A method for producing acetoin by microbial fermentation
A technology of acetoin and inoculum, which is applied in the field of bioengineering and can solve problems such as environmental pollution by resistance genes
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Embodiment 1
[0018] Example 1: Knockout of acuB gene
[0019] From the B. subtilis168 genome sequence published on the NCBI website, the sequence position of acuB was determined, and a gene fragment of 800 bp in size before and after acuB was downloaded as the upstream and downstream homology arms. Using the B. subtilis 6-7 genome as a template, P1 and P2 were used as primers to amplify the upstream homology arm of acuB, and P5 and P6 were used as primers to amplify the downstream homology arm of acuB. Using the p7Z6 plasmid as a template (the p7Z6 plasmid was disclosed in X, Yan, H.-J, etc. "Cre / lox System and PCR-Based Genome Engineering in Bacillus subtilis", publication date 2008), P3 and P4 were primers for PCR amplification. The lox71-zeo-lox66 fragment was added. Using overlap extension PCR technology, acuB upper and lower homology arm gene fragments and lox71-zeo-lox66 fragments were fused and amplified to obtain a recombinant gene fragment of about 2.2kb; the strain B. subtilis 6...
Embodiment 2
[0032] Example 2: Knockout of acoB gene
[0033] Determine the sequence position of acoB from the B. subtilis168 genome sequence published on the NCBI website, and download a gene fragment of 800 bp before and after acoB as the upstream and downstream homology arms. The genome of B. subtilis 6-7 was used as template, P7 and P8 were used as primers to amplify the upstream homology arm of acoB, and P11 and P12 were used as primers to amplify the downstream homology arm of acoB. The lox71-zeo-lox66 fragment was amplified by PCR using the p7Z6 plasmid as a template and P9 and P10 as primers. Using overlap extension PCR technology to fuse and amplify the three gene fragments of the upper and lower homology arm gene fragments of acoB and the lox71-zeo-lox66 fragment to obtain a recombinant gene fragment of about 2.2 kb; the bacterial strain B.subtilis prepared in Example 1 6-7ΔacuB was prepared as a competent cell, and the obtained recombinant gene fragment was introduced into B. s...
Embodiment 3
[0046] Embodiment 3: knockout of rex gene
[0047] Determine the sequence position of rex from the B. subtilis168 genome sequence published on the NCBI website, and download a gene fragment of 800 bp before and after rex as the upstream and downstream homology arms. Using the B. subtilis 6-7 genome as a template, P13 and P14 were used as primers to amplify rex upstream homology arms, and P17 and P18 were used as primers to amplify rex downstream homology arms. The lox71-zeo-lox66 fragment was amplified by PCR using the p7Z6 plasmid as a template and P15 and P16 as primers. Use overlap extension PCR technology to fuse and amplify the upper and lower homology arm gene fragments of acuB and the lox71-zeo-lox66 fragment to obtain a recombinant gene fragment of about 2.2 kb; the bacterial strain B.subtilis 6-7ΔacuBΔacoB prepared in Example 2 Prepare to be competent, introduce the recombinant gene fragment into B. subtilis 6-7ΔacuBΔacoB competent cells, spread on 30mg / L, zeo-resist...
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