[보고서]시스템대사공학을 이용한 Poly(lactic acid) 공중합체 폴리에스터 합성 재조합 미생물 플랫폼 시스템 개발

박시재

보고서 정보

주관연구기관

이화여자대학교
Ewha Womans University

연구책임자

박시재

참여연구자

이승환

보고서유형

최종보고서

발행국가

대한민국

언어

한국어

발행년월

2021-05

과제시작연도

2021

주관부처

과학기술정보통신부
Ministry of Science and ICT

등록번호

TRKO202200011647

과제고유번호

1711129830

사업명

기후변화대응기술개발(R&D)

DB 구축일자

2022-09-29

키워드

바이오리파이너리.대사공학.시스템대사공학.바이오화학산업.재조합미생물.바이오폴리에스터.Biorefinery.Metabolic engineering.Systems metabolic engineering.Bio-based chemical industry.Recombinant microorganism.Biopolyester.PLA.PLA copolymer.

표 Metabolic pathways for the production of P(2HB-co-LA) developed in this study. The overall metabolic pathways consisting of inherent metabolic pathways and introduced metabolic pathways for the production of P(2HB-co-LA) in E. coli are shown. Citramalate pathway is constructed by the expression of the evolved M. jannaschii cimA3.7 gene and E. coli leuBCD genes to synthesize 2-hydroxybutyrate from glucose
표 Lists of bacterial strains and plasmids used in P(2HB-co-LA) polymer synthesis in E. coli
$Biosynthesis of P(2HB-co-LA) in recombinant E. coli strains harboring p619C1437-pct540 by adding sodium 2-hydroxybutyrate and sodium lactate into the culture medium. Recombinant E. coli XL1-Blue, WL3110, BL21(DE3) strains were examined for the synthesis of P(2HB-co-LA) in different culture medium as follows: (A) 2 g/L of sodium 2-hydroxybutyrate was added to the culture medium (B) 2 g/L of sodium 2-hydroxybutyrate and 2 g/L of sodium lactate were added to the culture medium. (Symbols are: striped bar, lactate monomer fraction in polymer; open bar, 2HB monomer fraction in polymer; filled diamond, polymer content)$ 표 Biosynthesis of P(2HB-co-LA) in recombinant E. coli strains harboring p619C1437-pct540 by adding sodium 2-hydroxybutyrate and sodium lactate into the culture medium. Recombinant E. coli XL1-Blue, WL3110, BL21(DE3) strains were examined for the synthesis of P(2HB-co-LA) in different culture medium as follows: (A) 2 g/L of sodium 2-hydroxybutyrate was added to the culture medium (B) 2 g/L of sodium 2-hydroxybutyrate and 2 g/L of sodium lactate were added to the culture medium. (Symbols are: striped bar, lactate monomer fraction in polymer; open bar, 2HB monomer fraction in polymer; filled diamond, polymer content)
$Biosynthesis of P(2HB-co-LA) in recombinant E. coli XL1-Blue strain harboring p619C1437-pct540 cultured in chemically defined medium containing different concentration of sodium 2-hydroxybutyrate. Sodium 2-hydroxybutyrate was added to the culture medium to support the synthesis of 2HB-CoA by Pct540. Lactate, synthesized by inherent lactate synthesis pathway from glucose, was converted into lactyl-CoA by Pct540. 2HB-CoA and lactyl-CoA were polymerized into P(2HB-co-LA) by PhaC1437. (Symbols are: striped bar, lactate monomer fraction in polymer; open bar, 2HB monomer fraction in polymer; filled diamond, polymer content)$ 표 Biosynthesis of P(2HB-co-LA) in recombinant E. coli XL1-Blue strain harboring p619C1437-pct540 cultured in chemically defined medium containing different concentration of sodium 2-hydroxybutyrate. Sodium 2-hydroxybutyrate was added to the culture medium to support the synthesis of 2HB-CoA by Pct540. Lactate, synthesized by inherent lactate synthesis pathway from glucose, was converted into lactyl-CoA by Pct540. 2HB-CoA and lactyl-CoA were polymerized into P(2HB-co-LA) by PhaC1437. (Symbols are: striped bar, lactate monomer fraction in polymer; open bar, 2HB monomer fraction in polymer; filled diamond, polymer content)
