Synechococcus genetic engineering bacteria for biosynthesizing caryophyllene, and construction method and application thereof
A technology of genetically engineered bacteria and biosynthesis, which is applied in the fields of genetic engineering, biochemical equipment and methods, plant genetic improvement, etc., and can solve the problems that the output cannot meet the industrial demand.
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Embodiment 1
[0055] Construct the basic vectors pSI-SPE, pSII-CM and pSIII-KAN respectively
[0056] The genome of Synechococcus sp. UTEX 2973 was extracted using the bacterial genome extraction kit as a template, and the sequences shown in SEQ ID NO.19 and SEQ ID NO.20 were used as upstream and downstream primers for PCR amplification to obtain the upstream homology arms of the neutral site NSI Gene (SEQ ID NO.3); with the sequences shown in SEQ ID NO.21 and SEQ ID NO.22 as the upper and lower primers, PCR amplification was performed to obtain the homology arm gene (SEQ ID NO. 4); with the sequences shown in SEQ ID NO.23 and SEQ ID NO.24 as the upper and lower primers, carry out PCR amplification to obtain the homology arm gene (SEQ ID NO.5) upstream of the neutral site NSII; with SEQ ID The sequences shown in NO.25 and SEQ ID NO.26 are the upper and lower primers, and PCR amplification is carried out to obtain the homology arm gene (SEQ ID NO.6) downstream of the neutral site NSII; with ...
Embodiment 2
[0068] Construct vector pSI-ispA-gpps-idi1 sc Using the basic vector pSI-SPE, using SEQ ID NO.41 and SEQ ID NO.42 as the upstream and downstream primers of the pSI-SPE plasmid backbone, respectively, PCR amplification was performed to obtain promoter-free P CPC560pSI-SPE plasmid backbone.
[0069] Synechococcus UTEX 2973 genome was used as a template, and SEQ ID NO.43 and SEQ ID NO.44 were used as P psba1 The upstream and downstream primers of the promoter were used for PCR amplification to obtain P psba1 Promoter (SEQ ID NO. 18).
[0070] The Escherichia coli MG1655 genome extracted by the bacterial genome extraction kit was used as a template, and SEQ ID NO.45 and SEQ ID NO.46 were respectively used as the upstream and downstream primers of the geranyl pyrophosphate / farnesyl pyrophosphate synthase gene ispA, Perform PCR amplification to obtain the gene ispA (SEQ ID NO.14).
[0071] The codon-optimized geranyl pyrophosphate gene gpps connected to the universal vector pUC5...
Embodiment 3
[0078] Construction of vector pSII-idi1 sc The basic vector pSII-CM was used, and SEQ ID NO.53 and SEQ ID NO.54 were respectively used as the upstream and downstream primers of the pSII-CM plasmid backbone for PCR amplification to obtain the pSII-CM plasmid backbone.
[0079] The Saccharomyces cerevisiae BY4741 genome extracted by the yeast genome extraction kit was used as a template, and SEQ ID NO.51 and SEQ ID NO.52 were respectively used as the upstream and downstream primers of the isopentenyl diphosphate δ-isomerase gene idi1 for PCR amplification. The gene idi1 (SEQ ID NO.15) was obtained.
[0080] The pSII-CM plasmid backbone and gene idi1 were assembled using the assembly kit to obtain the vector construction vector pSII-idi1 sc .
[0081] Dilute the obtained PCR products according to the ratio of 10ng / 1000bp / μl, take 1μl of each PCR product, 1.5μl of sterile water, 1μl of 5×CE MultiS Buffer, MultiS 0.5μl, react at 37°C for 30min. After the reaction was completed...
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