[보고서]3-hydroxypropionic acid 생산을 위한 미생물 전환공정 연구

박성훈

3-hydroxypropionic acid 생산을 위한 미생물 전환공정 연구
Development of microbial conversion process for production of 3-hydroxypropionic acid 원문보기

보고서 정보
주관연구기관	부산대학교 Busan National University
연구책임자	박성훈
참여연구자	설은희 , 아속소마순다 , 고연주 , 김창만 , 리앙윤시아오 , 무게쉬.S , 순달아세칼바라지 , 아이날라 사티쉬 쿠마르 , 응웬티찐 , 응웬화이남 , 저우성팡
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2015-05
과제시작연도	2014
주관부처	미래창조과학부 Ministry of Science, ICT and Future Planning
등록번호	TRKO201800009244
과제고유번호	1711015348
사업명	글로벌프론티어사업
DB 구축일자	2018-05-26
키워드	대장균.비타민 B12 생합성.대사공학.합성생물학.3-HP.Escherichia coli.metabolic engineering.synthetic biotechnology.Pseudomonas denitrificans.Vitamin B12 biosynthesis.3-HP.
DOI	https://doi.org/10.23000/TRKO201800009244

초록 ▼

⦁ 본 연구의 목표는 중요한 플랫폼 화합물인 3-hydroxypropionic acid (3-HP)의 생산을 위한 원천 기술, 즉 유전자, 효소, 발현 시스템, 생산균주, 공정 등을 개발하는 것이었음.
⦁ 이를 위하여 대장균, Pseudomonas denitrificans 등을 대상으로 유전체, 전사체, 대사체 등을 분석하고 글리세롤 대사가 재설계된 3-HP 생산 균주를 개발하였고, promoter engineering 등 합성생물학 기법을 이용하여 글리세롤의 3-HP 전환 경로 및 비타민 B12 생합성이 효율적으로 재설계된 3-HP 생산 균주를 개발하였음.
⦁ 또한 시스템 수준의 정보와 합성생물학 기법을 바탕으로 고농도 3-HP에 대해 내성(acid tolerance)을 갖는 균주를 개발하고, 더 나아가 글리세롤 대비 높은 생성물 수율과 고농도 배양이 가능한 균주와 공정을 개발함으로써 관련 기업체에 기술을 이전하고 공동으로 상업화 연구를 수행할 수 있는 기틀을 마련하였음.
⦁ 정량적으로는 3-HP 생산 농도 60g/L, 3-HP 생산성 1.3 g/L/h, 글리세롤 대비 3-HP 생산 수율 0.5 이상을 달성하였음.

(출처 : 보고서 요약서 3p)

Abstract ▼

The objective of this study is to develop the strains and processes for production of an important platform chemical, 3-hydroxypropionic acid (3-HP) from glycerol. The study aims to synthesize, redesign and optimize 3-HP production pathway in such strains as E. coli and P. denitrificans by using genomic, transcriptomic approaches. The study also includes synthesis and optimization of biosynthetic pathway of vitamin B12, which is an important cofactor for a key enzyme glycerol dehydratase, in those strains. The study also aims to increase the resistance of these strains against to higher 3-HP concentration. Ultimate purpose of this study is to establish proper and economical biological process for the commercial production of 3-HP.

The scope of this study can be summarized as follows:
1. Development of pathway for effective production of 3-HP through genome-wide analysis of P. denitrificans
● Cloning of P. denitrificans vitamin B12 biosynthetic genes and expression in E. coli.
● Screening of 3-HP tolerance genes and expression in E. coli
● Redesigning carbon metabolic pathways and optimization of 3-HP pathway.

2. Development of strain and process for production of 3-HP
● Development of high concentration production technologies through the improved 3-HP tolerance
● Investigation of carbon and energy metabolism through transcriptome and metabolome analysis
● Development of high efficient P. denitrificans strains and evaluation of the performance

3. Optimization of strain and process for 3-HP production with high efficiency
● Increase of 3-HP production titer by improving the cultivation technology and tolerance to 3-HP
● Improvement of 3-HP production yield through the optimization of carbon and energy metabolism.
● Improvement of 3-HP productivity by applying the high-density cultivation, stabilizing and optimizing the strain and culture conditions.

This study can deliver many outcomes. Academically, this study enables us to better understand P. denitrificans metabolism at system level. It further enables us to understand many enzymes and gene expressions related with glycerol metabolism and vitamin B12. We also expect that the strains and processes developed through this study are commercially useful and the technologies can be transferred to a proper company which is interested in commercial production of 3-HP and acrylic acid. 3-HP will show a wide variety of industrial applications as a platform chemical to replace many petroleum-derived products.

