Analysis Of Sucrose Metabolism Pathway In Bacillus Licheniformis

Bacillus licheniformis is a kind of safe productive strain with good protein secretion effect,simple nutritional conditions and high enzyme expression.The commonly used carbon source is glucose.Sucrose is the most widely distributed non-reducing disaccharide in nature,and some microorganisms can use it as a carbon source for biotransformation of high value-added products.The sucrose metabolic pathway in B.licheniformis is not yet clear,and it is not known whether sucrose can be used as a carbon source to produce bulk industrial products in the future.In this paper,B.licheniformis CBB0302 will be used as the research object,the related enzymes and coding genes of sucrose metabolism pathway in B.licheniformis will be predicted through bioinformatics analysis.Single gene deletion and superimposed gene deletion were carried out respectively to obtain the corresponding mutant strain,then the growth of the mutants was monitored in an inorganic salt medium with sucrose as the sole carbon source.In order to research the sucrose metabolic pathway in B.licheniformis,and a new theoretical basis for the utilization of carbon source in B.licheniformis was developed.The main findings are as follows:（1）The sucrose metabolism pathway in B.licheniformis is divided into fructan synthesis pathway,sucrose phosphorylation pathway and sucrose transport pathway.Through bioinformatics analysis and experimental verification,it can be concluded that The fructan synthesis pathway is an extracellular fructansucrase encoded by the sacB gene that catalyzes the formation of fructan from sucrose and releases glucose into the glycolysis（EMP）pathway.Sucrose phosphorylation pathway is that the membrane protein encoded by the sac P gene transports sucrose into cells while phosphorylating it,and then decomposes sucrose 6-phosphate into glucose 6-phosphate and fructose through the glycoside hydrolase encoded by the sac A gene and then enters the EMP pathway.Sucrose transport pathway is the membrane protein encoded by the ybb F gene transports sucrose into the cell,and then the glycoside hydrolase encoded by the gan C,yug T and fru A genes decomposes the sucrose into glucose and fructose and then enters the EMP pathway.（2）The fructan synthesis pathway is the main sucrose metabolism pathway in B.licheniformis.By monitoring the growth of the mutant strain in the inorganic salt medium with sucrose as the sole carbon source,the highest OD₆₀₀ value of the original strain CBB0302 was 3.44,and the highest OD₆₀₀ value of CBB0302（△sacB）was only 2.46.Compared with deleting other genes,Deletion of the sacB gene significantly weakened the sucrose metabolism in B.licheniformis.Therefore,the fructan synthesis pathway was the main sucrose metabolism pathway in B.licheniformis.（3）When the sacB gene and the sac operon were deleted at the same time,the capacity of sucrose metabolism in B.licheniformis was stronger.At the same time,the sacB gene and sac operon were deleted,that is,the CBB0302（△sacB△sac）mutant had the highest OD₆₀₀ value of 6.35 in the inorganic salt medium with sucrose as the sole carbon source,and there was basically no lag period from 24 h.On the basis of the sacB gene and sac operon were deleted at the same time,the mutant strains CBB0302（△sacB△sac△ybb F）,CBB0302（△sacB△sac△gan C）and CBB0302（△sacB△sac△yug T）still use sucrose,and the highest OD₆₀₀value is between 5.92-6.62,while CBB0302（△sacB△sac△fru A）can no longer utilize sucrose completely.Therefore,fru A gene is a key gene of B.licheniformis sucrose transport pathway.