Cellulose oligosaccharide transporter as well as preparation method and application thereof

Abstract

The invention provides a cellooligosaccharide transporter as well as a preparation method and application thereof, the preparation method of the cellooligosaccharide transporter engineering strain comprises the following steps: step 1, taking a wild type SP-LacY as an initial bacteriophage, taking a visual bacteriophage-assisted continuous directed evolution system engineering bacterium containing an AP1 plasmid, an AP2 plasmid and an MP1 plasmid as a host, and preparing a cellooligosaccharide transporter engineering strain; culturing phage spots by using LB or M9 soft agar containing cellooligosaccharides as a culture medium; step 2, continuing to perform multi-round evolution culture on the obtained phage plaque, sequencing the phage plaque obtained in each round of evolution to verify the mutant accumulation trend, stopping evolution until no new mutant is formed, and finally obtaining proteins with different mutation sites; and step 3, combining different mutation sites of the protein obtained in the step 2 to construct different LacY mutant expression vectors, and transforming the LacY mutant expression vectors into host bacteria to obtain the LacY mutant. The cellooligosaccharide transporter provided by the invention can better ferment and utilize cellulose and cellooligosaccharide.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a cello-oligosaccharide transporter and a preparation method and application thereof. Background technique [0002] Cellulose is the most abundant natural polymer compound in the world, most of which are synthesized by green plants through photosynthesis. Currently using cellulosic to produce industrial by-products, such as alcohol, firstly, the cellulosic must be physically and chemically treated or pretreated with cellulase to degrade the cellulosic into glucose, which is then used by microorganisms to produce industrial by-products; in fact, the pretreated cellulosic The degradation products also contain a large amount of cello-oligosaccharides such as cellobiose and cellotriose. However, due to the lack of specific cello-oligosaccharide transporters in microorganisms, microorganisms cannot efficiently utilize cello-oligosaccharides, resulting in waste of resources...

AI Technical Summary

Problems solved by technology

At present, the use of fiber to produce industrial by-products, such as alcohol, first requires physical and chemical treatment of the fiber or pretreatment with cellulase to degrade the fiber into glucose, which is then used by microorganisms to produce industrial by-products; in fact, the pre-treated fiber The degradation products also contain a large amount of cellooligosaccharides such as cellobiose and cellotriose. However, due to the lack of specific cellooligosaccharide transporters in microorganisms, microorganisms cannot efficiently utilize cellooligosaccharides, resulting in waste of resources and low fiber utilization.

[0003] LacY is a lactose or galactose transporter, and its transport efficiency for cellooligosaccharides is very low, and no specific transporter for cellooligosaccharides has been found in bacteria. Therefore, in the field of fiber utilization, especially the use of bacterial fermentation Cellulose is restricted in obtaining industrial by-products. If a transporter can be developed to efficiently transport cellooligosaccharides into cells for use by bacteria, the above problems can be solved.

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