Metabolic Engineering Of Bacillus Amyloliquefaciens For Enhancing The Synthesis Of Levan

Levan is a kind of fructose homopolymer,which can be hydrolyzed by acid to generate levan-type fructo-oligosaccharide probiotics.Due to its excellent properties,levan has been used in the fields of medicines,industries,foods,cosmetics,et al.Bacillus amyloliquefaciens LL3 can simultaneously synthesize?-PGA(?-polyglutamic acid)and levan using sucrose as substrate.NK-?LP,a mutant with?-PGA synthetase-deficient,was used as parental strain to optimize metabolic pathway for levan overproduction.Two strategies were carried out to modify the metabolism of NK-?LP:transcriptional regulation and levansucrase secretion.To enhance levan production,six bacillus genus strong promoters were selected.Among which,the promoter P_grac was found to be the best in terms of the relative transcriptional level of sac B,which showed a 14-fold improvement in relative to the parental strain.The recombinant strain?LP/pHT-P_grac produced 24.3 g/L levan,which was 2.07-fold higher than that of parental strain.To optimize the overall metabolic ability of the strain,five regulatory factors were respective overexpressed downstream of the P_gracpromoter.During the whole fermentation period,the growth profile of?LP/pHT-abr B overexpressing the transition regulatory factor AbrB was superior than that of NK-?LP.?LP/pHT-ccpA,a strain overexpressed the key factor of carbon metabolism,produced the highest levan production,which was 97.7%higher than that of NK-?LP.To improve the extracellular transport of levansucrase,eight signal peptides were selected to replace the original signal peptide of sac B and placed downstream of P_gracpromoter.The recombinant strain?LP/pHTSPync M showed a 8.57 and 2.68-fold improvement in the extracellular activity of levansucrase compared to NK-?LP and ?LP/pHT-P_grac,respectively.And the secretion efficiency of ?LP/pHTSPync M was elevated from 69.88%to 89.89%.Besides,the levan production was 2.18-fold higher than that of the parental strain.In order to overcome the secretory pressure of bacillus cell wall on levansucrase,the genes dacA,dacC and dacF,which mediate peptidoglycan cross-linking were removed from NK-?LP,respectively.As a result,the growth profiles and cell dry weight of the three mutants were affected to a certain extent,while the extracellular levansucrase activity,secretion efficiency and levan production were all improved.Among which,the levan production of the mutant with dacF-deficient was 60.3%higher than that of the parental strain.