표 NMR analysis (1D 1H) of P(2HB-co-LA) synthesized in recombinant E. coli XL1-Blue (p619C1437-pct540)
표 Amino acid similarity of butyryl-CoA transferase with Clostridium propionicum Pct540. These butyryl-CoA trasnferases
$SDS-PAGE of the purified his-tagged butyrl-CoA transferase from Roseburia sp., Anaerostipes caccae, Faecalibacterium prausnitzii and Eubacterium hallii. The lanes are MW, molecular weight marker (kDa); T, totalproteins; F, flow-through; W, washed fraction; E, eluted fraction$ 표 SDS-PAGE of the purified his-tagged butyrl-CoA transferase from Roseburia sp., Anaerostipes caccae, Faecalibacterium prausnitzii and Eubacterium hallii. The lanes are MW, molecular weight marker (kDa); T, totalproteins; F, flow-through; W, washed fraction; E, eluted fraction
표 Relative activities of butyryl-CoA transferases with different substrates. The enzyme activities of (A)Roseburia sp. butyryl-CoA transferase, B)E. hallii butyryl-CoA transferase, (C)F. prausnitzii butyryl-CoA transferase, and (D) A. caccae butyryl-CoA transferase were represented by relative activities towards butyrate as a positive control (100% relative activity). The four butyryl-CoA transferase showed various degree of activity towards 3HB, LA and 2HB
표 Growth of recombinant E.coli XL1 Blue expressing PhaC1437 and butyrl-CoA transferase from A. caccae (purple square), E.hallii (red triangle), and F. prausnitzii (orange star) Roseburia sp. (blue diamond) was compared with the growth of E.coli XL1 Blue expressing PhaC1437 and C. propionicum propionyl-CoA transferase (green circle) in (A) LB medium and (B) MR medium containing 20g/L glucose
$Biosynthesis of PHAs containing 3HB, 2HB, and LA as monomer fractions in recombinant E. coli XL1-Blue expressing PhaC1437 and various butyryl-CoA transferases following addition of 2 g/L sodium 3HB and varying concentrations of 2HB. 2* refers to the addition of 2g/L 2HB without supplementation of 3HB to the culture medium. Filled diamonds are PHA content (wt%). Blue, red and orange bars are 3HB, LA, and 2HB monomer fractions, respectively. (A) Roseburia sp. butyrl-CoA transferase, (B) E. hallii butyrl-CoA transferase, (C) F. prausnitzii butyrl-CoA transferase, and (D) A. caccae butyrl-CoA transferase$ 표 Biosynthesis of PHAs containing 3HB, 2HB, and LA as monomer fractions in recombinant E. coli XL1-Blue expressing PhaC1437 and various butyryl-CoA transferases following addition of 2 g/L sodium 3HB and varying concentrations of 2HB. 2* refers to the addition of 2g/L 2HB without supplementation of 3HB to the culture medium. Filled diamonds are PHA content (wt%). Blue, red and orange bars are 3HB, LA, and 2HB monomer fractions, respectively. (A) Roseburia sp. butyrl-CoA transferase, (B) E. hallii butyrl-CoA transferase, (C) F. prausnitzii butyrl-CoA transferase, and (D) A. caccae butyrl-CoA transferase