(출처 : SUMMARY 7p)

목차 Contents

표지 ... 1
제 출 문 ... 2
보고서 요약서 ... 3
요 약 문 ... 4
S U M M A R Y ... 7
C O N T E N T S ... 8
목차 ... 9
제 1 장 연구개발과제의 개요 ... 10
1. 목적 ... 10
2. 필요성 ... 10
3. 범위 ... 10
제 2 장 국내외 기술개발 현황 ... 11
1. 국내외 연구현황 ... 11
2. 선행연구의 내용 및 결과 ... 12
3. 현기술상태의 취약성 ... 13
4. 앞으로의 전망 ... 14
제 3 장 연구개발수행 내용 및 결과 ... 15
제 4 장 목표달성도 및 관련분야에의 기여도 ... 133
제 5 장 연구개발결과의 활용계획 ... 135
1. 추가연구의 필요성 ... 135
2. 기업화 추진 방안 ... 136
제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 136
제 7 장 참고문헌 ... 136
끝페이지 ... 146

표/그림 (86)

표 : Structure of coenzyme B12
표 Cob operons in the chromosome of Pseudomonas denitrificans are arranged in two different gene clusters.
표 Oxygen dependent and oxygen independent coenzyme B12 biosynthetic pathway
표 : Construction of the plasmid pRcob.
표 Construction of the plasmid pAcob.
표 : Construction of the plasmid pCcob
$SDS-PAGE analysis of cell-free soluble extract of recombinant E. coli harboring pAcob, pCcob and pRcob plasmids. L1 and L2 are soluble fraction of E. coli BL21 DE3 and E. coli BL21DE3 harboring pAcob, pCcob and pRcob plasmids, respectively.$ 표 SDS-PAGE analysis of cell-free soluble extract of recombinant E. coli harboring pAcob, pCcob and pRcob plasmids. L1 and L2 are soluble fraction of E. coli BL21 DE3 and E. coli BL21DE3 harboring pAcob, pCcob and pRcob plasmids, respectively.
표 : Coenzyme B12 bioassay. Crude cell extracts of P. denitrificans, E. coli BL21 DE3 WT,recombinant E. coli BL21 DE3 overexpressing cob genes where seived through 0.2 μm filters before adding on bioassay plates containing S. typhimurium ΔmetE ΔcbiB.
표 E. coli strains used in the production of high concentrations of organic acids
표 Glycerol dehydratase and aldehyde dehydrogenase (KGSADH) activities in the crude cell extract of various recombinant strains.
표 SDS-PAGE analysis of cell-free soluble extract of various E. coli strains at different time points with DhaB, GdrAB and KGSADH overexpression.
표 Time-course profile of cell growth and pH by various E. coli strains with and without (25 g/L) 3-HP.
표 Time-course profile of cell growth, glycerol consumption and production of 3-HP and 1,3-PDO from glycerol by various recombinant E. coli strains overexpressing DhaB, GdrAB and KGSADH.
표 Time-course profile of cell growth, glycerol consumption and production of 3-HP and 1,3-PDO from glycerol by various recombinant E. coli strains overexpressing DhaB, GdrAB and KGSADH.
표 Time-course profile on glycerol consumption, cell growth, 3-HP, 1,3-PDO, 3-HPA and acetate production during glycerol fed-batch cultivation of recombinant E. coli W3110 DUBGK overexpressing DhaB, GdrAB and KGSADH.
표 Carbon distribution of glycerol metabolism by the recombinant E. coli W3110 DUBGK overexpressing DhaB, GdrAB and KGSADH, cultivated in bioreactor with 1 vvm of continuous air supply.
표 Time-course profile on glycerol consumption, cell growth, 3-HP, 1,3-PDO, 3-HPA and acetate production during glycerol fed-batch cultivation of recombinant E. coli W DUBGK overexpressing DhaB, GdrAB and KGSADH.
표 Carbon distribution of glycerol metabolism by the recombinant E. coli W DUBGK overexpressing DhaB, GdrAB and KGSADH, cultivated in bioreactor with 1 vvm of continuous air supply.
표 Apparent enzymes involved in 3-hydroxypropionic acid degradation in P. denitrificans.
표 Analysis of relative mRNA levels for 3hibdhI, 3hibdhII, 3hibdhIII and 3hibdhIV in wild type P. denitrificans ATCC13867. P. denitrificans ATCC13867 was cultured in M9 medium without (black bar) or with 3-HP supplementation (grey bar) at 30 mM and harvested in exponential growth period.
표 Enzyme activity in recombinant E. coli BL21(DE3).
표 Western blot analysis of total and cell-free soluble extracts from in vitro translation studies.
표 Enzyme activities of the in vitro translated enzymes
표 Resting cell experiments with P. denitrificans mutatnt strains.
표 Resting cell assay with P. denitrificans Δ3hpdh mutatnt strains.
표 Resting cell assay with P. denitrificans Δ3hpdhΔ3hibdhIV mutatnt strains.
