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Development of Escherichia coli Platform for Tyrosine-derivative Production Using Aromatic Amino Acid Hydroxylases
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| Title: | Development of Escherichia coli Platform for Tyrosine-derivative Production Using Aromatic Amino Acid Hydroxylases |
| Other Titles: | 芳香族アミノ酸水酸化酵素を用いたチロシン関連化合物生産のための大腸菌の構築 |
| Authors: | Sheng, Ning Browse this author |
| Issue Date: | 25-Mar-2024 |
| Publisher: | Hokkaido University |
| Abstract: | Aromatic compounds derived from tyrosine (Tyr) are important and diverse chemicals used for industrial and commercial applications. Although these compounds can be obtained by extraction from natural producers such as plants, their growth is slow and their content is low. To overcome these problems, many of them have been chemically synthesized from petroleum-based feedstocks. However, because of the environmental burden and depleting availability of feedstock, microbial cell factories are attracting much attention as sustainable and environmentally friendly processes. To develop microbial cell factories for Tyr and Tyr-derivative production, I constructed simple and convenient Tyr-producing Escherichia coli (E. coli) platforms with phenylalanine (Phe) hydroxylase (PheH), which converted Phe to Tyr with O2 and tetrahydromonapterin (MH4) as a cofactor, by engineering their genes with plasmid-based and chromosome-integrated methods. For effective Tyr production from Phe, a MH4 regeneration system consist of reduction of the oxidized form of the cofactor, quinonoid dihydromonapterin, with NADH by pterin-4α-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR), was also constructed. The Tyr titer of the plasmid-based E. coli platform expressing the genes encoding PheH, PCD, and DHPR was 25.5 mM (4.63 g/L) in a medium containing 30.3 mM (5.00 g/L) Phe with a test tube. The strain was successfully used to produce a industrially attractive compound, tyrosol, with a yield of 11.5 mM (1.58 g/L) by installing additional Tyrdecarboxylase (TDC) and tyramine oxidase (TYO) genes on a plasmid. Chromosomal engineering of E. coli has an advantage over the use of plasmids because it increases genetic stability without antibiotic feeding to the culture media and relieves the metabolic burden associated with plasmid maintenance. Therefore, I constructed a Tyr-producing platform strain by integration of multiple T7 promoter- controlled genes encoding PheH1, PCD, and SKIK-tagged DHPR on different chromosome loci (strainY3). The strain produced 28.6 ± 1.1 mM Tyr (5.19 g/L; 94.4% conversion from Phe). Then, I developed a tyrosol-producing platform from strain Y3 by expressing selected tyrosine decarboxylase-, tyramine oxidase (TYO)-, and medium-chain dehydrogenase/reductase (YahK)-encoding genes, all of which were controlled by T7 promoter and integrated into the chromosome. However, the strain produced a melanin-like pigment as a byproduct, which is suggested to be formed from 4-hydroxyphenylacetaldehyde (a TYO product/YahK substrate). By using a culture medium containing a high concentration of glycerol, which was reported to enhance NADH supply required for YahK activity, the final titer of tyrosol reached 2.42 g/L in test tube-scale cultivation with a concomitant decrease in the amount of pigment. In conclusion, I developed E. coli platforms for production of Tyr and Tyr-related compounds from Phe at multi-gram-per-liter levels in test-tube cultivation by plasmid- based and chromosome-engineered methods. The platforms would be useful for production of Tyr-derivatives at multi-grams-per-liter levels in test tubes. |
| Conffering University: | 北海道大学 |
| Degree Report Number: | 甲第15876号 |
| Degree Level: | 博士 |
| Degree Discipline: | 工学 |
| Examination Committee Members: | (主査) 教授 松本 謙一郎, 教授 渡慶次 学, 教授 大利 徹, 准教授 南 篤志, 助教 佐藤 康治 |
| Degree Affiliation: | 総合化学院（総合化学専攻） |
| Type: | theses (doctoral) |
| URI: | http://hdl.handle.net/2115/92147 |
| Appears in Collections: | 学位論文 (Theses) > 博士 （工学）
課程博士 (Doctorate by way of Advanced Course) > 総合化学院(Graduate School of Chemical Sciences and Engineering)
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