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Metabolic engineering of Escherichia coli for production of natural sweeteners, diterpenoid steviol glycosides

초록/요약

The increasing cost of energy and raw materials for building complex chemical structures, combined with increasing concerns about limited fossil fuels and global environmental problems, suggest that biosynthesis using engineered microbial cells will become a sustainable process for obtaining valuable chemicals [1, 2]. Together with "-omics" approaches, many efforts aimed at a systematic analysis of host microorganisms have been made to design optimized pathways into heterologous hosts. Ultimately, the goal is to construct microbial cell factories for the biosynthesis of SGs as sweeteners and to deliver a mature platform for large-scale commercial fermentation. ent-Kaurene is a dedicated precursor pool that is responsible for synthesizing natural sweeteners such as steviol glycosides. As described in Chapter 2, two ent-kaurene genes encoding ent-copalyl pyrophosphate synthase (CPPS) and ent-kaurene synthase (KS) from Stevia rebaudiana were modularly constructed and expressed for ent-kaurene production in Escherichia coli. To enhance ent-kaurene production in E. coli, six geranylgeranyl pyrophosphate synthases (GGPPS) from various microorganisms and eight E. coli strains were compared by measuring ent-kaurene production. GGPPS from Rhodobacter sphaeroides produced the highest concentration of ent-kaurene, approximately 41 mg/L, in E. coli strain MG1655 expressing CPPS and KS. The ent-kaurene production was further increased to 106.6 mg/L by overexpressing farnesyl pyrophosphate (FPP) synthase (IspA) and isopentenyl diphosphate (IPP) isomerase (idi) from E. coli. Finally, the highest titer of ent-kaurene (492.8 mg/L) with a specific yield of ent-kaurene of 143.5 mg/g dry cell weight was obtained by culturing E. coli strain MG1655 coexpressing the ent-kaurene module, IDI, and IspA in a 1-L bioreactor containing 20 g/L glycerol. Microbial production of the diterpenoid sweetener steviol glycoside (SG) has a potentially high commercial value in the development of healthy sweeteners [3]. As described Chapter 3, S. rebaudiana derived native UGTx having the of second uridine diphosphate (UDP)-dependent glycosyltransferase (UGT) were obtained. A combinatorial steviol glycoside synthetic pathway consisting of three expression modules was introduced into E. coli MG1655. One module in pUCM-idi-ispA is the precursor pathway consisting of idi from E. coli and ispA from E. coli. Another module in pSTV29-CPPSSR-KSSR-GGPPSRS-KO*SR-KAHSR is the steviol synthetic pathway consisting of GGPPSRS from R. sphaeroides and CPPSSR, KSSR, KO*SR, and KAHSR from S. rebaudiana. The other module in pBBR-74G1SR-85C2SR-UGTxSR contains the glycosylation pathway genes UGT85C2SR, UGTxSR, and UGT74G1SR from S. rebaudiana. Furthermore, to increase stevioside production, I constructed deletions of ushA, pfkA, pfkB, and pgi from wild-type E. coli to increase the UDP-glucose and NADPH levels. A complete biosynthetic pathway for SGs was modularly assembled and engineered, and SGs were successfully produced in recombinant E. coli (mutant strain M8, MG1655 △ushA △pfkA △pfkB) . The most valuable component, stevioside, reached 32.5 mg/L, which is the highest yield of SG biosynthesis in microorganisms. Finally, the highest stevioside (41.7 mg/L) concentration was achieved in a culture of E. coli strain M8 coexpressing the stevioside synthetic pathway in a 1-L bioreactor containing 20 g/L glucose. This dissertation shows that diverse engineering approaches can be applied for the high-level production of the natural sweetener, steviol glycoside, using E. coli as a microbial host.

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목차

LIST OF TABLES…………...……………………...........................…..….……....v
LIST OF FIGURES……………………...……..…………………….……..……..vi
ABSTRACT……………………………………………………………………..…ix

Chapter 1. General Introduction……………………………………………….....1

1.1 Metabolic engineering of microbial cell factories for biosynthesis of terpenoids…………………………………………………………………….....…2
1.2 Isoprenoids biosynthetic pathway………..……….……………..……………6
1.3 Steviol glycosides………………………………...………………….…….…10
1.4 Aims of This study …………..………………………….……………………14

Chapter 2. Metabolic engineering of the Stevia rebaudiana ent-kaurene biosynthetic pathway in recombinant Escherichia coli……………………………………………….………..………..16

2.1 Abstract……………………………………………………….…………...…17
2.2 Introduction………………..…………………..……………………………..18
2.3 Materials and Methods………………………………………………………..22
2.3.1. Bacterial strains and culture conditions..………………………………….22
2.3.2. Cloning of genes encoding CPPS and KS from S. rebaudiana…………....22
2.3.3. Expression and purification of CPPS and KS from S. rebaudiana
in E. coli…………………………………………………………………………23
2.3.4. Activity assay of recombinant CPPS, KS, and GGPPS…………………...23
2.3.5. Construction of synthetic expression modules…………………………....24
2.3.6. Analysis of ent-kaurene by GC-MS………………………………….…25
2.3.7. Comparison of proteome in MG1655 and JM109 by 2-DE ……………....25
2.3.8. Bioreactor study ………………………………………………………….27
2.3.9. Measurement of cell growth………..……………………………………27
2.4 Results…………………………….………………………………………….30
2.4.1. Expression and in vitro activity assay of ent-kaurene pathway enzymes, CPPS and KS in E. coli…………...…….…………………….……………….…30
2.4.2. Comparison of various GGPPSs for ent-kaurene production in
E. coli……………………………………………………………………………34
2.4.3. Comparison of ent-kaurene production among various E. coli strain.…..…36
2.4.4. Effect of isoprenoid precursors on ent-kaurene production…………….…41
2.4.5. The ent-kaurene production in a bioreactor…………….…………………43
2.5 Discussion………………………….…………………………………….…..46



Chapter 3. Metabolic engineering of the Stevia rebaudiana stevioside biosynthetic pathway using a new glucosyltransferase in recombinant Escherichia coli……………………………….…...….51

3.1. Abstract…………………………………………..…………………….……52
3.2. Introduction…………….………………………………..………………..…54
3.3. Materials and Methods……………………………………………………….59
3.3.1. Plant materials, RNA isolation and reverse transcription…..….………..…59
3.3.2. Bacterial strains and plasmids……………..…………………..….………59
3.3.3. Media and culture conditions…………………………………..….………60
3.3.4. Phylogenetic tree and In vitro analysis of UGTx…..…………..….………61
3.3.5. Product extraction and analytical methods…….…..…………..….………61
3.3.6. Construction of knockout strains…………….…..…………..….……...…63
3.3.7. Measurements of the NADP and NADPH concentrations during exponential
growth phase.……………………………………………………………………64
3.3.8. Bioreactor study.………………………….…..…………..….………64
3.4. Results …………..…………..……….…………………………………...…72
3.4.1. Confimation of KO from S. rebaudiana.…………………………….……72
3.4.2. Construction of knockout strains..…………………………….……….…74
3.4.3. Production of ent-kaurenoic acid in knockout strains..…………….……78
3.4.4. Functional characterization of UGTx.…………………………...….……81
3.4.5. Biosynthesis of SGs and pathway optimization in knockout E. coli………85
3.4.6. Bioreactor study..…………………………...……………………….……88
3.5. Discussion …………..………..……….………………………………..……90

4. Conclusion……………….……………………………………………………93
5. References……………………...……………….………………………………94
ABSTRACT IN KOREAN………………………………..…….……………….103

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