大腸菌에서 Propioante 代謝의 遺傳學的 및 分子生物學的 特性硏究 = Genetic and molecular characterization of propionate metabolism in Escherichia coli

다국어 초록 (Multilingual Abstract)

[ABSTRACT]
Genetic and Molecular Characterization of Propionate
Metabolism in Escherichia coli

Bo Min Kim
Department of biology
Graduate School of
Sung Kuyn Kwan University

Polyhydroxyalkanoates (PHAs) is a class of biopolymers naturally produced by microbes as storage materials for carbon and reducing equivalents. These natural polyesters have received a considerable interest because of its potential to be developed as biodegradable plastics substituting petrochemical-based plastics. Biopol [poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)] is a biodegradable PHA plastics of promising physical properties for general usage. For the production of PHBV, glucose and propionate are supplied to fermentation of a Ralstonia eutropha or other metabolically engineered microorganisms. Therefore, understanding catabolism of propionate in the cell is important for its application to the PHBV production using microbial fermentation. Understanding of propionate catabolism has only emerged in the recent past and is still under active investigation.

As an effort to understand propionate catabolism of Escherichia coli, in this study, we have performed experiments basically three different directions: 1) Isolation and characterization of mutants defective propionate catablism, 2) Isolation and characterization of mutants resistant to high concentration of propionate in the medium, and 3) Regulation of propionate catabolism.

1) Isolation of characterization of mutants defective propionate catabolism
Wild type E. coli strain MG1655 was mutagenized by Tn10 and total of 3 independent mutants defective in propionate utilization (Prp- ) as a sole carbon source. The Prp- phenotype was 100% linked with the Tcr phenotype in the mutants. Physical mapping of the Tn10s using inverse PCR technique positively identified two loci out of the threes: rpoS (encoding stationary phase sigma factor, RpoS) and accA (encoding acetyl-CoA carboxylase). These results suggest that both rpoS and accA are required for utilization of propionate as a sole carbon source in E. coli.

2) Isolation and characterization of mutants resistant to high concentration of propionate
Among the transposone Tn10 mutants of MG1655 were screened and total of 15 independent mutants (propionate-resistant, Prpr) able to grow high concentration (80mM) of propionate in the minimal medium were isolated. Five different alleles were positively identified by inverse PCR technique. These include yedJ of unknown function, gatA encoding galatitol-specific enzyme IIA of phosphoenolpyruavte trasnferase system (gatA), cspD encoding an DNA replication inhibitor, nad encoding a protein involved in biosynthesis of nicotineamide dinucleotide (NAD), and b2380 of unknow function. Though further detailed studies needs to be conducted, but these results suggest that multiple factors in various cellular physiology are involved in the resistance to the high concentration of propionate in the medium.

3) Regulation of propionate catabolism
Genetic factors influencing propionate catabolism in E. coli were identified by both lacZ operon fusion and mobility shift assay. A lacZ fusion was constructed in the putative E. coli prpBCDE operon homologous to prpBCDE previously identified to encode enzymes involved in propionate catabolism in Salmonella typhimurium. Mobility shift assay was conducted using a probe containing a putative promoter region of the E. coli prpBCDE. From these experiments, multiple transcriptional factors were found to alter the expression of the E. coli prpBCDE operon. These includes crp encoding catabolite repressor protein, arcA encoding the regulator of aerobic respiratory control (Arc) two component system, ompR encoding the regulator of the two component system of osmoregulation. These results suggest catabolism of propionate in E. coli is regulated by multiple global regulators.

목차 (Table of Contents)

• 목차
• List of table = ⅲ
• List of Figures = ⅳ
• Ⅰ. 서론 = 1
• 1. PHAs(Polyhyoxyalkanonates ) 란? = 1
• 2. PHA(folyhydroxyalkanonates)와 PHBV에 대한 연구목적 및 이해 = 3
• 3. PHB와 PHBV의 특징 = 4
• 4. PHBV를 연구하는 목적 = 8
• Ⅱ. 재료 및 방법 = 11
• 1. 사용균주, bacterlophages and plasmid = 12
• 2. 배양 및 배양 조건 = 13
• 3. Bacteriophage 배양 및 Iysate 준비 = 14
• 4. Pl-형질 도입 (transduction) = 15
• 5. β -galactosidase activity 측정 = 16
• 6. 형질전환 (transformation) = 17
• 7. TnIO transposon pool of 이용 = 18
• 8. Inverse PCR = 20
• 9. Northern blotting assay = 22
• 10. EMSA(Electrophorectic Mobility Shift Assay) = 22
• 11. Bioinfomatic techniques = 23
• 12. Construction of a null mutation in prpc by gene disruption = 23
• Ⅲ. 결과 = 24
• 1. Isolation and characterization of mutant pro- defective in propionate utilization = 24
• 1.1 Isolation of mutant prp- (defective in propionate utilization) = 32
• 1.2.Characterization of pro- mutant(defective in propionate utilization) = 25
• 2.Isolation and Pkaracterization of mutants resistant to high concentration proplonate and glucose = 33
• 2-1.glucose+ propionate를 잘 쓰는 mutants = 33
• 2-2.propionate resistant mutant (prpR) = 40
• 3.Regulation o( propionate catabolism in E.coli = 36
• 4.Metabolic engineering of host cells for enhanced copolymer production = 48
• Ⅳ. 고찰 = 67
• Ⅴ. 적요 = 73
• Ⅶ.reference = 76
• Abstract = 82