Process for activating gene expression in bacteria

Details Process for activating gene expression in bacteria Process for activating gene expression in bacteria

1996-04-01

1998-10-13

Degen, Nancy

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

435 691, 435713, 530350, 536 231, C12P 100

Patent

active

058210776

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to a process for activating bacterial gene expression and in particular to a process for activating cryptic gene expression, that is, the expression of genes which are not normally expressed or silent. More particularly, the present invention relates to a process for the activation of cryptic gene expression to produce bioactive compounds including antibiotics such as carbapenems.
The carbapenem antibiotics constitute a diverse group of .beta.-lactam antibiotics characterised by potent anti-bacterial and .beta.-lactamase--resistent activity. More than forty different carbapenems are known, most of which are produced by the actinomycetes, particularly Streotomyces spp (Ratcliffe and Albers-Schonberg, 1982; Brown 1984; Williamson 1986; all cited in Bainton et al 1992).
Carbapenems have been isolated from the Gram-negative bacterium Serratia marcescens and Erwinia carotovora by Parker et al. (1982) and in Azospirilium spp UK 1521 by Kintaka et al. (1985); all cited in Bainton et al 1992.
The regulatory mechanisms involved in the control of carbapenem biosynthesis remain largely unknown. However, Bainton et al (1992) have recently shown that carbapenom biosynthesis 1s regulated by the regulatory factor N-(3-oxohexanoyl)-L-homoserine lactone (HSL). This compound was previously only known for its role in auto-induction of bioluminescence in the marine bacterium Vibrio fischer. HSL is also structurally related to the A- and I-factors which are known to regulate production of antibiotics in some Streptomyces species.
In order to examine the biosynthetic and regulatory mechanisms involved in the production of the .beta.-lactam antibiotic 1-carbapen-2-em-3-carboxylic acid by Erwinia carotovora, blocked mutants were obtained with a carbapenem non-producing phenotype (Caf) as described by Bainton et al (1992). These mutants fell into two distinct groups: group 1 mutants secreted a low molecular mass diffusible factor which restored carbapenem biosynthesis in group 2 mutants, but not vice versa. This factor was shown to be HSL. Class 1 mutants produced HSL and were thus thought to be defective in carbapenenem biosynthatic genes.
In order to study class 1 mutants a chromosomal DNA cosmid library of Erwinia carotovora ATCC39048 was constructed in Escherichia coli. The cosmids produced were used in standard complementation studies to find a esquence which could restore the carbapenem antibiotic production in the class 1 mutants.
One cosmid (cWU142) was presumed to contain the carbapenem biosynthetic genes. Restriction fragments of this cosmid were sub-cloned and a 3.8 Kb EcoRI fragment was found to complement 7 out of 8 class 1 mutants. This fragment was sequenced and shown to comprise 2 Kb of cosmid DNA and 1.8 Kb of Erwinia DNA. This was extremely unexpected because known antibiotic biosynthetic gene sequences are much longer than 1.8 Kb.
The 1.8 Kb gene sequence was found to encode CarR a homologue of the LuxR regulatory protein from the Lux operon system associated with the bioluminescence phenotype of the marine bacterium V. fischeri.
By analogy with the V. fischeri Lux system, the inventors postulated that when HSL is made, it binds to CarR which can then act as a transcriptional activator of the carbapenem biosynthetic genes. Thus, the inventors reasoned that 7 out of 8 of the class 1 mutants are not, as expected, defective in genes required for synthesis of carbapenem, but in a gene encoding a regulatory protein, CarR, needed to switch on the carbapenem biosynthetic genes. Without the CarR gene product the carbapenem biosynthetic genes are not expressed, that is, they remain silent or cryptic.
By similar methods of those described for the construction of the Erwinia carotovora cosmid library, we have also made a pSF6 chromosomal library of a strain of Serratia marcescens that makes the same carbapenem (1-carbapen-2-em-3-carboxylic acid) as the Erwinia strain, but does not make HSL. We packaged this cosmid library into bacteriophage Lambda and used this lysate to transduce the class 1

REFERENCES:
Lewin, B., ed., 1983, Genes, John Wiley and Sons, see especially Chapter 15, pp. 247-249, 1983.

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