Enhancement of 1,3-Dihydroxyacetone Production from Gluconobacter oxydans by Combined Mutagenesis

Xi  Lin; Sha  Liu; Guangrong  Xie; Jing  Chen; Penghua  Li; Jianhua  Chen

doi:10.4014/jmb.1604.04019

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Research article

Biotechnology and Bioengineering (BB) | Enzyme, Protein Engineering, and Metabolic Engineering

Enhancement of 1,3-Dihydroxyacetone Production from Gluconobacter oxydans by Combined Mutagenesis

Xi Lin ¹, Sha Liu ¹, Guangrong Xie ¹, Jing Chen ¹, Penghua Li ¹ and Jianhua Chen ^1*

School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China

Received: April 7, 2016; Accepted: July 20, 2016

J. Microbiol. Biotechnol. 2016; 26(11): 1908-1917

Published November 28, 2016 https://doi.org/10.4014/jmb.1604.04019

Abstract

Wild strain L-6 was subjected to combined mutagenesis, including UV irradiation, atmospheric and room temperature plasma, and ion beam implantation, to increase the yield of 1,3-dihydroxyacetone (DHA). With application of a high-throughput screening method, mutant Gluconobacter oxydans I-2-239 with a DHA productivity of 103.5 g/l in flask-shake fermentation was finally obtained with the starting glycerol concentration of 120 g/l, which was 115.7% higher than the wild strain. The cultivation time also decreased from 54 h to 36 h. Compared with the wild strain, a dramatic increase in enzyme activity was observed for the mutant strain, although the increase in biomass was limited. DNA and amino acid sequence alignment revealed 11 nucleotide substitutions and 10 amino acid substitutions between the sldAB of strains L-6 and I-2-239. Simulation of the 3-D structure and prediction of active site residues and PQQ binding site residues suggested that these mutations were mainly related to PQQ binding, which was speculated to be favorable for the catalyzing capacity of glycerol dehydrogenase. RT-qPCR assay indicated that the transcription levels of sldA and sldB in the mutant strain were respectively 4.8-fold and 5.4-fold higher than that in the wild strain, suggesting another possible reason for the increased DHA productivity of the mutant strain.

Keywords

1,3-Dihydroxyacetone, Glycerol, Bioconversion, Fermentation, Enzyme activity, Combined mutagenesis

