N-acetylneuraminic acid (NeuAc) has applications as nutritional chemicals and pharmaceutical intermediates in the fields of health care products and medicine. To increase NeuAc production in Bacillus subtilis, NeuAc-biosensor was used to regulate the expression of antibiotic resistance genes, and cell growth was correlated with NeuAc synthesis in the presence of antibiotic, which was further used for adaptive evolution to promote the efficiency of NeuAc synthesis. The results showed that high-yielding strains could grow under conditions of higher antibiotic concentration. The adaptive evolution was carried out by gradually increasing the concentration of antibiotics. When NeuAc-Biosensor was used to regulate spc and erm for dual resistance (spectinomycin and erythromycin) adaptive evolution, the false positive rate (the proportion of strains with no increase in NeuAc production) was 46.7%, which was significantly lower than that (73.3% and 67.7%) of the strains obtained by the evolution of using single antibiotic of spectinomycin or erythromycin, respectively. Fermentation experiments verified that the production of NeuAc in evolutionary strain reached (3.16±0.19) g/L, which was 31.7% higher than the original strain. In order to further solve the problem of plasmid loss in recombinant B. subtilis during fermentation, the essential gene folB (encoding dihydroneopterin aldolase) was inserted into the recombinant plasmid carrying the gene encoding the key enzyme of NeuAc synthesis pathway with deletion of folB in the genome. The plasmid loss rate dropped from 34.1% to 11.8%. In this study, the yield and stability of NeuAc production was improved by recombinant B. subtilis, which lays a foundation for the industrial production of NeuAc by recombinant B. subtilis.
QIAN Lei
,
LIU Yanfeng
,
LI Jianghua
,
LIU Long
,
DU Guocheng
. Regulating the synthesis of N-acetylneuraminic acid based on adaptive evolution and plasmid stability modification in Bacillus subtilis[J]. Food and Fermentation Industries, 2021
, 47(5)
: 1
-6
.
DOI: 10.13995/j.cnki.11-1802/ts.025580
[1] CHENG J, ZHUANG W, TANG C, et al.Efficient immobilization of AGE and NAL enzymes onto functional amino resin as recyclable and high-performance biocatalyst[J].Bioprocess and Biosystems Engineering, 2017, 40(3):331-340.
[2] ZHU D, ZHAN X, WU J, et al.Efficient whole-cell biocatalyst for Neu5Ac production by manipulating synthetic, degradation and transmembrane pathways[J].Biotechnology Letters, 2017, 39(1):55-63.
[3] LUNDGREN B R, BODDY C N.Sialic acid and N-acylsialic acid analog production by fermentation of metabolically and genetically engineered Escherichia coli[J].Organic and Biomolecular Chemistry, 2007, 5(12):1 903-1 909.
[4] DROUILLARD S, MINE T, KAJIWARA H, et al.Efficient synthesis of 6′-sialyllactose, 6, 6′-disialyllactose, and 6′-KDO-lactose by metabolically engineered E.coli expressing a multifunctional sialyltransferase from the Photobacterium sp.JT-ISH-224[J].Carbohydr Res, 2010, 345(10):1 394-1 399.
[5] TEN BRUGGENCATE S J, BOVEE-OUDENHOVEN I M, FEITSMA A L, et al.Functional role and mechanisms of sialyllactose and other sialylated milk oligosaccharides[J].Nutrition Reviews, 2014, 72(6):377-389.
[6] LIU Y, ZHU Y, MA W, et al.Spatial modulation of key pathway enzymes by DNA-guided scaffold system and respiration chain engineering for improved N-acetylglucosamine production by Bacillus subtilis[J].Metabolic Engineering, 2014, 24:61-69.
[7] KANG J, GU P, WANG Y, et al.Engineering of an N-acetylneuraminic acid synthetic pathway in Escherichia coli[J].Metabolic Engineering, 2012, 14(6):623-629.
[8] UHDE A, BRUHL N, GOLDBECK O, et al.Transcription of sialic acid catabolism genes in Corynebacterium glutamicum is subject to catabolite repression and control by the transcriptional repressor NanR[J].Journal of Bacteriology, 2016, 198(16):2 204-2 218.
[9] SIEDLER S, STAHLHUT S G, MALLA S, et al.Novel biosensors based on flavonoid-responsive transcriptional regulators introduced into Escherichia coli[J].Metabolic Engineering, 2014, 21:2-8.
[10] ZHANG X, LIU Y, LIU L, et al.Modular pathway engineering of key carbon-precursor supply-pathways for improved N-acetylneuraminic acid production in Bacillus subtilis[J].Biotechnology and Bioengineering, 2018, 115(9):2 217-2 231.
[11] KALIVODA K A, STEENBERGEN S M, VIMR E R, et al.Regulation of sialic acid catabolism by the DNA binding protein nanr in Escherichia coli[J].Journal of Bacteriology, 2003, 185(16):4 806-4 815.
[12] WANG Z, ZHUANG W, CHENG J, et al.In vivo multienzyme complex coconstruction of N-acetylneuraminic acid lyase and N-acetylglucosamine-2-epimerase for biosynthesis of N-acetylneuraminic acid[J].Journal of Agricultural and Food Chemistry, 2017, 65(34):7 467-7 475.
[13] SHI S, CHOI Y W, ZHAO H, et al.Discovery and engineering of a 1-butanol biosensor in Saccharomyces cerevisiae[J].Bioresource Technology, 2017, 245(Pt B):1 343-1 351.
[14] SANDBERG T E, SALAZAR M J, WENG L L, et al.The emergence of adaptive laboratory evolution as an efficient tool for biological discovery and industrial biotechnology[J].Metabolic Engineering, 2019, 56:1-16.
[15] ZHANG R, YANG Y, WANG J, et al.Synthetic symbiosis combining plasmid displacement enables rapid construction of phenotype-stable strains[J].Metabolic Engineering, 2019, 55:85-91.
[16] ZHANG X, CAO Y, LIU Y, et al.Development and optimization of N-acetylneuraminic acid biosensors in Bacillus subtilis[J].Biotechnology and Applied Biochemistry, 2020, 67(4):693-705.
[17] GIBSON D G, BENDERS G A, ANDREWS-PFANNKOCH C, et al.Complete chemical synthesis, assembly, and cloning of a mycoplasma genitalium genome[J].Science, 2008, 319(5867):1 215-1 220.
[18] 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.
[19] ZHAO L, TIAN R, SHEN Q, et al.Pathway engineering of Bacillus subtilis for enhanced N-Acetylneuraminic acid production via whole-cell biocatalysis[J].Biotechnology Journal, 2019, 14(7).DOI:10.1002/biot.201800682.
[20] RAMAN S, ROGERS J K, TAYLOR N D, et al.Evolution-guided optimization of biosynthetic pathways[J].Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(50):17 803-17 808.
[21] NYERGES A, CSORGO B, NAGY I, et al.Conditional DNA repair mutants enable highly precise genome engineering[J].Nucleic Acids Res, 2014, 42(8):e62.
[22] SHUKAL S, CHEN X, ZHANG C.Systematic engineering for high-yield production of viridiflorol and amorphadiene in auxotrophic Escherichia coli[J].Metabolic Engineering, 2019, 55:170-178.
