Exploring the capacity of bacteria for natural product biosynthesis
Başlık çevirisi mevcut değil.
- Tez No: 597743
- Danışmanlar: PROF. JIXUN ZHAN
- Tez Türü: Doktora
- Konular: Biyomühendislik, Biyoloji, Bioengineering, Biology
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 2019
- Dil: İngilizce
- Üniversite: Utah State Unıversıty
- Enstitü: Yurtdışı Enstitü
- Ana Bilim Dalı: Belirtilmemiş.
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: Belirtilmemiş.
Özet
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Özet (Çeviri)
Bacteria are one of the richest sources of pharmaceutically relevant compounds. Nevertheless, the production level of those important compounds in the original hosts is often quite low, and sometimes it is so low that we cannot detect them with current analytical techniques. Researchers have developed various solutions, such as heterologous expression, co-cultivation of different bacteria, optimization of fermentation conditions, discovery of new species, engineering of biosynthetic enzymes, and manipulating regulatory elements. The main goal of my doctoral dissertation research is to engineer natural product biosynthetic pathways for the generation of novel compounds and enhanced production of known medicinally valuable compounds. I first investigated the glycosylation steps in the biosynthesis of two antifungal angucyclines, Sch47554 and Sch47555 using two different strategies. The heterologous co-expression of the aglycone and sugar biosynthetic genes with schS7 in Streptomyces lividans K4 led to the production of a novel C-glycosylated rabelomycin derivative. Gene inactivation of schS9 and schS10 revealed that subsequent glycosylation steps take place in a sequential manner in which SchS9 first attaches either a L-aculose or L-amicetose moiety to the 4'-OH of the C-glycosylated aglycone, then SchS10 transfers a L-aculose moiety to the 3-OH of the angucycline core. In this research, I isolated two novel angucycline derivatives and gained new insights into the glycosylation steps in the biosynthesis of Sch47554 and Sch47555. Next, I engineered the regulatory elements in Streptomyces sp. SCC-2136 through overexpression and targeted gene disruption approaches for enhanced production of pharmaceutically important angucyclines. Gene disruption of schA4 and schA16 led to a significant increase in the titer of Sch47554, while the titer was dramatically decreased in Streptomyces sp. SCC-2136/ΔschA21. Overexpression strains produced consistent results with the gene disruption strains. The highest titer of Sch47554 was achieved in Streptomyces sp. SCC-2136/ΔschA4 (27.94 mg/L), which is significantly higher than the wild type. Further, I confirmed the functions of these three regulatory elements. SchA4 and SchA16 are repressors, while SchA21 acts as an activator. Last, I isolated a carotenoid-producing endophytic bacterium from the leaves of Taxus chinensis, which was identified as Pseudomonas sp. 102515 based on the 16S rRNA gene sequence. Analysis of its secondary metabolites revealed that this endophytic strain produces a major product zeaxanthin diglucoside, a promising antioxidant natural product that belongs to the family of carotenoids. Based on the genome of a closely related Pseudomonas strain, I amplified a complete carotenoid (Pscrt) biosynthetic gene cluster from Pseudomonas sp. 102515. The functions of PsCrtI and PsCrtY in the biosynthesis of zeaxanthin diglucoside were characterized as phytoene desaturase and lycopene cyclase, respectively. The entire Pscrt biosynthetic gene cluster was successfully reconstituted in E. coli BL21(DE3) and Pseudomonas putida KT2440. The engineered strain of P. putida KT2440 produced zeaxanthin diglucoside at 144 ± 4 mg/L in SOC medium supplemented with 0.5% glycerol at 23 C, while the titer of zeaxanthin diglucoside in E. coli BL21(DE3) was very low. The production of zeaxanthin diglucoside in Pseudomonas sp. 102515 was improved through the optimization of fermentation conditions such as medium, cultivation temperature and growth time. The highest titer under the optimized conditions reached 206 ± 6 mg/L. To further enhance the production, I introduced an expression plasmid that harbors the Pscrt biosynthetic gene cluster into Pseudomonas sp. 102515, yielding an efficient producing strain of zeaxanthin diglucoside. The titer in this engineered strain reached 380 ± 12 mg/L, which is 85% higher than the wild type.
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