표 NMR analysis (1H and 13C) of P(3HB-co-2HB-co-LA) terpolymer synthesized from recombinant E. coli XL1-Blue expressing PhaC1437 and E. hallii butyryl-CoA transferase cultured in MR medium containing 20 g/L glucose supplemented with 1 g/L 3HB and 1 g/L 2HB. (A) 500 MHz 1H NMR spectrum (B) 13C NMR spectrum with the chemical shift assignment
$Metabolic pathway for the production of PHAs having 3HB, 2HB and LA monomer fractions. The genes are M. jannaschii citramalate synthase (cimA3.7), E. coli 3-isopropylmalate dehydrogenase (leuB), E. coli isopropyl malate (IPM) isomerase (leuCD), L. lactis subsp. lactis Il1403 2-hydroxyacid dehydrogenase (panE), R. eutropha β-ketothiolase and acetoacetyl-reductase (phaAB), Pseudomonas sp. 6-19 PHA synthase (phaC1437), and bct genes from Roseburia sp., E. hallii, F. prausnitzii, and A. caccae. Red arrows indicate butyryl-CoA transferase$ 표 Metabolic pathway for the production of PHAs having 3HB, 2HB and LA monomer fractions. The genes are M. jannaschii citramalate synthase (cimA3.7), E. coli 3-isopropylmalate dehydrogenase (leuB), E. coli isopropyl malate (IPM) isomerase (leuCD), L. lactis subsp. lactis Il1403 2-hydroxyacid dehydrogenase (panE), R. eutropha β-ketothiolase and acetoacetyl-reductase (phaAB), Pseudomonas sp. 6-19 PHA synthase (phaC1437), and bct genes from Roseburia sp., E. hallii, F. prausnitzii, and A. caccae. Red arrows indicate butyryl-CoA transferase
$Biosynthesis of P(3HB-co-2HB-co-LA) in recombinant E. coli XL1-Blue harboring Pseudomonas sp. 6-19 phaC1437, M. jannaschii cimA3.7, E.coli leuBCD ,L. lactis subsp. lactisIl 1403 panE, and R. eutropha phaAB together with Roseburia sp. or E.hallii bct gene. Filled diamonds are PHA content. Blue, red and orange bars are 3HB, LA, and 2HB monomer fractions, respectively. All the experiments were carried out in triplicates$ 표 Biosynthesis of P(3HB-co-2HB-co-LA) in recombinant E. coli XL1-Blue harboring Pseudomonas sp. 6-19 phaC1437, M. jannaschii cimA3.7, E.coli leuBCD ,L. lactis subsp. lactisIl 1403 panE, and R. eutropha phaAB together with Roseburia sp. or E.hallii bct gene. Filled diamonds are PHA content. Blue, red and orange bars are 3HB, LA, and 2HB monomer fractions, respectively. All the experiments were carried out in triplicates
$Time profiles of batch fermentations of recombinant E.coli XL1-Blue expressing Pseudomonas sp. 6-19 phaC1437, M. jannaschii cimA3.7, E.coli leuBCD, L. lactis subsp. lactis Il1403 panE , and R.eutropha phaAB together with E. hallii bct gene (blue) or C. propionicum pct540 gene (red). (A) The cell growth (OD600) and glucose concentration (g/L) are shown as circle and diamond, respectively. (B) The PHA content, 3HB, 2HB and LA monomer fractions are shown as circle, diamond, triangle and square, respectively$ 표 Time profiles of batch fermentations of recombinant E.coli XL1-Blue expressing Pseudomonas sp. 6-19 phaC1437, M. jannaschii cimA3.7, E.coli leuBCD, L. lactis subsp. lactis Il1403 panE , and R.eutropha phaAB together with E. hallii bct gene (blue) or C. propionicum pct540 gene (red). (A) The cell growth (OD600) and glucose concentration (g/L) are shown as circle and diamond, respectively. (B) The PHA content, 3HB, 2HB and LA monomer fractions are shown as circle, diamond, triangle and square, respectively
표 Summary of Batch fermentation
표 Strains and Plasmid used in this study
