Research of 1,3-Dihydroxyacetone Production by Overexpressing Glycerol Transporter and Glycerol Dehydrogenase

Abstract

1,3-Dihydroxyacetone (DHA), a natural ketose, is widely used in the chemical, cosmetic, and pharmaceutical industries. The current method for DHA production is Gluconobacter oxydans (G. oxydans) fermentation, but the high concentration of glycerol in the fermentation broth inhibits cells growth. To overcome this obstacle, in this study, we overexpressed the glycerol transporter (GlpFp) by the use of promoters P _tufB, P _gmr, P _glp1, and P _glp2 in G. oxydans 621H. The results show that the glycerol tolerances of strains overexpressing GlpF were all much better than that of the control strain. The glycerol dehydrogenase gene (Gdh) was overexpressed by the promoters P _tufB and P _gdh, which increased the DHA titer by 12.7% compared with that of the control group. When GlpF and Gdh genes were co-overexpressed in G. oxydans 621H, the OD600 value of the engineered strains all increased, but the DHA titers decreased in different degrees, as compared with strains that overexpressed only Gdh. This study provides a reference for future research on DHA production.

Keywords

G. oxydans 621H / 1,3-dihydroxyacetone / Glycerol / Glycerol transporter / Glycerol dehydrogenase

References

[1] Meybeck A. A spectroseopic study of reaction products of dihydroxyacetone with aminoacids. J Soc Cosmet Chem, 1977, 28: 25-35.

[2] Bobin MF, Martii MC, Cotte J. Effect of color adjuvants on the tanning effect of dihydroxyacetone. J Soc Cosmet Chem, 1984, 35: 265-272.

[3] Fesq H, Brockow K, Strom K, et al. Dihydroxyacetone in a new formulation: a powerful therapeutic option in vitiligo. Dermatology, 2001, 203(3): 241-243.

[4] Stanko RT, Robertson RJ, Spina RJ, et al. Enhancement of arm exercise endurance capacity with dihydroxyacetone and pyruvate. J Appl Physiol, 1990, 68(1): 119

[5] Ivy JL. Effect of pyruvate and dihydroxyacetone on metabolism and aerobic endurance capacity. Med Sci Sports Exerc, 1998, 30(6): 837-843.

[6] Schlifke AC. Can pyruvate and dihydroxyacetone(DHA) improve athletic performance. Nutr Bytes, 1999, 5: 1-5.

[7] Niknahad H, Ghelichkhani E. Antagonism of cyanide poisoning by dihydroxyacetone. Toxicol Lett (Shannon), 2002, 132(2): 95-100.

[8] Cummings TF. The treatment of cyanide poisoning. Occup Med, 2004, 54(2): 82-85.

[9] Oh CH, Hong JH. Short synthesis and antiviral evaluation of c-fluoro-branched cyclopropyl nucleosides. Nucleosides, Nucleotides Nucleic Acids, 2007, 26(4): 403-411.

[10] Henderson PW, Kadouch DJ, Singh SP, et al. A rapidly resorbable hemostatic biomaterial based on dihydroxyacetone. J Biomed Mater Res Part A, 2010, 93(2): 776-782.

[11] Hekmat D, Bauer R, Fricke J. Optimization of the microbial synthesis of dihydroxyacetone from glycerol with Gluconobacter oxydans. Bioprocess Biosyst Eng, 2003, 26(2): 109-116.

[12] Green SR, Whalen EA, Molokie E. Dihydroxyacetone: production and uses. Biotechnol Bioeng, 1961, 3(4): 351-355.

[13] Tanamool V, Hongsachart P, Soemphol W. Bioconversion of biodiesel-derived crude glycerol to 1, 3-dihydroxyacetone by a potential acetic acid bacteria. Sains Malays, 2018, 47(3): 481-488.

[14] Dikshit PK, Moholkar VS. Kinetic analysis of dihydroxyacetone production from crude glycerol by immobilized cells of Gluconobacter oxydans MTCC 904. Bioresour Technol, 2016, 216: 948-957.

[15] Claret C, Bories A, Soucaille P. Glycerol inhibition of growth and dihydroxyacetone production by Gluconobacter oxydans. Curr Microbiol, 1992, 25(3): 149-155.

[16] Dikshit PK, Kharmawlong GJ, Moholkar VS. Investigations in sonication–induced intensification of crude glycerol fermentation to dihydroxyacetone by free and immobilized Gluconobacter oxydans. Biores Technol, 2018, 256: 302-311.

