Abstract: A menaquinone-7 (MK-7) high-producing strain needs to be isolated to increase MK-7 production, in order to meet a requirement of MK-7 given the low MK-7 content in food products. This article focuses on developing MK-7 high-producing strains via screening and mutagenesis by an atmospheric and room temperature plasma (ARTP) mutation breeding system. We isolated an MK-7-producing strain Y-2 and identified it as Bacillus amyloliquefaciens, which produced (7.1±0.5) mg/L of MK-7 with maize meal hydrolysate as carbon source. Then, an MK-7 high-producing strain B. amyloliquefaciens H.β.D.R.-5 with resistance to 1-hydroxy-2-naphthoic acid, β-2-thienylalanine, and diphenylamine was obtained from the mutation of the strain Y-2 using an ARTP mutation breeding system. Using strain H.β.D.R.-5, efficient production of MK-7 was achieved ((30.2±2.7) mg/L). In addition, the effects of nitrogen sources, prenyl alcohols, and MgSO₄ on MK-7 production were investigated, suggesting that soymeal extract combined with yeast extract, isopentenol, and MgSO₄ was beneficial. Under the optimized condition, the MK-7 production and biomass-specific yield reached (61.3±5.2) mg/L and 2.59 mg/L per OD₆₀₀ unit respectively in a 7-L fermenter. These results demonstrated that strain H.β.D.R.-5 has the capacity to produce MK-7 from maize meal hydrolysate, which could reduce the substrate cost.

带有二苯胺和结构类似物抗性的芽孢杆菌突变株以玉米水解液为底物合成甲萘醌-7的研究

[1]Armougom, F., Bittar, F., Stremler, N., et al., 2009. Microbial diversity in the sputum of a cystic fibrosis patient studied with 16S rDNA pyrosequencing. Eur. J. Clin. Microbiol. Infect. Dis., 28(9):1151-1154.

[2]Berenjian, A., Mahanama, R., Talbot, A., et al., 2011. Efficient media for high menaquinone-7 production: response surface methodology approach. New Biotechnol., 28(6): 665-672.

[3]Chen, J.N., Yang, W.S., Dick, K., et al., 2008. Tip-enhanced Raman scattering of p-thiocresol molecules on individual gold nanoparticles. Appl. Phys. Lett., 92:093110.

[4]Chen, Z.M., Li, Q., Liu, H.M., et al., 2010. Greater enhancement of Bacillus subtilis spore yields in submerged cultures by optimization of medium composition through statistical experimental designs. Appl. Microbiol. Biotechnol., 85(5):1353-1360.

[5]Fernandez, F., Collins, M.D., 1987. Vitamin K composition of anaerobic gut bacteria. FEMS Microbiol. Lett., 41(2): 175-180.

[6]Fujii, H., Sagami, H., Koyama, T., et al., 1980. Variable product specificity of solanesyl pyrophosphate synthetase. Biochem. Biophys. Res. Commun., 96(4):1648-1653.

[7]AQSIQ (General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China), SAC (Standardization Administration of the People’s Republic of China), 2007. GB/T 20885-2007: Glucose Syrup.

[8]Howard, L.M., Payne, A.C., 2006. Health Benefits of Vitamin K₂: A Revolutionary Natural Treatment for Heart Disease and Bone Loss. Basic Health Publications, California.

[9]Kim, Y.K., Kim, S.M., Kim, J.Y., et al., 2011. The culture filtrates from Bacillus subtilis natto lowers blood pressure via renin-angiotensin system in spontaneously hypertensive rats fed with a high-cholesterol diet. J. Korean Soc. Appl. Biol. Chem., 54(6):959-965.

[10]Li, H.G., Ofosu, F.K., Li, K.T., et al., 2014. Acetone, butanol, and ethanol production from gelatinized cassava flour by a new isolates with high butanol tolerance. Bioresour. Technol., 172:276-282.

[11]Li, H.P., Wang, L.Y., Li, G., et al., 2011. Manipulation of lipase activity by the helium radio-frequency, atmospheric-pressure glow discharge plasma jet. Plasma Proc. Polym., 8(3):224-229.

[12]Liu, Y., Zhang, Z.M., Qiu, H.W., et al., 2014. Surfactant supplementation to enhance the production of vitamin K₂ metabolites in shake flask cultures using Escherichia sp. mutant FM3-1709. Food Technol. Biotechnol., 52(3): 269-275.

[13]Lorenzi, V., Muselli, A., Bernardini, A.F., et al., 2009. Geraniol restores antibiotic activities against multidrug-resistant isolates from Gram-negative species. Antimicrob. Agents Chemother., 53(5):2209-2211.

[14]Patil, S.R., Dayanand, A., 2006. Optimization of process for the production of fungal pectinases from deseeded sunflower head in submerged and solid-state conditions. Bioresour. Technol., 97(18):2340-2344.

