Expression of Escherichia coli promoters in Brevibacterium lactofermentum using the shuttle vector pEB003

Morinaga, Yasushi; Tsuchiya, M; Miwa, Kiyoshi; Sano, Konosuke

doi:10.1016/0168-1656(87)90027-7

Cited by 37 publications

(15 citation statements)

References 15 publications

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“…On the other hand, some high-GC gram-positive bacteria seem to be less specific than B. subtilis in this respect. For example, Corynebacterium glutamicum ("Brevibacterium lactofermentum"), a gram-positive host with a high GC content (15), readily utilizes certain E. coli promoters (17). The expression of membrane proteins of gram-negative origin in gram-positive organisms has not been investigated at all to date.…”

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confidence: 99%

Lactose permease of Escherichia coli catalyzes active beta-galactoside transport in a gram-positive bacterium

1993

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The following several lines of evidence demonstrate that lactose permease (LacY) (2,10,16). On the other hand, some high-GC gram-positive bacteria seem to be less specific than B. subtilis in this respect. For example, Corynebacterium glutamicum ("Brevibacterium lactofermentum"), a gram-positive host with a high GC content (15), readily utilizes certain E. coli promoters (17). The expression of membrane proteins of gram-negative origin in gram-positive organisms has not been investigated at all to date. This scenario is even more complex than that with soluble proteins because, besides efficient transcription and translation, the heterologous polypeptide must also be targeted to the membrane of the host cell and assume correct overall folding and orientation of the transmembrane segments.We have previously (4) found that the introduction of the E. coli lac operon into C. glutamicum conferred the ability to utilize lactose upon the recombinant organism. The presence of lacY in addition to lacZ was an absolute prerequisite for growth on lactose, whereas expression of lacZ alone was not sufficient. This work has provided genetic and phenotypic evidence for the functional expression of the lactose carrier (LacY) in C. glutamicum. We now show that LacY, an integral membrane protein with 12 hydrophobic membrane-spanning segments, is indeed integrated into the cytoplasmic membrane of C. glutamicum, and we provide biochemical evidence demonstrating that it catalyzes the active transport of P-galactosides in this heterologous membrane environment. The two C. glutamicum strains used in this study were R163(pECLlx) and R163(pECL3x), which bear recombinant plasmids with the entire E. coli lac operon (pECL1x) or only the lacZ gene (pECL3x) under control of a mutant lac promoter which works efficiently in C. glutamicum (4).Localization and integrity of the E. coli lac gene products expressed in C. glutamicum. The cellular localization of the lacZ and lacY gene products expressed in C. glutamicum R163 was investigated. For this purpose, recombinant C. glutamicum * Corresponding author. R163 cells were disrupted by a twofold passage through a French pressure cell at 6.9 MPa at 40C in the presence of DNase I, phenylmethylsulfonyl fluoride, MgSO4, and dithiothreitol at final concentrations of 45 jig/ml, 150 ,uM, 7.5 mM, and 1.5 mM, respectively. The crude extract was freed of debris (two times, 20 min, 9,000 x g, 4°C) and fractionated by high-speed centrifugation (two times, 2 h, 105,000 x g, 20C).The success of the fractionation procedure was verified by measuring the activities of the marker enzymes L-glutamate dehydrogenase and succinate dehydrogenase as described by others (3, 12) (data not shown). More than 95% of the P-galactosidase activity was found to be located in the cytoplasmic fraction of the lacZ-bearing strains, as expected. Additionally, immunoblot experiments demonstrated the cytoplasmic and membrane localization of ,-galactosidase and lactose permease, respectively, in C. glutamicum R163 (pECLlx) and showed t...

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confidence: 99%

Lactose permease of Escherichia coli catalyzes active beta-galactoside transport in a gram-positive bacterium

1993

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“…Also, C. glutamicum does not have the disadvantage of seVere genetic instabilities, as are known to occur in Streptomyces species. One major advantage over low-G+C gram-positive hosts such as Bacillus species or Staphylococcus species is the rather broad acceptance of heterologous expression signals, including gram-negative promoters (23), observed for C. glutamicum ("Brevibacterium lactoferinentum").…”

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Expression, secretion, and processing of staphylococcal nuclease by Corynebacterium glutamicum

