Deletion of luxS gene mediated byλRed gene recombination technology reduces biofilm formation and stress resistance of Lactobacillus fermentum

This study investigated the effects of the luxS gene on the biofilm formation of Lactobacillus fermentum and its stress resistance.Deletion of the luxS gene was related to the physiological characteristics of biofilm development,strain resistance,bacterial morphology,biofilm morphology,functional group types,the main components of the biofilm,and the expression of related genes.Therefore,a luxS gene-deficient strain was constructed usingλRed gene recombination technology,and the biofilm production and stress resistance of the WT andΔluxS strains were compared.Deletion of the gene reduced the biofilm formation of L.fermentum and its resistance to acid,bile salt,high temperatures,and a hypertonic environment.Further,this work found that its deletion affects the types of functional groups in biofilms,and at the same time downregulate the expressions of the fabI,cysE,argR,and purD genes,thus reducing the levels of fatty acids,exopolysaccharide,protein,and eDNA in biofilms.A correlation analysis found that protein in biofilms was the most important factor affecting biofilm formation.In conclusion,the luxS gene in L.fermentum is involved in the regulation of biofilm formation and stress resistance.These findings provide a theoretical basis for the study of the mechanism of biofilm formation in beneficial bacteria.

Effects of aroP gene disruption on L-tryptophan fermentation

The production of L-tryptophan through che- mical synthesis, direct fermentation, bioconversion and enzymatic conversion has been reported. However, the role of the transport system for the aromatic amino acids in L- tryptophan producing strains has not been fully explored. In this study, the atop gene of the L-tryptophan producing Escherichia coli TRTH strain was disrupted using Red recombination technology and an atoP mutant E. coli TRTH AaroP was constructed. Fed-batch fermentation of E. coli TRTH △aroP was carried out in 30-L fermentor to investigate the L-tryptophan production. Compared with E. coli TRTH, the atoP mutant was able to maintain a higher growth rate during the exponential phase of the fermentation and the L-tryptophan production increased by 13.3%.

Direct cloning and transplanting of large DNA fragments from Escherichia coli chromosome

We applied a resistance split-fusion strategy to increase the in vivo direct cloning efficiency mediated by Red recombination. The cat cassette was divided into two parts： cma （which has a homologous sequence with cmb） and cmb, each of which has no resistance separately unless the two parts are fused together. The crab sequence was integrated into one flank of a target clon- ing region in the chromosome, and a linear vector containing the cma sequence was electroporated into the cells to directly capture the target region. Based on this strategy, we successfully cloned an approximately 48 kb DNA fragment from the E. coli DH1-Z chromosome with a positive frequency of approximately 80%. Combined with double-strand breakage-stimulated homologous recombination, we applied this strategy to successfully replace the corresponding region of the E. coli DH36 chromosome and knock out four non-essential genomic regions in one step. This strategy could provide a powerful tool for the heterologous expression of microbial natural product biosynthetic pathways for genome assembly and for the functional study of DNA sequences dozens of kilobases in length.