| Bacterial cellulose(BC)is a type of cellulose synthesized by microorganisms,and its basic unit consists of glucose units linked by β-1,4-glycoside bonds.BC exhibits good biocompatibility,an extremely high-water retention capacity,and excellent mechanical properties.Because of these characteristics,BC is widely used in cosmetics,papermaking,medical materials,and other fields.Different fields have different requirements for the structural characteristics of BC.To meet these needs,chemical modification or regulation of fermentation conditions has traditionally been used to regulate BC structure.However,chemical modifications often lead to high costs and reduce biocompatibility.The regulation of the BC structure under fermentation conditions is limited by the inherent genetic properties of the strain.Based on this situation,the model production strain of BC(Komagataeibater xylinus CGMCC 2955)was studied in the following aspects.(1)In order to maintain the excellent characteristics of BC in the process of regulating the structure of BC while breaking through the limitation of BC structure by the genetic characteristics of the strain,this study intends to genetically modify the BC producing strain and regulate the expression of genes related to BC synthesis through genetic tools,and then regulate the structure of BC in the process of fermentation.However,because few genetic tools are available,and no genetic tools are available to regulate gene expression in K.xylinus,a genetic tool based on CRISPR/dCas9 was constructed to regulate gene expression levels.The expression of the pgm gene in K.xylinus was regulated by CRISPR/dCas9,resulting in a 51 % decrease in the expression level of the pgm and 82.5 % decrease in the BC yield.(2)To identify the target genes that can regulate the structure of BC,this study combined the differentially expressed genes with the phenotype of K.xylinus producing BC with different structures under different oxygen tensions,it is revealed that gal U is a potential target gene that regulates the structure of BC.CRISPR/dCas9 was used to regulate gal U expression in K.xylinus,and a range of strains with different gal U expression levels were obtained.The characterization of the BC structure showed that the BC porosity varied from 64.39 % to 90.66 % and was negatively correlated with gal U expression level;the crystallinity varied from 56.25 % to 85.99 % and was positively correlated with gal U expression level.This finding makes it possible to modulate the BC structure during one-step fermentation.(3)Changes in gal U expression level will significantly affect the BC production.In order not to affect the yield of BC,this study selected bcs D,which is involved in the synthesis of BC,but not necessary for the synthesis of BC,as the target site of CRISPR/dCas9.A series of strains with different expression levels of bcs D gene were obtained by targeting the bcs D.Characterizing the structure of BC found that with the decrease of bcs D expression level,the crystallinity of BC decreased from 93.75 %to 74.26 %;when the expression level of bcs D was lower than 55.34 % of the control strain,the porosity increased from about 65 % to 80 %,However,the production of BC did not change significantly due to the difference in the expression of bcs D.(4)Differences in the effects of gal U and bcs D expression levels on BC structure suggest that different genes have distinct effects on BC structure.To regulate BC structure from distinct dimensions,more genes related to BC structure need to be regulated.To achieve this,potential BC structure-related genes need to be identified,and the effects of these genes on BC structure need to be elucidated.This requires editing of genes in the genome.However,no genetic tools are currently available for rapid editing of the genome of K.xylinus.To efficiently edit the genome,we developed a CRISPR/Cas9-based gene-editing system in K.xylinus.Using this tool,bcs Z was selected as the target to delete genome fragments of different lengths in K.xylinus.The results showed that the gene editing tool could delete at least 10 kb genome fragments in K.xylinus,and the editing efficiency was more than 97 %,the plasmid loss efficiency is above 87%.Using CRISPR/Cas9-mediated gene editing tools to replace the promoter of the gal U with the trc promoter increased the expression of gal U by 175%,and the BC production reached 5.75 g/L,an increase of 23.6%.The successful replacement of the promoter indicated that the gene editing tool could integrate the exogenous gene into the K.xylinus genome.(5)K.xylinus CGMCC 2955 contains four cellulose synthase(Bcs)operators.The Bcs operators contain bcs A,bcs B,and bcs C,which are necessary for BC synthesis,as well as bcs D,bcs H,bcs X,bcs Y,and bcs Z,which are related to BC synthesis.However,the effects of these proteins on BC structure have not been elucidated.To identify more genes associated with BC structure,the CRISPR/Cas9 gene editing tool was used to delete the genes associated with BC synthesis(bcs H,bcs X,bcs Y,and bcs Z)and have unknown effects on BC structure.By comparing the fermentation and BC structural properties of the wild-type strain with the strains that had bcs H,bcs X,bcs Y,and bcs Z deleted individually,it was found that the deletion of these genes enhanced the growth of K.xylinus.The polymerization capacity of BC produced by strains that deleted these genes individually during dynamic fermentation was significantly reduced.Deletion of bcs H or bcs X lowered the average diameter of cellulose filaments.The absence of bcs H or bcs X decreased the yield of BC by 53.85 % and 56.53 %,respectively,and the average diameter of cellulose filaments also decreased.Deletion of bcs Y increased the average diameter of cellulose filaments but had no significant effect on BC yield.Deletion of bcs Z reduced BC yield by13.32 %,and many prominent nodes appeared in cellulose filaments.These findings will help regulate the structure of BC in more dimensions,thereby increasing its application in more fields. |
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