표 Time course profile on cell growth and 3-HP consumption by P. denitrificans mutant strains. Mutant strains were cultivated in M9 medium supplemented with 3-HP as the sole carbon and energy source.
표 Production of 3-HP by recombinant P. denitrificans mutant strains overexpressing glycerol dehydratase.
표 Carbon distribution cultivation of recombinant P. denitrificans and deletion mutant overexpressing DhaB at 24h
표 Effect of the addition of lactic acid (A) and 3-HP (B) on the cell growth of E. coli W at pH 7.0 and 37 °C.
표 List of organic acids
표 (A) represents the specific growth rates of E. coli W as a function of the concentrations of lactic acid (▲) or 3-HP (˜) added in the medium at pH 7.0. (B) and (C) show the relative specific growth rates as a function of the concentration of undissociated lactic acid or 3-HP. (D) demonstrates the effect of temperature on the relative specific growth rate of E. coli W as 440 mM of lactic acid or 3-HP was added in the medium.
표 Effect of pH on the relative specific growth rate of E. coli W (A). The medium contained 440 mM of lactic acid (▲) or 3-HP (˜). The relative specific growth rate versus the concentration of undissociated lactic acid or 3-HP (B). The pH levels tested are denoted by grey symbols (pH 7.5), closed symbols (pH 7.0) and open symbols (pH 6.5).
표 Effect of the media on the specific growth rate as a function of the concentration of lactic acid or 3-HP at pH 7.0 and 37 °C (A and B). The relative growth rate versus the concentration of undissociated acid (C)
표 Relative specific growth rate of E. coli W as a function of the concentration of undissociated organic acids at pH 7.0 (A) and at pH 7.5 (B).
표 Glycerol metabolism in recombinant E. coli W developed for the production of 3-hydroxypropionic acid from glycerol. The pathways shown in gray were knocked-out.
표 Strains and plasmids used in this study
표 Time course profile of glycerol consumption, cell biomass, pH, 3-HP, 1,3-PDO, acetate and pyruvate production by recombinant E. coli W strains overexpressing glycerol dehydratase and aldehyde dehydrogenase.
표 Glycerol fed-batch bioreactor experiments with recombinant E. coli W mutant strains overexpressing DhaB, GdrAB and KGSADH and time course profile of DhaB and KGSADH activities from recombinant E. coli W strains.
표 Carbon distribution of E. coli W ΔpoxB DUBGK
표 Carbon distribution of E. coli W ΔpoxB ΔyqhD DUBGK
표 Bacterial strains and plasmids used in this study
표 Time course profile of E. coli W- DUBGK for its glycerol consumption, cell growth, 3-HP production and pH
표 Time course profile of E. coli W ΔglpK ΔgldA - DUBGK for its glycerol consumption, cell growth, 3-HP production and pH
표 Glycerol dehdyratase and KGSADH activities in the samples of EcW DUBGK and EcWΔ glpKΔgldA DUBGK, collected at 6 and 9 h from shake flask cultures supplemented with various concentrations of glucose.
표 Time course profile of glycerol consumption, 3-HP production and cell growth of EcW Δ glpKΔgldA DUBGK overexpressing glycerol dehydratase and KGSADH.
표 Time course profile of 3-HPA, acetate and 1,3-PDO production by recombinant EcW Δ glpKΔgldA DUBGK overexpressing glycerol dehydratase and KGSADH
표 Glycerol dehydratase and KGSADH activities and expression profile in the samples of EcW ΔglpKΔgldA DUBGK collected at various time intervals from glycerol and glucose fed batch bioreactor.
표 pUCPK’/PC3-dhaB-gdrAB, PC4-KGSADH plasmid construct to express glycerol dehydratase and KGSADH in P. denitrificans
표 Time course profile of glucose & glycerol consumption, cell growth, 3-HP production, pH and 1,3-PDO production by Pd Δ3hpdhΔ3hibdhIV (pUCPK’/PC3-dhaB-gdrAB, PC4-KGSADH) cultivated with and without coenzyme B12.
표 Specific enzyme activity of glycerol dehydratase and KGSADH in crude cell extracts of Pd Δ3hpdhΔ3hibdhIV pUCPK’/PC3-dhaB-gdrAB, PC4-KGSADH) collected at various time points.
표 Carbon distribution in recombinant Pd Δ3hpdhΔ3hibdhIV (pUCPK’/PC3-dhaB-gdrAB, PC4-KGSADH) overexpressing glycerol dehydratase and KGSADH
표 Specific enzyme activity of glycerol dehydratase (DhaB) and KGSADH measured in the samples collected from various time intervals (6, 12, 21, 33, 39, 48, 60 h) of bioreactor A and B.