표 Time profile of miscanthus hydrolysate solution utilization (blue 10 g/L glucose from miscathus; red, 20g/L glucose from miscanthus; green, 20 g/L pure glucose) by R. eutropha 2649. Cell growth is shown in triangle, whereas the glucose utilization is shown as circle
표 PHB production by R. eutropha 2649 using miscanthus hydrolysate solution as carbon source. The final concentration of PHB produced from 10 g/L glucose (miscanthus hydrolysate solution), 20 g/L glucose (miscanthus hydrolysate solution) and 20 g/L pure glucose were 3.93 g/L, 3.78 g/L and 4.14 g/L, respectively, suggesting that miscanthus hydrolysate can be used as an alternative cheaper carbon substrate of pure glucose for polymer production
표 Construction of plasmids expressing the phaC1437 gene and newly screened propionyl-CoA transferase
표 Bacterial strains and plasmids used in this study
표 Metabolic pathways for P(3HB-co-LA) biosynthesis in recombinant E. coli XL1-Blue expressing substituted propionyl-CoA transferase and newly screened propionyl-CoA transferase developed in this study. Lactyl-CoA and 3HB-CoA are utilized by PHA synthase for the polymerization to produce P(3HB-co-LA)
표 Biosynthesis of P(3HB) and P(3HB-co-LA) by flask cultures of recombinant E. coli XL1-Blue
표 Metabolic pathways for P(3HB) biosynthesis in recombinant E. coli from sucrose
표 Strains and plasmids used in P(3HB) synthesis from sucorse
표 PHA content and DCW of E. coli strains
표 PHA content and DCW of E. coli strains from batch-fermentation
표 Strains and plasmids used in P(3HB-co-LA) synthesis from sucrose
표 Metabolic pathways for the production of P(3HB-co-LA) from sucrose
표 Results of flask cultures of recombinant E. coli strains for P(3HB-co-LA) biosynthesis from sucrose
표 Results of batch cultures of recombinant E.coli XL1-Blue for P(3HB-co-LA) biosynthesis from sucrose (Filled diamond: Dry cell weight, Open square: 3HB concentration, Open column: P(3HB-co-LA) content. Closed column : LA content
표 PHA synthase mutant의 특징
표 Comparison of FACS signal in the cells expressing the engineeredd-PLA synthases (PhaC1437, PhaC1437PHBI and PhaC1437PLAI)
표 Enzymatic activity verification by in vitroassay. Two-enzyme in vitroassay system for investigating the d-PLA polymerization activity of PhaC1437 and PhaC1437PLAI. Time course of the released free CoA from d-LA-CoA by purified PHA synthases in optical enzyme assay. d-LA-CoA was synthesized from d-lactate and acetyl-CoA by purified propionyl-CoA transferase (PCT540). Blue circles, orange squares and gray triangles represent the amount of released CoA in the reaction with PhaC1437PLAI, PhaC1437 and no enzyme, respectively. Error bars represent the s.d. of experiments conducted in duplicates
표 HPLC-MS analysis of free CoA and d-LA-CoA formed by PhaC1437, PhaC1437PLAI and no enzyme reactions. The molecular weights of free CoA and d-LA-CoA were 765.9 g mol-1and 837.9 g mol-1, respectively
표 Themolecular weights of d-PLA synthesized in recombinant E. coliXB15 expressing PhaC1437, PhaC1437PHBIandPhaC1437PLAI, respectively
표 Metabolic pathways for the production of P(3HB-co-LA) from sucrose
표 Time profiles of flask-cultures of recombinant R. eutropha NCIMB 11599 (pKM212-SacC) in sugarcane molasses based MR medium initially containing (A) 10 g/L of sucrose and (B) 20 g/L of sucrose for the synthesis of P(3HB)
