Method for producing cellooligosaccharides from lignocellulose
A technology of lignocellulose and cellulosic oligosaccharides, which is applied in the field of preparing cellulosic oligosaccharides, can solve the problems of high cost, low efficiency, and low yield, and achieve the effects of controlling the degree of polymerization, reducing production costs, and increasing product concentration
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Embodiment 1
[0028] Example 1: Knockout of exocellulase from Clostridium thermocellum cellulosomes
[0029]Through seamless cloning, the tdk expression cassette (promoter comprising the gapDH gene) and the pyrF expression cassette (comprising the pyrF self-promoter) were cloned into the plasmid pHK (Mohr, G., Hong, W., Zhang, J., Cui, G.-Z., Yang, Y., Cui, Q., et al. (2013) A targetron system for gene targeting in thermophiles and its application in Clostridium thermocellum, PLoS One 8:e69032.) downstream of the antibiotic gene cat, And through the design of primers, NheI and XbaI restriction sites were added between tdk and pyrF expression cassettes, and EagI and MluI restriction sites were added downstream of pyrF for cloning of homology arm fragments, thereby constructing pHK-HR plasmid.
[0030] The cellulosic exonuclease Cel48S (encoded by the nucleic acid sequence 3228088-3230229 in the genome CP002416.1) of the cellulosome of Clostridium thermocellum was selected for targeted knock...
Embodiment 2
[0034] Example 2: Knockout of exocellulase from Clostridium thermocellum cells
[0035] The difference from Example 1 is that the exocellulase Cel9K (encoded by the nucleic acid sequence 2113813-2111293 in the genome CP002416.1) of the cellulosome of Clostridium thermocellum was selected for targeted knockout gene. The upstream homology arm HR-up is the nucleic acid sequence from 2110625 to 2111125 in the Clostridium thermocellum DSM1313 genome (the sequence number in the NCBI database is CP002416.1), the downstream homology arm HR-down is the nucleic acid sequence from 2113814 to 2114314 in the DSM1313 genome, and the middle The homology arm HR-short is the nucleic acid sequence from 2110825 to 2111125 in the DSM1313 genome. Knockout plasmids do not contain the gene of interest. The constructed plasmid was transformed into the DSM1313 mutant strain of pyrF deletion, and the Cel9K knockout strain ΔCel9K was obtained according to the three-step screening method.
Embodiment 3
[0036] Example 3: Modification of exocellulase of Clostridium thermocellum cellulosomes
[0037] The difference from Example 1 is that the knockout plasmid contains the target gene Cel9-48 (encoded by the nucleic acid sequence 1968724 to 1973904 in the genome CP001393.1). The constructed plasmid was transformed into the DSM1313 mutant strain of pyrF deletion, and the Cel9-48 replaced Cel48S strain ΔCel48S::Cel9-48 according to the three-step screening method.
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