Cited by 22 publications

(8 citation statements)

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“…Based on the information described above, the phosphateresponsive promoter P PHO89 and the process involving it are extremely attractive for industrial applications. Phosphate-responsive promoters have been exploited in other microorganisms, such as E. coli (6,18) and S. cerevisiae (27). However, a growth limitation other than sugar limitation in E. coli and S. cerevisiae triggers acetic or alcoholic fermentation, leading to arrest of cell growth and lower productivity (11,28).…”

Section: Discussionmentioning

confidence: 99%

“…These results imply that the metabolic activities and cell viability are maintained even in stationary phase in a phosphate-limited environment, which could be exploited for the production of recombinant proteins. Also, it was reported previously that transcriptional activities of the promoters of phosphate-responsive genes, such as the genes encoding alkaline phosphatase in Escherichia coli (6,18) and acid phosphatase in Saccharomyces cerevisiae (32), are regulated by phosphate and are highly activated by the simple and cheap technique of lowering the phosphate concentration in the culture medium.…”

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confidence: 99%

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Phosphate-Responsive Promoter of a Pichia pastoris Sodium Phosphate Symporter

Ahn

Hong

Park

et al. 2009

Appl Environ Microbiol

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To develop a functional phosphate-regulated promoter in Pichia pastoris, a phosphate-responsive gene, PHO89, which encodes a putative sodium (Na ؉ )-coupled phosphate symporter, was isolated. Sequencing analyses revealed a 1,731-bp open reading frame encoding a 576-amino-acid polypeptide with 12 putative transmembrane domains. The properties of the PHO89 promoter (P PHO89 ) were investigated using a bacterial lipase gene as a reporter in 5-liter jar fermentation experiments. P PHO89 was tightly regulated by phosphate and was highly activated when the cells were grown in a phosphate-limited external environment. Compared to translation elongation factor 1␣ and the glyceraldehyde-3-phosphate dehydrogenase promoter, P PHO89 exhibited strong transcriptional activity with higher specific productivity (amount of lipase produced/cell/h). Furthermore, a cost-effective and simple P PHO89 -based fermentation process was developed for industrial application. These results demonstrate the potential for efficient use of P PHO89 for controlled production of recombinant proteins in P. pastoris.Pichia pastoris is a methylotrophic yeast species that is increasingly used as a host system for heterologous protein expression for both academic and industrial purposes. To date, more than 500 proteins have been cloned and expressed using this system (19; http://faculty.kgi.edu/cregg/index htm), and it has provided the protein production platform for several structural genomics programs (25,35).In this system, most recombinant proteins have been produced using the alcohol oxidase I promoter (P AOX1 ), which is completely repressed when cells are grown on glucose and is induced in the presence of methanol (31). However, there are circumstances in which the promoter may not be suitable; the principal problem is that the highly volatile and inflammable compound methanol is required for transcription (29). Use of methanol for the induction of gene expression is often not permitted in the production of food products since methanol is a petroleum by-product (13, 33). Also, P AOX1 -based fermentation requires relatively long times and sophisticated feeding strategies for maintenance of the induction phase. These properties hamper the industrial applicability of this approach. Therefore, a promoter that does not require methanol is attractive for expression of foreign genes in P. pastoris. Potential promoters that are alternatives to P AOX1 include the glutathione-dependent formaldehyde dehydrogenase promoter (P FLD1 ) (26), for which either methanol or methylamine can act as an inducer, and the glyceraldehyde-3-phosphate dehydrogenase promoter (P GAP ) (33), which is a strong constitutive promoter in various microorganisms. Recently, we also developed the translation elongation factor 1␣ promoter (P TEF1 ), with more highly growth-associated expression characteristics, as an alternative to P GAP (2). The current study describes the use of a phosphate-responsive promoter as an alternative to constitutive and inducible promoters to drive expres...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Phosphate-Responsive Promoter of a Pichia pastoris Sodium Phosphate Symporter

Ahn

Hong

Park

et al. 2009

Appl Environ Microbiol

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“…The plasmid constructs used are shown in Figure 1. pJP71 and pJP77 are derivatives of pACYC184 and have been described elsewhere (9), as has pSB43 (10). pBL1 18 and pBL121 are derivatives of pBR322 (1).…”

