The growth of Bacillus licheniformis DW2 and the bacitracin anabolism are competitive in energy and precursors utilization. In order to improve the efficiency of bacitracin synthesis by B. licheniformis DW2, the regulatory effect of ccpN knockout in two strains was studied in 20 L bioreactor. The results showed that the peak bacitracin production of DW2ΔccpN (1 337 U/mL) was 30.6% higher than that of DW2 (1 024 U/mL). However, the maximum biomass of DW2ΔccpN (3.39×1010 CFU/mL) was only 61% of DW2 (5.56×1010 CFU/mL). In addition, the extracellular amino acids content of DW2ΔccpN (36 h) and the total amount of overflow organic acids (22-27 h) were 43.8% and 24.7%-37.6% higher than that of DW2, respectively. Meanwhile, the transcriptome expression (TPM value) of exponential phase including energy metabolism (citZ, icd, zwf, cydA), amino acid transport (relA, brnQ, vpr, yvbW), pyruvate conversion (pycA, pckA, pyk) and hprK was also enhanced by 2.0-18.4 times than that of DW2, respectively. The results suggested that in the strain DW2ΔccpN ,the overflow metabolism was enhanced, providing more carbon sources and energy molecules in the logarithmic period; the stringent response was increased, reducing the regulatory activity of CodY, decreasing the cell growth rate and increasing the amino acid transport capacity and the supplies of ATP and NADPH were improved, which ultimately enhanced bacitracin synthesis efficiency, providing important references for bacitracin fermentation production.
[1] SHU C C, WANG D, GUO J, et al.Analyzing AbrB-knockout effects through genome and transcriptome sequencing of Bacillus licheniformis DW2[J].Frontiers in Microbiology, 2018, 9:307.
[2] DRABLØS F, NICHOLSON D G, RØNNING M.EXAFS study of zinc coordination in bacitracin A[J].Biochimica et Biophysica Acta, 1999, 1431(2):433-442.
[3] HANCOCK R E.Mechanisms of action of newer antibiotics for Gram-positive pathogens[J].The Lancet.Infectious Diseases, 2005, 5(4):209-218.
[4] WANG Q, ZHENG H, WAN X, et al.Optimization of inexpensive agricultural by-products as raw materials for bacitracin production in Bacillus licheniformis DW2[J].Applied Biochemistry and Biotechnology, 2017, 183(4):1 146-1 157.
[5] 刘铁军, 吴飞.葡萄糖浓度对杆菌肽发酵过程的影响[J].医学信息(中旬刊), 2011, 24(9):4 659-4 660.
LIU T J, WU F.Effect of glucose concentration on baciartcin fermentation process[J].Medical Information, 2011, 24(9):4 659-4 660.
[6] GÖRKE B, STÜLKE J.Carbon catabolite repression in bacteria:Many ways to make the most out of nutrients[J].Nature Reviews Microbiology, 2008, 6(8):613-624.
[7] SCHUMACHER M A, SPREHE M, BARTHOLOMAE M, et al.Structures of carbon catabolite protein A-(HPr-Ser46-P) bound to diverse catabolite response element sites reveal the basis for high-affinity binding to degenerate DNA operators[J].Nucleic Acids Research, 2011, 39(7):2 931-2 942.
[8] 张清, 朱杉, 崔乃香, 等.能量代谢工程促地衣芽胞杆菌DW2高效合成杆菌肽[J].生物工程学报, 2020, 36(6):1 126-1 137.
ZHANG Q, ZHU S, CUI N X, et al.Enhanced production of bacitracin via energy metabolism engineering in Bacillus licheniformis DW2[J].Chinese Journal of Biotechnology, 2020, 36(6):1 126-1 137.
[9] 李阳, 吴非, 蔡冬波, 等.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[J].生物工程学报, 2018, 34(6):916-927.
LI Y, WU F, CAI D B, et al.Enhanced production of bacitracin by knocking out of amino acid permease gene yhdG in Bacillus licheniformis DW2[J].Chinese Journal of Biotechnology, 2018, 34(6):916-927.
[10] TÄNNLER S, FISCHER E, LE COQ D, et al.CcpN controls central carbon fluxes in Bacillus subtilis[J].Journal of Bacteriology, 2008, 190(18):6 178-6 187.
