Improvement of levan production in Bacillus amyloliquefaciens through metabolic optimization of regulatory elements

Abstract

Levan is a functional homopolymer of fructose with considerable applications in food, pharmaceutical and cosmetic industries. To improve the levan production in Bacillus amyloliquefaciens, the regulatory elements of sacB (encoding levansucrase) expression and levansucrase secretion were optimized. Four heterologous promoters were evaluated for sacB expression, and the Pgrac promoter led to the highest level for both sacB transcription and levansucrase enzyme activity. The levan production in the corresponding recombinant strain ΔLP-pHTPgrac reached 30.5 g/L, which was 114% higher than that of the control strain NK-ΔLP. In a further step, eight signal peptides were investigated (with Pgrac as the promoter for sacB expression) for their effects on the levansucrase secretion and levan production. The signal peptide yncM was identified as the optimal one, with a secretion efficiency of approximately 90%, and the levan production in the corresponding recombinant strain ΔLP-Y reached 37.4 g/L, which was 161% higher when compared with the control strains NK-ΔLP. Finally, fed-batch fermentation was carried out in 5-L bioreactors for levan production using the recombinant strain ΔLP-Y. A final levan concentration of 102 g/L was achieved, which is very close to the ever reported highest levan production level from the literature. To our best knowledge, this is the first report of the improvement of levan production through metabolic optimization for sacB expression and levansucrase secretion. The results from this study provided essential insights for systematically metabolic engineering of microbial cell factories for enhanced biochemical production.

Bacillus amyloliquefaciens levan and its silver nanoparticles with antimicrobial properties

“…In the 13 Consistent with other reports on microbial levans [21,22], the above analysis suggested that the exopolysaccharide from B. amyloliquefaciens PB6 existed as a levan structure having b-(2,6) linked fructofuranosyl backbone and some b-(1,2!6) linked fructofuranosyl branches with a D-fructosyl or D-glucosyl residue at the end of the main chain. Gu et al [23] reported that, through metabolic optimisation of regulatory elements, the levan production in the recombinant B. amyloliquefaciens strain could be improved to 102 g/L, which was close to the reported highest levan production level in the literature. Considering that B. amyloliquefaciens PB6 is a non-engineered strain and its exopolysaccharide yield was 104 g/L, the confirmation of levan structure in this study implied that levan could be produced from B. amyloliquefaciens PB6 with high safety and a high yield.…”

Factors Influencing Recombinant Protein Secretion Efficiency in Gram-Positive Bacteria: Signal Peptide and Beyond

“…A promoter is defined as the region of DNA sequence that initiates the gene transcription (Wrighton, 2018). Promoters are often used together with signal peptides as regulatory elements for the expression and production of recombinant proteins (Guan et al, 2016; Gu et al, 2017; Maffei et al, 2017; Cui et al, 2018). Zhang et al (2016) performed an experimental screen of 138 signal peptides from B. subtilis for the production of an alkali-tolerant xylanase (XynBYG) from Bacillus pumilus BYG.…”

Evaluation of the Potential Use of Levan Polysaccharide in Paper Conservation

“…Levan is a fructose-based homopolysaccharide, a fructan that is mainly composed of β-D-fructofuranose residues linked by β- (2)(3)(4)(5)(6) glycosidic bonds (see Figure 1). It serves both structural and functional roles and stands out from other natural polymers by its unique combination of properties such as strong adhesivity, self-assembly into spherical colloids in water, very low intrinsic viscosity, high biocompatibility, and health benefits, which in turn make this unusual polysaccharide a valuable polymer for various applications in cosmetic, pharmaceutical, food, and other industries [1].…”

“…It serves both structural and functional roles and stands out from other natural polymers by its unique combination of properties such as strong adhesivity, self-assembly into spherical colloids in water, very low intrinsic viscosity, high biocompatibility, and health benefits, which in turn make this unusual polysaccharide a valuable polymer for various applications in cosmetic, pharmaceutical, food, and other industries [1]. Whereas levan is also produced by a limited number of plant species at a low degree of polymerization (DP 10-100), longer levan chains (>DP 10 3 -10 4 ) offering a broader spectrum of applications are basically produced by miscellaneous microorganisms both extracellularly and at high titers from sucrose by the hydrolysis and transfructosylation action of levansucrase enzyme [2,3]. Besides mesophilic levan producers from a wide range of genera that include Acetobacter, Bacillus, Erwinia, Gluconobacter, Microbacterium, Pseudomonas, and Zymomonas, as well as many acetic acid and lactic acid bacteria, Halomonas cultures hold distinctive industrial potential due to their added advantages such as high yield and nonsterile production capacity under high salinity [4,5].…”