Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin, 300071, China.
Biosystems Engineering Department, Auburn University, Auburn, AL, 36849, USA.
Appl Microbiol Biotechnol. 2017 May;101(10):4163-4174. doi: 10.1007/s00253-017-8171-2. Epub 2017 Feb 14.
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.
低聚果糖是果糖的功能同聚物，在食品、制药和化妆品行业有广泛的应用。为了提高解淀粉芽孢杆菌中低聚糖的产量，优化了 sacB（编码蔗聚糖酶）表达和蔗聚糖酶分泌的调控元件。评估了四个异源启动子用于 sacB 表达，Pgrac 启动子导致 sacB 转录和蔗聚糖酶酶活水平最高。相应重组菌株 ΔLP-pHTPgrac 的低聚糖产量达到 30.5 g/L，比对照菌株 NK-ΔLP 高 114%。在进一步的步骤中，研究了八个信号肽（以 Pgrac 为 sacB 表达的启动子）对蔗聚糖酶分泌和低聚糖产量的影响。确定了 yncM 信号肽为最佳信号肽，分泌效率约为 90%，相应重组菌株 ΔLP-Y 的低聚糖产量达到 37.4 g/L，比对照菌株 NK-ΔLP 高 161%。最后，在 5-L 生物反应器中使用重组菌株 ΔLP-Y 进行分批补料发酵生产低聚糖。最终低聚糖浓度达到 102 g/L，非常接近文献中报道的最高低聚糖产量水平。据我们所知，这是首次通过代谢优化 sacB 表达和蔗聚糖酶分泌来提高低聚糖产量的报道。本研究结果为系统代谢工程微生物细胞工厂提供了重要的见解，以增强生物化学生产。