표 Venn diagram representing single nucleotide mutations identified in glycerol assimilatory genes of E. coli W and BL21 (DE3) ΔldhAΔpta-ackΔyqhDΔpoxBΔfrdA
표 Summary of nucleotide changes in glycerol assimilatory genes of various E. coli strains adapted in the presence of 3-HP.
표 Bacteria strains used in this study
표 Growing cells 3-HP assimilation (A) and resting cell 3-HP degradation (B) by the P. denitrificans strain.
표 A scheme showing 3-HP metabolic pathways. In P. denitrificans, the oxidative pathway, not the reductive pathway is responsible for 3-HP assimilation to cellular constituents.
표 Relative mRNA levels for the 3-hydroxypropinate catabolism genes in wild type P. denitrificans ATCC13867. P. denitrificans cells were cultivated in M9 medium supplemented with 3-HP at 25 mmol/L (grey bar) or without 3-HP (black bar) and harvested during the exponential growth period.
표 Genetic arrangement of 3-HP catabolism genes in P. denitrificans. 3-HP catabolic genes (3hpdh, 3hibdhIV and mmsadh) are divergently located from lysR family transcription regulatory gene. -35 and -10 consensus sequences are in bold. A hypothetical scheme on a typical structural of a divergent promoter regulated by a LysR family transcriptional regulator is illustrated
표 A proposed schematic model of LysR family transcriptional regulator dependent transcription regulation.
표 Organization of gene clusters involved in 3-HP degradation pathway among various microorganisms in comparison with Pseudomonas denitrificans ATCC 13861. (A) 3-HIBDH, (B) 3-HPDH
표 Comparison of enzyme (C4-LysR, MMSADH and 3HIBDH) sequence similarity among different organisms
표 Comparison of enzyme (C3-LysR, and 3HPDH) sequence similarity among different organisms
표 Multiple sequence alignment of the N-terminal HTH containing region of LysR’s found in various microorganisms. Amino acid residues highlighted in yellow are well conserved in all microorganisms.
표 Docking poses and protein-ligand interactions of LysR’s with 3-HP. (A) 3-HIBDH, (B) 3-HPDH
표 Resting cell degradation of 3-HP in various microorganisms.
표 pUCPK’/PC3-gdrAB-dhaB, PC4-KGSADH plasmid construct to express glycerol dehydratase and KGSADH in P. denitrificans
표 Time course profile of glucose (Fig. 1-O2 (Fig. 1-O3 & S3), 12 μmol/L coenzyme B12 was added to the culture medium. 100mM of glycerol was added at 3h of cultivation.
표 Specific enzyme activity of glycerol dehydratase and KGSADH in crude cell extracts of Pd Δ3hpdhΔ3hibdhIV (pUCPK’/PC3-dhaB-gdrAB, PC4-KGSADH) and Pd Δ3hpdhΔ3hibdhI( pUCPK’/ PC3- gdrAB-dhaB, PC4-KGSADH)collected at various time points.
표 Time course profile of glycerol, glucose consumption, biomass, and 3-HP production in fed-batch bioreactor studies with recombinant Pd Δ3hpdhΔ3hibdhIVΔ3hibdhI (pUCPK’/PC3-dhaB-gdrAB, PC4-KGSADH) (A) and Pd Δ3hpdhΔ3hibdhIVΔ3hibdhI (pUCPK’/PC3-gdrAB-dhaB, PC4-KGSADH) (B) overexpressing glycerol dehydratase and KGSADH.
표 Effect of aeration on 3-HP Production
표 Activities of DhaB (●) and KGSADH (○)
표 NAD+ (dark gray) and NADH (white) levels at 3 hour
표 Effect of carbon sources on 3-HP production
표 Activities of DhaB (●) and KGSADH (○)
표 Changes in NAD+, NADH and total NAD(H) for glycerol (▼), glucose (▲), glutamate
표 Effect of other amino acids on 3-HP production: (A) lysine, (B) valine and (C) casamino acid: Glycerol (▼), 3-HP (●), OD (○), and pH (×).
표 Enzyme activities and the amount of NAD+ and NADH at 3 hour
표 Comparison of cell viability at 10 hour.
표 Cell viability at 10 hour.
표 Effect of Nox: (A) E. coli W2, (B) E. coli W20, (C) E. coli W20 + glutamate: Glycerol (▼), 3-HP (●), and OD (○).
표 Enzyme activities
표 Expressed DhaB, KGSADH, and Nox on SDS-PAGE: The strains were the control (E.coli W Δglpk ΔgldA), W1, W2, and W20 (without Nox overexpression).
표 Effect of 3-HP in the presence of glutamate on 3-HP production. Concentration of 3-HP
표 Enzyme activities at 3 hour

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연합인증

3-hydroxypropionic acid 생산을 위한 미생물 전환공정 연구
Development of microbial conversion process for production of 3-hydroxypropionic acid 원문보기