References

Ameyama M, Matsushita K, Ohno Y, Shinagawa E, Adachi O. 1981. Existence of a novel prosthetic group, PQQ, in membrane-bound, electron transport chain-linked, primary dehydrogenases of oxidative bacteria. FEBS Lett. 130: 179-183.
Ameyama M, Shinagawa E, Matsushita K, Adachi O. 1985. Solubilization, purification and properties of membranebound glycerol dehydrogenase from Gluconobacter industrius. Agric. Biol. Chem. 49: 1001-1010.
Bicker M, Endres S, Ott L, Vogel H. 2005. Catalytical conversion of carbohydrates in subcritical water: a new chemical process for lactic acid production. J. Mol. Catal. A Chem. 239: 151-157.
Black CS, Nair GR. 2013. Bioconversion of glycerol to dihydroxyacetone by immobilized Gluconacetobacter xylinus cells. Int. J. Chem. Eng. Appl. 4: 310-314.
Brenner DJ, Krieg NR, Staley JT, Garrity GM. 2005. Genus VIII. Gluconacetobacter, pp. 72-73. In Brenner DJ, Krieg NR, Staley JT (eds.), Bergey’s Manual of Sytematic Bacteriology, 2nd Ed. Springer Science+Business Media, New York.
Chen J, Chen JH, Zhou CL. 2008. HPLC method for determination of dihydroxyacetone and glycerol in fermentation broth and comparison with a visible spectrophotometric method to determine dihydroxyacetone. J. Chromatogr. Sci. 46: 912-916.
Enders D, Voith M, Lenzen A. 2005. The dihydroxyacetone unit - a versatile C(3) building block in organic synthesis. Angew. Chem. Int. Ed. Engl. 44: 1304-1325.
Gätgens C, Degner U, Bringer-Meyer S, Herrmann U. 2007. Biotransformation of glycerol to dihydroxyacetone by recombinant Gluconobacter oxydans DSM 2343. Appl. Microbiol. Biotechnol. 76: 553-559.
Guo T, Tang Y, Xi YL, He AY, Sun BJ, Wu H, et al. 2011. Clostridium beijerinckii mutant obtained by atmospheric pressure glow discharge producing high proportions of butanol and solvent yields. Biotechnol. Lett. 33: 2379-2383.
Hekmat D, Bauer R, Fricke J. 2003. Optimization of the microbial synthesis of dihydroxyacetone from glycerol with Gluconobacter oxydans. Bioprocess Biosyst. Eng. 26: 109-116.
Hekmat D, Bauer R, Neff V. 2007. Optimization of the microbial synthesis of dihydroxyacetone in a semi-continuous repeated-fed-batch process by in situ immobilization of Gluconobacter oxydans. Process Biochem. 42: 71-76.
Hoshino T, Sugisawa T, Shinjoh M, Tomiyama N, Miyazaki T. 2003. Membrane-bound D-sorbitol dehydrogenase of Gluconobacter suboxydans IFO 3255 - enzymatic and genetic characterization. Biochim. Biophys. Acta 1647: 278-288.
Hu ZC, Liu ZQ, Xu JM, Zheng YG, Shen YC. 2012. Improvement of 1,3-dihydroxyacetone production from Gluconobacter oxydans by ion beam implantation. Prep. Biochem. Biotechnol. 42: 15-28.
Hu ZC, Zheng YG. 2009. A high throughput screening method for 1,3-dihydroxyacetone-producing bacterium by cultivation in a 96-well microtiter plate. J. Rapid Methods Autom. Microbiol. 17: 233-241.
Hu ZC, Zheng YG. 2011. Enhancement of 1,3-dihydroxyacetone production by a UV-induced mutant of Gluconobacter oxydans with DO control strategy. Appl. Biochem. Biotechnol. 165: 11521160.
Lapenaite I, Kurtinaitiene B, Razumiene J, Laurinavicius V, Marcinkeviciene L, Bachmatova I, et al. 2005. Properties and analytical application of PQQ-dependent glycerol dehydrogenase from Gluconobacter sp. 33. Anal. Chim. Acta 549: 140-150.
Li G, Li HP, Wang LY, Wang S, Zhao HX, Sun WT, et al. 2008. Genetic effects of radio-frequency, atmospheric-pressure glow discharges with helium. Appl. Phys. Lett. 92: 221504.
Liu RM, Liang LY, Ma JF, Ren XY, Jiang M, Chen KQ, et al. 2013. An engineering Escherichia coli mutant with high succinic acid production in the defined medium obtained by the atmospheric and room temperature plasma. Process Biochem. 48: 1603-1609.
Liu YP, Sun Y, Tan C, Li H, Zheng XJ, Jin KQ, Wang G. 2013. Efficient production of dihydroxyacetone from biodieselderived crude glycerol by newly isolated Gluconobacter frateurii. Bioresour. Technol. 142: 384-389.
Ma L, Lu W, Xia Z, Wen J. 2010. Enhancement of dihydroxyacetone production by a mutant of Gluconobacter oxydans. Biochem. Eng. J. 49: 61-67.
Miyazaki T, Tomiyama N, Shinjoh M, Hoshino T. 2002. Molecular cloning and functional expression of D-sorbitol dehydrogenase from Gluconobacter suboxydans IFO3255, which requires pyrroloquinoline quinone and hydrophobic protein SldB for activity development in E. coli. Biosci. Biotechnol. Biochem. 66: 262-270.
Nabe K, Izuo N, Yamada S, Chibata I. 1979. Conversion of glycerol to dihydroxyacetone by immobilized whole cells of Acetobacter xylinum. Appl. Environ. Microbiol. 38: 1056-1060.
Nie GJ, Yang XR, Liu H, Wang Li, Gong GH, Jin W, Zheng ZM. 2013. N+ ion beam implantation of tannase-producing and Aspergillus niger and optimization of its process parameters under submerged fermentation. Ann. Microbiol. 63: 279-287.
Raška J, Skopal F, Komers K, Machek J. 2007. Kinetics of glycerol biotransformation to dihydroxyacetone by immobilized Gluconobacter oxydans and effect of reaction conditions. Collect. Czech. Chem. Commun. 72: 1269-1283.
Roy A, Kucukural A, Zhang Y. 2010. I-TASSER: a unified platform for automated protein structure and function prediction. Nat. Protoc. 5: 725-738.
Ruch FE, Lin EC. 1975. Independent constitutive expression of the aerobic and anaerobic pathways of glycerol catabolism in Klebsiella aerogenes. J. Bacteriol. 124: 348-352.
Silva GPD, Mack M, Contiero J. 2009. Glycerol: a promising and abundant carbon source for industrial microbiology. Biotechnol. Adv. 27: 30-39.
Toyama H, Chen ZW, Fukumoto M, Adachi O, Matsushita K, Mathews FS. 2005. Molecular cloning and structural analysis of quinohemoprotein alcohol dehydrogenase ADHIIG from Pseudomonas putida HK5. J. Mol. Biol. 352: 91-104.
Wang LY, Huang ZL, Li G, Zhao HX, Xing XH, Sun WT, et al. 2009. Novel mutation breeding method for Streptomyces avermitilis using an atmospheric pressure glow discharge plasma. J. Appl. Microbiol. 108: 851-858.
Wang Q, Feng LR, Wei L, Li HG, Wang L, Zhou Y. 2014. Mutation breeding of lycopene-producing strain Blakeslea trispora by a novel atmospheric and room temperature plasma (ARTP). Appl. Biochem. Biotechnol. 174: 452-460.
Xu S, Wang X, Du G, Zhou J, Chen J. 2014. Enhanced production of L-sorbose from D-sorbitol by improving the mRNA abundance of sorbitol dehydrogenase in Gluconobacter oxydans WSH-003. Microb. Cell Fact. 13: 146.
Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y. 2014. The I-TASSER Suite: protein structure and function prediction. Nat. Methods 12: 7-8.
Yang J, Zhang Y. 2015. I-TASSER server: new development for protein structure and function predictions. Nucleic Acids Res. 43: 174-181.
Yang W, Zhou Y, Zhao ZK. 2013. Production of dihydroxyacetone from glycerol by engineered Escherichia coli cells co-expressing gldA and nox genes. Afr. J. Biotechnol. 12: 4387-4392.
Zong H, Zhan Y, Li X, Peng L, Feng F, Li D. 2012. A new mutation breeding method for Streptomyces albulus by an atmospheric and room temperature plasma. Afr. J. Microbiol. Res. 6: 3154-3158.