1992

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The gene for staphylococcal nudease (SNase), an extracellular enzyme of Staphylococcus aureus, was introduced into Corynebacterium gbiamicum. The heterologous gene was expressed in this host organism, and SNase was efficiently exported to the culture medium. Amino-terminal sequencing of SNase secreted by C. glutamicum revealed that the signal peptide was apparently cleaved off at precisely the same position as in the original host, S. aureus. As with S. aureus, a second smaller form of SNase (A form), whose appearance is presumably the result of a secondary processing step, was found in the culture medium of the recombinant C. glutamicum strain. The A form was one residue shorter than the mature nuclease A produced by S. aureus. Variation of the sodium chloride concentration in the growth medium had a marked influence on the location and the processing of SNase by C. glutamicum. In a complex growth medium containing 4% sodium chloride, SNase was exclusively located in the supernatant, but a significant amount of the enzyme remained cell associated if the strain was grown in a low-salt medium. Also, high salt concentrations seemed to inhibit processing of the high-molecular-weight form of SNase (B form) to the smaller A form. Similarities and differences in the export and modes of processing of SNase by three different, nonrelated gram-positive host organisms are discussed. Finally, a versatile Escherichia coli-C. gluamicum tae-lacl expression shuttle vector was constructed. With this vector, it was possible to achieve isopropyl-o-D-galactopyranoside (ITG)-inducible overexpression and secretion of SNase in C. gtamicum, whereby the expression level was dependent on the concentration of the inducer.Corynebacterium glutamicum is a gram-positive bacterium with a high G+C content of DNA and is closely related to other so-called "amino acid-producing corynebacteria" (12). These organisms are used for the industrial production of certain amino acids. Recent progress in the development of more sophisticated genetic tools for C glutamicum makes this species an interesting alternative gram-positive cloning host. Compared with other established high-G+C grampositive hosts such as certain Streptomyces species, C. glutamicum has some advantages; e.g., there are no complex cellular differentiation steps such as mycelium formation or sporulation during growth. Also, C. glutamicum does not have the disadvantage of seVere genetic instabilities, as are known to occur in Streptomyces species. One major advantage over low-G+C gram-positive hosts such as Bacillus species or Staphylococcus species is the rather broad acceptance of heterologous expression signals, including gram-negative promoters (23), observed for C. glutamicum ("Brevibacterium lactoferinentum").It is generally accepted that gram-positive bacteria are superior to other bacteria as protein secreters, the most prominent examples being certain Bacillus species. Nevertheless, knowledge about protein export by gram-positive bacteria is still very poor. This is especially ...

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“…Expression issues have been mostly solved using several E. coli promoters that have been observed to remain functional in corynebacteria (82). A recent study demonstrated that numerous promoters of C. glutamicum exhibit a functional structure similar to that of common bacterial promoters, with extensive similarity existing between the Ϫ10 hexamers of C. glutamicum and E. coli promoters (93).…”

Section: Vectorsmentioning

confidence: 99%

Manipulating Corynebacteria, from Individual Genes to Chromosomes

Vertès

Inui

Yukawa

2005

Appl Environ Microbiol

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Corynebacterium glutamicum is an industrial organism with a long history of use for the production of various fine chemicals. The C. glutamicum-mediated production of L-glutamic acid by fermentation was one of the very first industrial processes of the biotechnology era. This fermentation originated in Japan from the discovery in 1957 by Kinoshita et al. (cited by Kumagai [68]) that, under suitable conditions, this soil bacterium is able to secrete L-glutamic acid in significant amounts (66). This process successfully replaced less cost-effective methods based on hydrolysis by concentrated hydrochloric acid of soy or various other plant proteins. Glutamic acid was traditionally extracted from seaweed to serve as a seasoning agent and remains to this day a very important food flavor additive, the worldwide production of which approximates 1.5 million tons per year (43). Industrial glutamic acid fermentation was implemented early on by major Japanese companies, thus building over time exquisite manufacturing knowledge at various industrial scales up to approximately 5,000 hectoliters (43). In the 1970s, efficient C. glutamicum L-lysine and L-threonine producers were generated by random mutagenesis and positively selected by phenotypic resistance to lysine or threonine analogs. The advent of molecular biology enabled a new wave of development in which this industrial know-how was leveraged, not only to improve the performance of the existing lysine and threonine production processes, but also to enable the production of other amino acids and vitamins (29,43,51,68). The utilization of recombinant DNA techniques, combined with metabolic and carbon flux analyses (67, 99), facilitated the identification of metabolic bottlenecks and their bypassing by expressing or repressing the corresponding genes to develop further improved amino acid industrial production processes. The intrinsic characteristics of this food grade microbial workhorse include its lack of pathogenicity and its lack of spore-forming ability, both desirable traits as listed by the U.S. Center for Biologics Evaluation and Research and the U.S. Center for Drug Evaluation and Research guidelines, as well as its high growth rate, its relatively limited growth requirements, the ability of several strains not to undergo autolysis under conditions of repressed cell division (50), the absence of native extracellular protease secretion that makes corynebacteria suitable hosts for protein expression (10, 100), and the relative stability of the corynebacterial genome itself (85). These intrinsic attributes, combined with an up-to-date set of genetic-engineering tools, make this organism ideal for the development of robust industrial processes that are increasingly competitive compared to Escherichia coli-, Bacillus subtilis-, or yeast-based processes. As a result, corynebacterial fermentations have become increasingly relevant to a wide range of industrial sectors, including food, feed, cosmetic, pharmaceutical, and chemical companies.Nonetheless, the significance...

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Expression of Escherichia coli promoters in Brevibacterium lactofermentum using the shuttle vector pEB003

Cited by 37 publications

References 15 publications

Lactose permease of Escherichia coli catalyzes active beta-galactoside transport in a gram-positive bacterium

Lactose permease of Escherichia coli catalyzes active beta-galactoside transport in a gram-positive bacterium

Expression, secretion, and processing of staphylococcal nuclease by Corynebacterium glutamicum

Manipulating Corynebacteria, from Individual Genes to Chromosomes

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