표 iosynthesis of P(3HB) by flask-cultures of recombinant R. eutropha NCIMB 2649 (pKM212-SacC) in sugarcane molasses based MR-N medium initially containing (A) 10 g/L of sucrose and (B) 20 g/L of sucrose for the synthesis of P(3HB)
표 Time profiles of flask-cultures of (A) recombinant R. eutropha NCIMB 11599 (pKM212-SacC), (B) R. eutropha 437-540 (pKM212-SacC), and (C) R. eutropha 437-540 (pKM212-EcLdhA-SacC) in sugarcane molasses based MR medium initially containing 5 g/L of sucrose for the synthesis of P(3HB). During the cultures, an appropriate amount of molasses solution was supplemented to the broth at every 24, 48, and 72h
표 Biosynthesis of P(3HB) and P(3HB-co-LA) by flask-cultures of recombinant R. eutropha NCIMB 2649 (pKM212-SacC), R. eutropha 437-540 (pKM212-SacC), and R. eutropha 437-540 (pKM212-EcLdhA-SacC) in sugarcane molasses based MR-N medium initially containing 5 g/L of sucrose. During the cultures, an appropriate amount of molasses solution was supplemented to the broth at every 24, 48, and 72 h
표 Composition of Sugarcane molasses
표 Biosynthesis of P(3HB) and P(3HB-co-LA) by flask-cultures of recombinant R. eutropha NCIMB 2649 (pKM212-SacC), R. eutropha 437-540 (pKM212-SacC), and R. eutropha 437-540 (pKM212-EcLdhA-SacC) in charcoal-treated sugarcane molasses based MR-N medium initially containing 20 g/L of sucrose
표 Time profiles of flask-cultures of (A) recombinant R. eutropha NCIMB 11599 (pKM212-SacC), (B) R. eutropha 437-540 (pKM212-SacC) and (C) R. eutropha 437-540 (pKM212-EcLdhA-SacC) in charcoal-treated sugarcane molasses based MR medium initially containing 20 g/L of sucrose for the synthesis of P(3HB)
표 Time profiles of batch fermentation of (A) recombinant R. eutropha NCIMB 11599 (pKM212-SacC), (B) R. eutropha 437-540 (pKM212-SacC), and (C) R. eutropha 437-540 (pKM212-EcLdhA-SacC) in charcoal-treated sugarcane molasses based MR-N medium initially containing 20 g/L of sucrose for the synthesis of P(3HB)
표 PduP 기반 PHA 생합성 대사경로
표 PduP 기반 모노머 합성 카세트 플라스미드
표 PduP 기반 PHA 생산 플라스크 배양 결과
표 Ethylene glycol로부터 PLGA 생산 대사경로
표 Ethylene glycol로부터 PGA 생산 대사경로
표 96시간 glycolate 생산 플라스크 배양 time profile
표 EG 이용 PLGA 생산 재조합 E. coli 플라스크 배양 결과
표 EG 이용 PLGA 생산 결과
표 5HV-containing PHA 생산 대사경로
표 본 연구에서 사용된 균주 및 플라스미드
표 5HV feeding을 통한 P(5HV) 생산 결과
표 5HV feeding을 통한 P(3HB-co-5HV) 생산 결과

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시스템대사공학을 이용한 Poly(lactic acid) 공중합체 폴리에스터 합성 재조합 미생물 플랫폼 시스템 개발
Development of recombinant bacterial platform system for the synthesis of poly(lactic acid)[PLA] copolymer polyesters based on systems biotechnology 원문보기

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시스템대사공학을 이용한 Poly(lactic acid) 공중합체 폴리에스터 합성 재조합 미생물 플랫폼 시스템 개발 Development of recombinant bacterial platform system for the synthesis of poly(lactic acid)[PLA] copolymer polyesters based on systems biotechnology 원문보기

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시스템대사공학을 이용한 Poly(lactic acid) 공중합체 폴리에스터 합성 재조합 미생물 플랫폼 시스템 개발
Development of recombinant bacterial platform system for the synthesis of poly(lactic acid)[PLA] copolymer polyesters based on systems biotechnology 원문보기

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