[17] Stasiak-Różańska L, Berthold-Pluta A, Dikshit PK. Valorization of waste glycerol to dihydroxyacetone with biocatalysts obtained from Gluconobacter oxydans. Appl Sci, 2018, 8(12): 2517

[18] Bauer R, Katsikis N, Varga S, et al. Study of the inhibitory effect of the product dihydroxyacetone on Gluconobacter oxydans in a semi-continuous two-stage repeated-fed-batch process. Bioprocess Biosyst Eng, 2005, 28(1): 37-43.

[19] Claret C, Bories A, Soucaille P. Inhibitory effect of dihydroxyacetone on Gluconobacter oxydans: kinetic aspects and expression by mathematical equations. J Ind Microbiol, 1993, 11(2): 105-112.

[20] Dikshit PK, Moholkar VS. Batch and repeated-batch fermentation for 1,3-dihydroxyacetone production from waste glycerol using free, immobilized and resting Gluconobacter oxydans cells. Waste Biomass Valoriz, 2018.

[21] Gätgens C, Degner U, Bringer-Meyer S, et al. Biotransformation of glycerol to dihydroxyacetone by recombinant Gluconobacter oxydans DSM 2343. Appl Microbiol Biotechnol, 2007, 76(3): 553-559.

[22] Habe H, Fukuoka T, Morita T, et al. Disruption of the membrane-bound alcohol dehydrogenase-encoding gene improved glycerol use and dihydroxyacetone productivity in Gluconobacter oxydans. Biosci Biotechnol Biochem, 2010, 74(7): 1391-1395.

[23] Calmes R, Deal SJ. Glycerol transport by Nocardia asteroides. Can J Microbiol, 1972, 18(11): 1703-1708.

[24] Richey DP, Lin EC. Importance of facilitated diffusion for effective utilization of glycerol by Escherichia coli. J Bacteriol, 1972, 112(2): 784-790.

[25] Saheb SA. Perméation du glycérol et sporulation chez Bacillus subtilis. Can J Microbiol, 1972, 18(8): 1307-1313.

[26] Stasiak-Różańska L, Błażejak S, Gientka I. Effect of glycerol and dihydroxyacetone concentrations in the culture medium on the growth of acetic acid bacteria Gluconobacter oxydans ATCC 621. Eur Food Res Technol, 2014, 239(3): 453-461.

[27] Claret C, Salmon JM, Romieu C, et al. Physiology of Gluconobacter oxydans during dihydroxyacetone production from glycerol. Appl Microbiol Biotechnol, 1994, 41(3): 359-365.

[28] Hedfalk K, Bill RM, Hohmann S, et al. Overexpression and purification of the glycerol transport facilitators, fps1p and GlpF, in Saccharomyces Cerevisiae and Escherichia coli. Mol Biol Physiol Water Solut Transp, 2000.

[29] Wei DZ, Ma XY, Zheng Y et al (2007) Gene engineering bacteria for producing dihydroxy acetone, constructing method, and application: CN, 101092604A. 2007-12-26 (in Chinese)

[30] Saito Y, Ishii Y, Hayashi H, et al. Cloning of genes coding for L-sorbose and L-sorbosone dehydrogenases from Gluconobacter oxydans and microbial production of 2-keto-L-gulonate, a precursor of L-ascorbic acid, in a recombinant G. oxydans strain. Appl Environ Microbiol, 1997, 63(2): 454-460.

[31] Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol, 1983, 166(4): 557-580.

[32] Boyer HW, Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol, 1969, 41(3): 459-472.

[33] Yang XP, Wei LJ, Lin JP, et al. Membrane-bound pyrroloquinoline quinone-dependent dehydrogenase in Gluconobacter oxydans M5, responsible for production of 6-(2-hydroxyethyl) amino-6-deoxy-L-sorbose. Appl Environ Microbiol, 2008, 74(16): 5250-5253.

[34] Kovach ME, Elzer PH, Steven Hill D, et al. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene, 1995, 166(1): 175-176.

[35] Figurski DH, Helinski DR. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci, 1979, 76(4): 1648-1652.

[36] Xue CY, Zhang XM, Yu ZR, et al. Up-regulated spinosad pathway coupling with the increased concentration of acetyl-CoA and malonyl-CoA contributed to the increase of spinosad in the presence of exogenous fatty acid. Biochem Eng J, 2013, 81: 47-53.

[37] Sugisawa T, Hoshino T. Purification and properties of membrane-bound D-sorbitol dehydrogenase from Gluconobacter suboxydans IFO 3255. Biosci Biotechnol Biochem, 2002, 66(1): 57-64.

[38] Li MH, Wu J, Liu X, et al. Enhanced production of dihydroxyacetone from glycerol by overexpression of glycerol dehydrogenase in an alcohol dehydrogenase-deficient mutant of Gluconobacter oxydans. Bioresour Technol, 2010, 101(21): 8294-8299.