Butanol synthase and method for producing butanol
A technology of supplemental alcohol and enzyme activity, applied in the directions of biochemical equipment and methods, botanical equipment and methods, enzymes, etc., can solve problems such as cost-effectiveness and complexity
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Embodiment 1
[0146] Transcriptome sequencing of pepperberry and caprylic anise plant material and leaves
[0147] Bitter anise and pepperberry plants were obtained from Bluebell Nursery (Leicestershire, UK). To analyze the composition of the terpene molecules, the leaves were harvested and subjected to solvent extraction using MTBE (methyl tertiary butyl ether). Extracts were analyzed by GCMS using an Agilent 6890 Series GC system connected to an Agilent 5975 mass detector. The GC was equipped with a 0.25mm id x 30m DB-1ms capillary column (Agilent). The carrier gas was He at a constant flow rate of 1 mL / min. The initial oven temperature was 50 °C (hold for 1 min), followed by a gradient of 10 °C / min to 300 °C. Injection was performed in a split / splitless injector set at 260°C and in splitless mode. Product identification is based on comparison of mass spectra and retention indices using authentic standards and an in-house mass spectral database. The leaves of both plants contained ...
Embodiment 2
[0149] Example 2. Functional expression and characterization of DlTps589 from pepperberry
[0150] The DNA sequence of the selected sesquiterpene synthase DlTps589 was codon optimized, synthesized in vitro and cloned in pJ444-SR expression material (DNA2.0, Menlo Park, CA, USA).
[0151] Heterologous expression of DlTps589 synthase was performed in KRX E. coli cells (Promega). A single colony of cells transformed with the pJ444SR-DlTps589 expression material was used to grow 5 ml of LB medium. After 5 to 6 hours of incubation at 37°C, the culture was transferred to a 20°C incubator and left for 1 hour to equilibrate. Then, expression of the protein was induced by adding 1 mM IPTG and 0.2% L-rhamnose, and the culture was incubated overnight at 20°C. The next day, cells were harvested by centrifugation, resuspended in 0.1 volume of 50 mM MOPSO pH 7, 10% glycerol and lysed by sonication. Extracts were clarified by centrifugation (30 min at 20,000 g) and the supernatant conta...
Embodiment 3
[0153] Example 3. In vivo production of (-)-butanol in E. coli cells using DlTps589
[0154]To evaluate the in vivo production of (-)-butanol in heterologous cells, E. coli cells were transformed with the pJ444SR-DlTps589 expression plasmid and the production of sesquiterpenes from the endogenous FPP pool was evaluated. To increase the productivity of the cells, a heterologous FPP synthase and enzymes from the intact heterologous mevalonate (MVA) pathway were also expressed in the same cells. The construction of expression plasmids containing the FPP synthase gene and genes for the complete MVA pathway is described in patent WO2013064411 or in Schalk et al (2013) J. Am. Chem. Soc. 134, 18900-18903. Briefly, expression plasmids containing two operons consisting of genes encoding enzymes for the complete mevalonate pathway were prepared. Synthesized in vitro (DNA2.0, Menlo Park, CA, USA) by Escherichia coli acetoacetyl-CoA thiolase (atoB), Staphylococcus aureus HMG-CoA syntha...
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