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New expression system from rhodococcus

A technology of Rhodococcus and Rhodococcus erythropolis, which is applied in the determination/inspection of bacteria and microorganisms, and the introduction of foreign genetic material using vectors, etc. It can solve the problems of negative effects on biotransformation efficiency and lack of specificity in response.

Inactive Publication Date: 2006-01-11
NV ORGANON
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the chemicals used to primarily block enzyme activity are often not reaction specific and may inhibit other important enzyme reactions, which may have a negative effect on biotransformation efficiency

Method used

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  • New expression system from rhodococcus
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1. Constitutive expression of kstD after deletion of the kstR non-marker gene

[0065] Construction of the mutagenic plasmid pREG104 for the deletion of the kstR non-marker gene, which encodes the transcriptional regulator of the kstD gene in Rhodococcus erythropolis SQ1 (encoding 3-sterone Δ 1 -Dehydrogenase KSTD1)( figure 1 ). Briefly, pSDH205 (Van der Geize, R. et al., 2000. Appl. Environ. Microbiol. 66:2029-2036) was digested with restriction enzymes NruI and BalI, followed by self-ligation to generate plasmid pREG103. The EcoRI DNA fragment of pREG103 containing the deletion of the kstR gene was then cloned into the EcoRI digested pK18mobsacB vector to generate pREG104. The kstR non-marker gene deletion mutant strain Hongchuanhong was isolated from Rhodococcus erythropolis SQ1 by the sacB counter-selection method as described (Van der Geize R. et al., 2001. FEMS Microbiol. Lett. 205: 197-202) using pREG104 coccus RG10. Authentic kstR gene deletion was c...

Embodiment 2

[0068] Example 2. Microbial steroids Δ 1 - Constitutive expression of KstD2 upon dehydrogenation

[0069] Using pREG104 ( figure 1 , see Example 1) Construction of kstR gene deletion mutant Rhodococcus erythropolis RG9 (Van der Geize, R. et al., 2002. Mol. Microbiol. 45: 1007-1018), denoted as Rhodococcus erythropolis RG17. Strain RG17 is thus a kstD kstD2kshA1 kstR quadruple gene deletion mutant that lacks 3-sterone Δ in addition to the transcriptional regulator of the kstD promoter. 1 - Dehydrogenase (KSTD1 and KSTD2) and 3-sterone 9α-hydroxylase (KSH) activity. Due to the kstD kstD2 kshA phenotype of this mutant, strain RG17 metabolizes 4-androstene-3,17-dione (AD), 1,4-androstadiene-3,17-dione (ADD) and 9α-hydroxy-4 -Androstene-3,17-dione (90HAD) was completely blocked.

[0070]A Rhodococcus expression vector was constructed for gene expression under the control of the kstD promoter of Rhodococcus erythropolis SQ1 (Van der Geize, R. et al., 2000. Appl. Environ. Microbi...

Embodiment 3

[0074] Example 3. Expression of the kshA isogene kshA2 complements the kshA mutant phenotype

[0075] Nucleotide sequencing of DNA fragments isolated from UV-induced complementation experiments of Rhodococcus mutants identified the identity of Rhodococcus erythropolis SQ1 (Van der Geize, R. et al., 2002. Mol. Microbiol. 45:1007-1018). Homologue of the kshA gene. Rhodococcus erythropolis SQ1 contains at least two kshA isogenes, denoted as kshA and kshA2.

[0076] The kshA gene deletion mutant of Rhodococcus erythropolis RG2-Rhodococcus erythropolis SQ1 (Van der Geize, R. et al., 2002. Mol. Microbiol. 45: 1007-1018) was shown not to supplement AD (0.25 g l -1 ) as the sole source of carbon and energy on a mineral agar plate (1 g l -1 NH 4 NO 3 , 0.25g l -1 K 2 HPO 4 , 0.25g l -1 MgSO 4 ·7H 2 O, 5mg·l -1 NaCl, 5mg·l -1 FeSO 4 ·7H 2 O (pH7.2), 1.5% agar). Thus, kshA2 was not expressed in RG2 under these growth conditions.

[0077] The kshA2 gene in pRESX is unde...

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Abstract

The present invention provides an isolated polynucleotide comprising the kstD promoter from Rhodococcus erythropolis. The polynucleotide can very advantageously be used as a controllable transcription activator. Said controlling function can be provided by providing said isolated polynucleotide with a nucleotide sequence encoding a transcription regulator of said promoter. In the present invention, such a transcription regulator may be externally induced, such as by introduction of steroidal compounds. In an alternative embodiment of the present invention the isolated polynucleotide may comprise the kstR gene or a homologue or a functional part thereof as the transcription regulator of the kstD promoter.

Description

field of invention [0001] The present invention relates to a promoter from Rhodococcus, more specifically from Rhodococcus erythropolis, its regulation and the use of this promoter and its regulation as an expression system in heterologous applications. Background of the invention [0002] Actinomycetes, and especially Rhodococcus bacteria, are known for their ability to metabolize complex molecules. Several Rhodococcus species are capable of degrading fuel, benzene, and even TNT, so they are widely studied in the field of microbiology involving biochemical pathways and cell factories. Among the microorganisms that oxidize natural and anthropogenic hydrocarbons and are active participants in the biogeochemical processes of the biosphere, such as those that contribute to the creation of a hydrocarbon-free atmosphere on Earth, Rhodococcus occupy a dominant position. [0003] Several Rhodococcus species also degrade natural phytosterols via steroid formation as pathway interme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/76C12N15/11C12N1/21C12Q1/68C07K14/36C12N15/74
CPCC12N15/74C07K14/36
Inventor R·范德盖泽G·赫塞尔斯L·戴克休伊曾P·范德梅登
Owner NV ORGANON
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