Metabolic engineering of Bacillus amyloliquefaciens for efficient production ofα-glucosidase inhibitor1-deoxynojirimycin

Owing to the feature of strongα-glucosidase inhibitory activity,1-deoxynojirimycin(1-DNJ)has broad application prospects in areas of functional food,biomedicine,etc.,and this research wants to construct an efficient strain for 1-DNJ production,basing on Bacillus amyloliquefaciens HZ-12.Firstly,using the temperature-sensitive shuttle plasmid T2(2)-Ori,gene ptsG in phosphotransferase system(PTS)was weakened by homologous recombination,and non-PTS pathway was strengthened by deleting its repressor gene iolR,and 1-DNJ yield of resultant strain HZ-S2 was increased by 4.27-fold,reached 110.72 mg/L.Then,to increase precursor fructose-6-phosphate(F-6-P)supply,phosphofructokinase was weaken,fructose phosphatase GlpX and 6-phosphate glucose isomerase Pgi were strengthened by promoter replacement,moreover,regulator gene nanR was deleted,1-DNJ yield was further increased to 267.37 mg/L by 2.41-fold.Subsequently,promoter of 1-DNJ synthetase cluster was optimized,as well as 5′-UTRs of downstream genes in synthetase cluster,and 1-DNJ produced by the final strain reached 478.62 mg/L.Last but not the least,1-DNJ yield of 1632.50 mg/L was attained in 3 L fermenter,which was the highest yield of 1-DNJ reported to date.Taken together,our results demonstrated that metabolic engineering was an effective strategy for 1-DNJ synthesis,this research laid a foundation for industrialization of functional food and drugs based on 1-DNJ.

Microbial synthesis of bacitracin:Recent progress,challenges,and prospects

Microorganisms are important sources of various natural products that have been commercialized for human medicine and animal healthcare.Bacitracin is an important antibacterial natural product predominantly pro-duced by Bacillus licheniformis and Bacillus subtilis,and it is characterized by a broad antimicrobial spectrum,strong activity and low resistance,thus bacitracin is extensively applied in animal feed and veterinary medicine industries.In recent years,various strategies have been proposed to improve bacitracin production.Herein,we systematically describe the regulation of bacitracin biosynthesis in genus Bacillus and its associated mechanism,to provide a theoretical basis for bacitracin overproduction.The metabolic engineering strategies applied for bacitracin production are explored,including improving substrate utilization,using an enlarged precursor amino acid pool,increasing ATP supply and NADPH generation,and engineering transcription regulators.We also present several approaches of fermentation process optimization to facilitate the industrial large-scale production of bacitracin.Finally,the challenges and prospects associated with microbial bacitracin synthesis are discussed to facilitate the establishment of high-yield and low-cost biological factories.

地衣芽胞杆菌DW2中敲除氨基酸转运蛋白基因yhdG提高杆菌肽产量

杆菌肽是微生物产生的由11种氨基酸残基组成的广谱性抗生素,前体物的供应可能是限制杆菌肽高产的重要因素。文中通过支链氨基酸(异亮氨酸、亮氨酸、缬氨酸)的添加实验考察了前体物质支链氨基酸对杆菌肽高产的影响,证实了异亮氨酸(Ile)和亮氨酸(Leu)的添加可以提高杆菌肽的效价,其中Ile的添加对杆菌肽效价提高的效果较为明显。随后,文中以地衣芽胞杆菌DW2为出发菌株,分别构建了支链氨基酸转运蛋白Yhd G的缺失和强化表达菌株。发酵结果表明,转运蛋白Yhd G缺失工程菌DW2△yhd G的杆菌肽效价达到917.35 U/m L,与原始菌DW2相比提高了11%,而强化Yhd G则会使杆菌肽效价下降25%。最后通过分析胞内胞外支链氨基酸含量,发现缺失转运蛋白Yhd G能够在发酵中后期显著提高胞内支链氨基酸含量,表明氨基酸转运蛋白Yhd G在地衣芽胞杆菌DW2中可能发挥着氨基酸输出的功能。综上,文中通过缺失转运蛋白Yhd G显著提高了地衣芽胞杆菌胞内支链氨基酸的供给水平,从而提高了杆菌肽效价,为杆菌肽高产菌株的构建提供了一种新的策略。