[11] MA W L, LIU Y F, SHIN H D, et al.Metabolic engineering of carbon overflow metabolism of Bacillus subtilis for improved N-acetyl-glucosamine production[J].Bioresource Technology, 2018, 250:642-649.
[12] 曾新年, 鲍帅帅, 李洪杰, 等.双氧水对地衣芽胞杆菌合成杆菌肽的影响[J].中国酿造, 2013, 32(3):94-97.
ZENG X N, BAO S S, LI H J, et al.Effects of H2O2 addition on bacitracin production by Bacillus licheniformis LC-11[J].China Brewing, 2013, 32(3):94-97.
[13] 刘道奇, 陈守文, 李俊辉, 等.混合碳源对地衣芽孢杆菌发酵合成杆菌肽的影响[J].食品与发酵工业, 2017, 43(9):52-57.
LIU D Q, CHEN S W, LI J H, et al.Effects of the mixed carbon resource addition on bacitracin biosynthesis by Bacillus licheniformis[J].Food and Fermentation Industries, 2017, 43(9):52-57.
[14] 黄松, 吴月娜, 刘梅, 等.茚三酮比色法测定青天葵中总游离氨基酸的含量[J].中国中医药信息杂志, 2010, 17(12):50-52.
HUANG S, WU Y N, LIU M, et al.Quantitative determination of total free-amino acid in Nervilia fordii(hance) schltr.by ninhydrin colorimetric method[J].Chinese Journal of Information on Traditional Chinese Medicine, 2010, 17(12):50-52.
[15] WON H I, WATSON S M, AHN J S, et al.Inactivation of the Pta-AckA pathway impairs fitness of Bacillus anthracis during overflow metabolism[J].Journal of Bacteriology, 2021, 203(9):e00660-e00620.
[16] 王境. 毕赤酵母和枯草杆菌产碱性蛋白酶的对比研究[D].太原:山西大学, 2019.
WANG J.Comparative study on alkaline protease produced by Pichia pastoris and Bacillus subtilis[D].Taiyuan:Shanxi University, 2019.
[17] SHIVERS R P, SONENSHEIN A L.Activation of the Bacillus subtilis global regulator CodY by direct interaction with branched-chain amino acids[J].Molecular Microbiology, 2004, 53(2):599-611.
[18] CAI D B, ZHU J, ZHU S, et al.Metabolic engineering of main transcription factors in carbon, nitrogen, and phosphorus metabolisms for enhanced production of bacitracin in Bacillus licheniformis[J].ACS Synthetic Biology, 2019, 8(4):866-875.
[19] WEN J H, ZHAO X Y, SI F M, et al.Surfactin, a quorum sensing signal molecule, globally affects the carbon metabolism in Bacillus amyloliquefaciens[J].Metabolic Engineering Communications, 2021, 12:e00174.
[20] 刘钊远. 增强地衣芽胞杆菌TCA循环代谢水平高产杆菌肽[D].武汉:湖北大学, 2018.
LIU Z Y.Enhance bacitracin production by increasing the TCA cycle in Bacillus licheniformis[D].Wuhan:Hubei University, 2018.
[21] ZHU S, CAI D B, LIU Z W, et al.Enhancement of bacitracin production by NADPH generation via overexpressing glucose-6-phosphate dehydrogenase zwf in Bacillus licheniformis[J].Applied Biochemistry and Biotechnology, 2019, 187(4):1 502-1 514.
[22] CAI D B, ZHANG B W, RAO Y, et al.Improving the utilization rate of soybean meal for efficient production of bacitracin and heterologous proteins in the AprA-deficient strain of Bacillus licheniformis[J].Applied Microbiology and Biotechnology, 2019, 103(12):4 789-4 799.
[23] ZHU J, CAI D B, XU H X, et al.Enhancement of precursor amino acid supplies for improving bacitracin production by activation of branched chain amino acid transporter BrnQ and deletion of its regulator gene lrp in Bacillus licheniformis[J].Synthetic and Systems Biotechnology, 2018, 3(4):236-243.
[24] MAJERCZYK C D, SADYKOV M R, LUONG T T, et al.Staphylococcus aureus CodY negatively regulates virulence gene expression[J].Journal of Bacteriology, 2008, 190(7):2 257-2 265.
[25] CHOWDHURY N, KWAN B W, WOOD T K.Persistence increases in the absence of the alarmone guanosine tetraphosphate by reducing cell growth[J].Scientific Reports, 2016, 6:20519.
