基于基因组重排技术选育D-核糖高产菌株
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摘要
以SFR-3A为出发菌株,构建获得一株具有正反向筛选标记的菌株SFR-3A-ZMS;并通过该菌株和菌株SFR-4进行基因组重排,实现高抗逆性D-核糖高产菌株SFRCP-100的快速高效选育。在酵母浸粉、玉米浆及硫酸铵添加量分别为7.5 g/L、3 g/L及7 g/L的优化氮源条件下,5 L发酵罐中D-核糖产量达到38.2 g/L,其转化率和整体生产效率分别达到35%和0.73 g/(L·h)。本实验成功添加正反向筛选标记,提高了基因组重排技术在枯草芽孢杆菌(Bacillus subtilis)优良菌株选育中的有效性和高效性。
Using strain SFR-3A as the starting strain, the strain SFR-3A-ZMS with positive and counter selectable markers was constructed. Through genome shuffling of constructed strain and strain SFR-4, a D-ribose high-producing strain SFRCP-100 with high resistance was quickly and efficiently bred. Under the optimized nitrogen condition of yeast powder 7.5 g/L, corn pulp 3 g/L and ammonium sulfate 7 g/L, the yield of D-ribose in 5-L bioreactors reached 38.2 g/L, and the conversion rate and overall production efficiency reached 35% and 0.73 g/(L·h). In the experiment, the positive and negative screening markers were successfully added to improve the effectiveness and efficiency of genome shuffling technology in the breeding of Bacillus subtilis.
Using strain SFR-3A as the starting strain, the strain SFR-3A-ZMS with positive and counter selectable markers was constructed. Through genome shuffling of constructed strain and strain SFR-4, a D-ribose high-producing strain SFRCP-100 with high resistance was quickly and efficiently bred. Under the optimized nitrogen condition of yeast powder 7.5 g/L, corn pulp 3 g/L and ammonium sulfate 7 g/L, the yield of D-ribose in 5-L bioreactors reached 38.2 g/L, and the conversion rate and overall production efficiency reached 35% and 0.73 g/(L·h). In the experiment, the positive and negative screening markers were successfully added to improve the effectiveness and efficiency of genome shuffling technology in the breeding of Bacillus subtilis.
引文
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[2]袁保辉,丁琪,刘桂芹,等.D-核糖的生理功能及其应用[J].中国生化药物杂志,2016,36(5):210-212.
[3]DE W P,VANDAMME E J.Production of D-ribose by fermentation[J].Appl Microbiol Biot,1997,48(2):141-148.
[4]PAULY D F,PEPINE C J.D-ribose as a supplement for cardiac energy metabolism[J].J Cardiovascul Pharmacol Therap,2000,5(4):249-258.
[5]MACCARTER D,VIJAY N,WASHAM M,et al.D-ribose aids advanced ischemic heart failure patients[J].Int J Cardiol,2009,137(1):79-80.
[6]PARK Y C,LEE H J,KIM C S,et al.Effects of oxygen supply and mixed sugar concentration on D-ribose production by a transketolase-deficient Bacillus subtilis SPK1[J].J Microbiol Biot,2013,23(4):560-564.
[7]常景玲,李慧,张志宏.D-核糖发酵液预处理研究[J].中国酿造,2008,27(14):60-62.
[8]CHENG J,ZHUANG W,LI N N,et al.Efficient biosynthesis of D-ribose using a novel co-feeding strategy in Bacillus subtilis without acid formation[J].Lett Appl Microbiol,2017,64(1):73-78.
[9]WULF P D,SOETAERT W,SCHWENGERS D,et al.Optimization of D-ribose production with a transketolase-affected Bacillus subtilis mutant strain in glucose and gluconic acid-based media[J].J Appl Microbiol,1997,83(1):25-30.
[10]王丽宁,赵妍,奚丽萍,等.基因组重排技术及其在真菌育种中的应用[J].食品工业科技,2014,35(18):362-365.
[11]ZHANG Y X,PERRY K,VINCI V A,et al.Genome shuffling leads to rapid phenotypic improvement in bacteria[J].Nature,2002,415(6872):644-646.
[12]MAGOCHA T A,ZABED H,YANG M,et al.Improvement of industrially important microbial strains by genome shuffling:Current status and future prospects[J].Bioresource Technol,2018,258(6):1-14.
[13]WANG Y,ZHANG G,ZHAO X,et al.Genome shuffling improved the nucleosides production in Cordyceps kyushuensis[J].J Biotechnol,2017,260(1):42-47.
[14]蒋承州.后基因组时代基因组重排在微生物菌种选育中的应用及展望[J].微生物学通报,2018,45(11):2494-2502.
[15]BIOTPELLETIER D,MARTIN V J.Evolutionary engineering by genome shuffling[J].Appl Microbiol Biot,2014,98(9):3877-3887.
[16]LUNA FLORES C H,PALFREYMAN R W,KR魻MER J O,et al.Improved production of propionic acid using genome shuffling[J].Biotechnol J,2017,12:1600120.
[17]YING Z,LIU J Z,HUANG J S,et al.Genome shuffling of Propionibacterium shermanii for improving vitamin B12 production and comparative proteome analysis[J].J Biotechnol,2010,148(2-3):139-143.
[18]赵祥颖,刘建军,张家祥,等.D-核糖性质、应用及其生产菌株的选育[J].食品与药品,2005,7(3):23-26.
[19]BELDA E,SEKOWSKA A,LE F F,et al.An updated metabolic view of the Bacillus subtilis 168 genome[J].Microbiology,2013,159(4):757-770.
[20]ZHAO C,ZHAO X Y,LIU J J,et al.Enhanced D-ribose production by genetic modification and medium optimization in Bacillus subtilis 168[J].Kor J Chem Eng,2018,35(5):1137-1143.
[21]LIN Z,DENG B,JIAO Z,et al.A versatile mini-maz F-cassette for markerfree targeted genetic modification in Bacillus subtilis[J].J Microbiol Meth,2013,95(2):207-214.
[22]SPIZIZEN J.Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate[J].Proceed Natl Acad Sci USA,1958,44(10):1072-1078.
[23]YU H J,YAN X,SHEN W L,et al.Efficient and precise construction of markerless manipulations in the Bacillus subtilis genome[J].J Microbiol Biot,2010,20(1):45-53.