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Reverse engineering industrial bacte...

Lum, Amy Melissa.

Reverse engineering industrial bacteria that overproduce antibiotics.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Reverse engineering industrial bacteria that overproduce antibiotics.
作者:	Lum, Amy Melissa.
面頁冊數:	103 p.
附註:	Adviser: Camilla M. Kao.
附註:	Source: Dissertation Abstracts International, Volume: 66-11, Section: B, page: 6119.
Contained By:	Dissertation Abstracts International66-11B.
標題:	Engineering, Chemical.
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3197472
ISBN:	9780542431715

Reverse engineering industrial bacteria that overproduce antibiotics.
Lum, Amy Melissa.

Reverse engineering industrial bacteria that overproduce antibiotics. - 103 p.

Adviser: Camilla M. Kao.

Thesis (Ph.D.)--Stanford University, 2006.

A putative regulator, eryR, for the erythromycin biosynthetic cluster was identified by DNA binding gel-shift assays. Increased shift activity in the overproducer strain suggested a connection between this regulator and the gene expression changes of the ery cluster in the overproducer. We purified this protein from crude cell extracts, and obtained peptide sequences through mass-spec de novo sequencing to extract the DNA sequence from the genome. The DNA sequence of eryR encodes an 18 kD protein which is homologous to BldD, a known developmental regulator in Streptomyces coelicolor. When produced heterologously in E. coli, EryR bound all of the erythromycin promoters confirming we had purified a dedicated regulator of the cluster. However, sequence comparison of eryR and its promoter region between the Sac. erythraea overproducer and wildtype strains revealed no mutations, suggesting the mutation causing overproduction may be at a higher level of regulation.

ISBN: 9780542431715Subjects--Topical Terms:

226989
Engineering, Chemical.

Reverse engineering industrial bacteria that overproduce antibiotics.
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245 1 0 $a Reverse engineering industrial bacteria that overproduce antibiotics.
300 $a 103 p.
500 $a Adviser: Camilla M. Kao.
500 $a Source: Dissertation Abstracts International, Volume: 66-11, Section: B, page: 6119.
502 $a Thesis (Ph.D.)--Stanford University, 2006.
520 # $a A putative regulator, eryR, for the erythromycin biosynthetic cluster was identified by DNA binding gel-shift assays. Increased shift activity in the overproducer strain suggested a connection between this regulator and the gene expression changes of the ery cluster in the overproducer. We purified this protein from crude cell extracts, and obtained peptide sequences through mass-spec de novo sequencing to extract the DNA sequence from the genome. The DNA sequence of eryR encodes an 18 kD protein which is homologous to BldD, a known developmental regulator in Streptomyces coelicolor. When produced heterologously in E. coli, EryR bound all of the erythromycin promoters confirming we had purified a dedicated regulator of the cluster. However, sequence comparison of eryR and its promoter region between the Sac. erythraea overproducer and wildtype strains revealed no mutations, suggesting the mutation causing overproduction may be at a higher level of regulation.
520 # $a Actinomycetes are an important source of antibiotics and natural products. These compounds are typically produced in small quantities in vivo , making overproducing strains necessary for manufacturing these products. Industrial strains are traditionally acquired through classical strain improvement programs involving years of random mutation and screening. This process is not only time consuming, but the accumulated mutations leading to overproduction are unknown. With the wealth of genomic sequence generated daily, there are now methods to study entire genomes at once, making it feasible to correlate overproduction with genotype. Our research goal was to discover overproduction mechanisms from these industrial strains such that they can be utilized in new strain engineering efforts.
520 # $a To identify potential mechanisms for antibiotic overproduction in existing industrial actinomycete strains, we used DNA microarrays to monitor transcription. In a Saccharopolyspora erythraea erythromycin overproducer from Kosan Biosciences, we found expression of the erythromycin polyketide synthase genes (eryA) was extended four-fold, which may contribute significantly to increased titers. The majority of the erythromycin biosynthetic cluster also followed this expression pattern, suggesting a possible mutation in a dedicated regulator of the cluster.
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