Recombinant microorganism capable of efficiently utilizing methanol and application thereof
A technology of recombinant microorganisms and methanol dehydrogenase, applied in the fields of genetic engineering and biological fermentation, can solve the problems of grain competition and price competition, and achieve the effect of increasing biomass, improving biological efficiency and broad application prospects.
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Embodiment 1
[0024] Embodiment 1 introduces methanol conversion pathway in Escherichia coli
[0025] In this example, the chassis strain X1 / mdh-das capable of utilizing methanol was obtained by introducing the plasmid pTrc99a-mdh-das into Escherichia coli W3110 (purchased from China Industrial Microorganism Culture Collection Center).
[0026] The construction method of plasmid pTrc99a-mdh-das: artificially synthesize mdh and das genes, the gene sequences of which are shown in SEQ ID No: 1 and SEQ ID No: 2, respectively. The mdh and das genes were inserted into the EcoRI / SmaI restriction site of plasmid pTrc99a (purchased from addgene) by Gibson assembly method to obtain plasmid pTrc99a-mdh-das.
[0027] The construction method of Escherichia coli X1 / mdh-das: The plasmid pTrc99a-mdh-das was electrotransformed into Escherichia coli W3110, and the resistant strain was obtained on the LB plate containing 100mg / L ampicillin, named X1 / mdh-das.
[0028] Escherichia coli X1 / mdh-das was cultured ...
Embodiment 2
[0029] Example 2 Further improving methanol utilization by transforming the metabolism of Escherichia coli itself
[0030] Methanol utilization pathways are limited by the original intracellular formaldehyde dissimilation pathway, most of which are converted to carbon dioxide instead of entering central carbon metabolism. In this embodiment, the formaldehyde dissimilation gene frmAB is firstly knocked out by Red recombination technology (the nucleic acid sequence of the frmA gene is shown in SEQ ID No: 3, and the nucleic acid sequence of the frmB gene is shown in SEQ ID No: 4), which can improve the integration of methanol into the central metabolic cycle Proportion. In addition, by knocking out the 6-phosphofructokinase gene pfkAB (the nucleic acid sequence of the pfkA gene is shown in SEQ ID No: 5, and the nucleic acid sequence of the pfkB gene is shown in SEQ ID No: 6) and the α-ketoglutarate dehydrogenase gene sucA (The nucleic acid sequence of the sucA gene is shown in S...
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