Bacillus subtilis is a generally recognized as safe (GRAS) organism, which has unique advantages in the fermentation field as a chassis cell. However, compared with the most widely used prokaryotic production host Escherichia coli and eukaryotic host Saccharomyces cerevisiae, B. subtilis is limited in the biotechnological research owing to the lack of genetic regulatory elements. In the present study, through comparison with bsrG/SR4, a new modulation via the small RNA (sRNA)-dependent operation system (MS-DOS) based on another type I toxin-antitoxin system bsrE/SR5 was constructed successfully, whose inhibition rate was 88.8%. This system provided a new post-transcriptional regulatory tool for B. subtilis gene regulation. Then, the truncation of SR5 was investigated. The SR5 mutant maintained high activity (83.8% inhibition rate) in the case of retaining only the terminator sequence. The truncated SR5 could further reduce the metabolic pressure of the bacteria, and thus could be directly added to the plasmid by designing primers since its fragment is short (only 37 nt), providing convenience for its application. The construction of the bsrE/SR5 post-transcriptional regulatory system enriches the MS-DOS regulatory system library which will provide more gene regulation tools for B. subtilis and promote the development of B. subtilis in basic and applied research.
XU Yaqing
,
TANG Yao
,
SUN Yuqian
,
YIN Guobin
,
WANG Yang
,
KANG Zhen
. Construction and optimization of bsrE/SR5 posttranscriptional regulation system in Bacillus subtilis[J]. Food and Fermentation Industries, 2021
, 47(24)
: 21
-27
.
DOI: 10.13995/j.cnki.11-1802/ts.027238
[1] JIN P, KANG Z, YUAN P H, et al.Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168[J].Metabolic Engineering, 2016, 35:21-30.
[2] GU Y, LYU X Q, LIU Y F, et al.Synthetic redesign of central carbon and redox metabolism for high yield production of n-acetylglucosamine in Bacillus subtilis[J].Metabolic Engineering, 2019, 51:59-69.
[3] SUN Y W, LIU C, TANG W Z, et al.Manipulation of purine metabolic networks for riboflavin production in Bacillus subtilis[J].ACS Omega, 2020, 5(45):29 140-29 146.
[4] FAN X G, WU H Y, JIA Z F, et al.Metabolic engineering of Bacillus subtilis for the co-production of uridine and acetoin[J].Applied Microbiology and Biotechnology, 2018, 102(20):8 753-8 762.
[5] YAN P P, WU Y Q, YANG L, et al.Engineering genome-reduced Bacillus subtilis for acetoin production from xylose[J].Biotechnology Letters, 2018, 40(2):393-398.
[6] FU J, HUO G X, FENG L L, et al.Metabolic engineering of Bacillus subtilis for chiral pure meso-2,3-butanediol production[J].Biotechnology for Biofuels, 2016, 9(10):90.
[7] 李洪康, 李由然, 李赢, 等.枯草芽孢杆菌产中性蛋白酶发酵条件优化[J].食品与发酵工业, 2016, 42(5):102-107.
LI H K, LI Y R, LI Y, et al.Optimization of fermentation conditions for the production of neutral protease by Bacillus subtilis[J].Food and Fermentation Industries, 2016, 42(5):102-107.
[8] MENG F Q, ZHU X Y, ZHAO H Z, et al.Improve production of pullulanase of Bacillus subtilis in batch and fed-batch cultures[J].Applied Biochemistry and Biotechnology, 2020, 193(1):296-306.
[9] ZHANG K, DUAN X G, WU J.Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system[J].Scientific Reports, 2016, 6(10):6 983-6 995.
[10] SONG Y F, NIKOLOFF J M, FU G, et al.Promoter screening from Bacillus subtilis in various conditions hunting for synthetic biology and industrial applications[J].PloS One, 2016, 11(7):e0158447.
[11] GUIZIOU S, SAUVEPLANE V, CHANG H J, et al.A part toolbox to tune genetic expression in Bacillus subtilis[J].Nucleic Acids Research, 2016, 44(15):7 495-7 508.
[12] YANG S, WANG Y, WEI C B, et al.A new sRNA-mediated posttranscriptional regulation system for Bacillus subtilis[J].Biotechnology and Bioengineering, 2018, 115(12):2 986-2 995.
[13] MORGENS D W, WAINBERG M, BOYLE E A, et al.Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens[J].Nature Communications, 2017, 8(1):299-311.
[14] WIEGERT T, SCHUMANN W.Ssra-mediated tagging in Bacillus subtilis[J].Journal of Bacteriology, 2001, 183(13):3 885-3 889.
[15] WU Y K, LIU Y F, LYU X Q, et al.CAMERS-B:CRISPR/Cpf1 assisted multiple-genes editing and regulation system for Bacillus subtilis[J].Biotechnology and Bioengineering, 2020, 117(6):1 817-1 825.
[16] YAN X, YU H J, HONG Q, et al.Cre/lox system and PCR-based genome engineering in Bacillus subtilis[J].Applied and Environmental Microbiology, 2008, 74(17):5 556-5 562.
[17] DURAND S, JAHN N, CONDON C, et al.Type I toxin-antitoxin systems in Bacillus subtilis[J].RNA Biology, 2012, 9(12):1 491-1 497.
[18] JAHN N, BRANTL S.One antitoxin-two functions:SR4 controls toxin mRNA decay and translation[J].Nucleic Acids Research, 2013, 41(21):9 870-9 880.
[19] JAHN N, BRANTL S.Heat-shock-induced refolding entails rapid degradation of bsrG toxin mRNA by Rnases Y and J1[J].Microbiology, 2016, 162(3):590-599.
[20] JAHN N, PREIS H, WIEDEMANN C, et al.BsrG/SR4 from Bacillus subtilis--the first temperature-dependent type I toxin-antitoxin system[J].Molecular Microbiology, 2012, 83(3):579-598.
[21] MEISSNER C, JAHN N, BRANTL S.In vitro characterization of the type I toxin-antitoxin system bsrE/SR5 from Bacillus subtilis[J].The Journal of Biological Chemistry, 2016, 291(2):560-571.
[22] MULLER P, JAHN N, RING C, et al.A multistress responsive type I toxin-antitoxin system:bsrE/SR5 from the B.subtilis chromosome[J].RNA Biology, 2016, 13(5):511-523.
[23] FEUCHT A, ERRINGTON J.Ftsz mutations affecting cell division frequency, placement and morphology in Bacillus subtilis[J].Microbiology, 2005, 151(6):2 053-2 064.