Section: Nucleic Acids Researchmentioning

confidence: 99%

Molecular organization ofsbcC, a gene that affects genetic recombination and the viability of DNA palindromes inEscherichia coliK-12

Naom

Morton²,

Leach

et al. 1989

Nucl Acids Res

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“…1. pSB43, pJP77, and pIN510 are described in detail elsewhere (3,23,28). pIN507 corresponds to pJP77 sbcC:: TnlOOO no.…”

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Identification of sbcD mutations as cosuppressors of recBC that allow propagation of DNA palindromes in Escherichia coli K-12

1992

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The function of an open reading frame (orf-45) located upstream of the sbcC gene of Escherichia coli was investigated. Mutations that inactivate sbcC improve the ability to propagate Ared gam phage that carry a palindromic sequence in their DNA. They also act with sbcB mutations as cosuppressors of the defects in recombination, DNA repair, and cell viability associated with recBC mutations. A 1,282-bp cassette encoding resistance to kanamycin was used to disrupt orf-45. The mutation, which has a polar effect on the expression of sbcC, allowed stable propagation of palindromic k phage even when the sbcC gene product was provided in trans. Additional nonpolar mutations in orf-45 were isolated on the basis of their ability to improve the growth of recBC sbcB strains. These mutations also confer resistance to mitomycin C, allow efficient recombination in Hfr crosses, and facilitate stable propagation of palindromic phage. It is concluded that the products of orf-45 and sbcC are functionally related. The orf-45 gene is therefore renamed sbcD.The sbcC gene of Escherichia coli was defined originally by mutations that arise spontaneously in recBC sbcB strains (16). These mutations act with mutations in sbcB to suppress the defects in recombination and DNA repair associated with the recBC genotype and are selected, therefore, during normal growth because of the improved viability. Subsequently, sbcC single mutants were shown to be good hosts for a Xred gam phage carrying a DNA sequence with palindromic symmetry (Xpal) (5). Phage of this type grow poorly in sbcC+ cells. Leach and Stahl (13) had observed this feature of the sbcC phenotype in their earlier work with recBC sbcB sbcC strains but, because isogenic strains were not constructed and used for comparisons, had attributed it to the absence of exonuclease V (RecBCD enzyme) and exonuclease I (the product of sbcB). This seemed reasonable since palindromes might form hairpins or cruciforms which could be recognized and cleaved by nucleases that normally resolve Holliday intermediates in genetic recombination. Kulkarni and Stahl (11) MATERIALS AND METHODSBacterial strains and X phages. The E. coli K-12 strains and A phages used are described in Table 1. Phage stocks were prepared on strain JC7623, which allows stable propagation of the 571-bp palindrome carried by XMMS1632.Plasmids. Plasmid constructs carrying DNA inserts from the sbcC-orf-45 region of the chromosome are shown in Fig. 1. pSB43, pJP77, and pIN510 are described in detail elsewhere (3,23,28). pIN507 corresponds to pJP77 sbcC:: TnlOOO no. 5 described by Naom et al. (23). pFG103 was made by cloning a BamHI-HindIII fragment of approximately 14.5 kb from pJP77 into pACYC184 (6). A 1,282-bp cassette (kan) encoding resistance to kanamycin was cloned from pUC4K (29) into the unique PstI site of pFG103 to give pFG103K. pFG106 was constructed by removing the Kmr insertion from pFG103K by digestion with EcoRI and inserting it into the EcoRI site of the temperature-sensitive replicon pHSG415 (10). The sbcC+ construc...

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Recombinant plasmids with genes for the biosynthesis of alkaline phosphatase of Escherichia coli

Cited by 22 publications

References 20 publications

Phosphate-Responsive Promoter of a Pichia pastoris Sodium Phosphate Symporter

Phosphate-Responsive Promoter of a Pichia pastoris Sodium Phosphate Symporter

Molecular organization ofsbcC, a gene that affects genetic recombination and the viability of DNA palindromes inEscherichia coliK-12

Identification of sbcD mutations as cosuppressors of recBC that allow propagation of DNA palindromes in Escherichia coli K-12

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