[15]Rosa-Putra, S., Hemmerlin, A., Epperson, J., et al., 2001. Zeaxanthin and menaquinone-7 biosynthesis in Sphingobacterium multivorum via the methylerythritol phosphate pathway. FEMS Microbiol. Lett., 204(2):347-353.

[16]Sagami, H., Ogura, K., Seto, S., 1977. Solanesyl pyrophosphate synthetase from Micrococcus lysodeikticus. Biochemistry, 16(21):4616-4622.

[17]Saito, Y., Ogura, K., 1981. Biosynthesis of menaquinones. Enzymatic prenylation of 1,4-dihydroxy-2-naphthoate by Micrococcus luteus membrane fractions. J. Biochem., 89(5):1445-1452.

[18]Sato, T., Yamada, Y., Ohtani, Y., et al., 2001a. Efficient production of menaquinone (vitamin K₂) by a menadione-resistant mutant of Bacillus subtilis. J. Ind. Microbiol. Biotech., 26(3):115-120.

[19]Sato, T., Yamada, Y., Ohtani, Y., et al., 2001b. Production of menaquinone (vitamin K₂)-7 by Bacillus subtilis. J. Biosci. Bioeng., 91(1):16-20.

[20]Song, J.Y., Liu, H.X., Wang, L., et al., 2014. Enhanced production of vitamin K₂ from Bacillus subtilis (natto) by mutation and optimization of the fermentation medium. Braz. Arch. Biol. Technol., 57(4):606-612.

[21]Takahashi, I., Ogura, K., Seto, S., 1980. Heptaprenyl pyrophosphate synthetase from Bacillus subtilis. J. Biol. Chem., 255:4539-4543.

[22]Tani, Y., Asahi, S., Yamada, H., 1985. Production of menaquinone (vitamin K₂)-5 by a hydroxynaphthoate-resistant mutant derived from Flavobacterium meningosepticum, a menaquinone-6 producer. Agric. Biol. Chem., 49(1): 111-115.

[23]Tsukamoto, Y., Kasai, M., Kakuda, H., 2001. Construction of a Bacillus subtilis (natto) with high productivity of vitamin K₂ (menaquinone-7) by analog resistance. Biosci. Biotechnol. Biochem., 65(9):2007-2015.

[24]Unnanuntana, A., Bonsignore, L., Shirtliff, M.E., et al., 2009. The effects of farnesol on Staphylococcus aureus biofilms and osteoblasts. An in vitro study. J. Bone Joint. Surg. Am., 91(11):2683-2692.

[25]Walther, B., Karl, J.P., Booth, S.L., et al., 2013. Menaquinones, bacteria, and the food supply: the relevance of dairy and fermented food products to vitamin K requirements. Adv. Nutr., 4:463-473.

[26]Wee, Y.J., Reddy, L.V.A., Ryu, W.H., 2008. Fermentative production of L(+)-lactic acid from starch hydrolyzate and corn steep liquor as inexpensive nutrients by batch culture of Enterococcus faecalis RKY1. J. Chem. Technol. Biotechnol., 83(10):1387-1393.

[27]Wu, W.J., Ahn, B.Y., 2011. Isolation and identification of Bacillus amyloliquefaciens BY01 with high productivity of menaquinone for cheonggukjang production. J. Korean Soc. Appl. Biol. Chem., 54(5):783-789.

[28]Xu, J.Z., Han, M., Zhang, J.L., et al., 2014. Metabolic engineering Corynebacterium glutamicum for the L-lysine production by increasing the flux into L-lysine biosynthetic pathway. Amino Acids, 46(9):2165-2175.

[29]Yanagisawa, Y., Sumi, H., 2005. Natto bacillus contains a large amount of water-soluble vitamin K (menaquinone-7). J. Food Biochem., 29(3):267-277.

[30]Zhang, X., Zhang, X.F., Li, H.P., et al., 2014. Atmospheric and room temperature plasma (ARTP) as a new powerful mutagenesis tool. Appl. Microbiol. Biotechnol., 98(12): 5387-5396.

[31]Zhang, X., Zhang, C., Zhou, Q.Q., et al., 2015. Quantitative evaluation of DNA damage and mutation rate by atmospheric and room-temperature plasma (ARTP) and conventional mutagenesis. Appl. Microbiol. Biotechnol., 99(13): 5639-5646.

[32]List of electronic supplementary materials

[33]Fig. S1 Biosynthetic pathway of MK-7 and regulation mechanism by inhibition of aromatic amino acids and diphenylamine (Armougom et al., 2009)

[34]Fig. S2 Mutation rate and lethality rate of B. amyloliquefaciens Y-2 by ARTP

[35]Fig. S3 Cell growth, MK-7 production, and sugar utilization of the mutant B. amyloliquefaciens H.β.D.R.-5 after several generations