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Article

Research article

J. Microbiol. Biotechnol. 2016; 26(11): 1908-1917

Published online November 28, 2016 https://doi.org/10.4014/jmb.1604.04019

Enhancement of 1,3-Dihydroxyacetone Production from Gluconobacter oxydans by Combined Mutagenesis

Xi Lin ¹, Sha Liu ¹, Guangrong Xie ¹, Jing Chen ¹, Penghua Li ¹ and Jianhua Chen ^1*

School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China

Received: April 7, 2016; Accepted: July 20, 2016

Abstract

Wild strain L-6 was subjected to combined mutagenesis, including UV irradiation,
atmospheric and room temperature plasma, and ion beam implantation, to increase the yield
of 1,3-dihydroxyacetone (DHA). With application of a high-throughput screening method,
mutant Gluconobacter oxydans I-2-239 with a DHA productivity of 103.5 g/l in flask-shake
fermentation was finally obtained with the starting glycerol concentration of 120 g/l, which
was 115.7% higher than the wild strain. The cultivation time also decreased from 54 h to 36 h.
Compared with the wild strain, a dramatic increase in enzyme activity was observed for the
mutant strain, although the increase in biomass was limited. DNA and amino acid sequence
alignment revealed 11 nucleotide substitutions and 10 amino acid substitutions between the
sldAB of strains L-6 and I-2-239. Simulation of the 3-D structure and prediction of active site
residues and PQQ binding site residues suggested that these mutations were mainly related to
PQQ binding, which was speculated to be favorable for the catalyzing capacity of glycerol
dehydrogenase. RT-qPCR assay indicated that the transcription levels of sldA and sldB in the
mutant strain were respectively 4.8-fold and 5.4-fold higher than that in the wild strain,
suggesting another possible reason for the increased DHA productivity of the mutant strain.

Keywords: 1,3-Dihydroxyacetone, Glycerol, Bioconversion, Fermentation, Enzyme activity, Combined mutagenesis

Abstract

References

Ameyama M, Matsushita K, Ohno Y, Shinagawa E, Adachi O. 1981. Existence of a novel prosthetic group, PQQ, in membrane-bound, electron transport chain-linked, primary dehydrogenases of oxidative bacteria. FEBS Lett. 130: 179-183.
Ameyama M, Shinagawa E, Matsushita K, Adachi O. 1985. Solubilization, purification and properties of membranebound glycerol dehydrogenase from Gluconobacter industrius. Agric. Biol. Chem. 49: 1001-1010.
Bicker M, Endres S, Ott L, Vogel H. 2005. Catalytical conversion of carbohydrates in subcritical water: a new chemical process for lactic acid production. J. Mol. Catal. A Chem. 239: 151-157.
Black CS, Nair GR. 2013. Bioconversion of glycerol to dihydroxyacetone by immobilized Gluconacetobacter xylinus cells. Int. J. Chem. Eng. Appl. 4: 310-314.
Brenner DJ, Krieg NR, Staley JT, Garrity GM. 2005. Genus VIII. Gluconacetobacter, pp. 72-73. In Brenner DJ, Krieg NR, Staley JT (eds.), Bergey’s Manual of Sytematic Bacteriology, 2nd Ed. Springer Science+Business Media, New York.
Chen J, Chen JH, Zhou CL. 2008. HPLC method for determination of dihydroxyacetone and glycerol in fermentation broth and comparison with a visible spectrophotometric method to determine dihydroxyacetone. J. Chromatogr. Sci. 46: 912-916.
Enders D, Voith M, Lenzen A. 2005. The dihydroxyacetone unit - a versatile C(3) building block in organic synthesis. Angew. Chem. Int. Ed. Engl. 44: 1304-1325.
Gätgens C, Degner U, Bringer-Meyer S, Herrmann U. 2007. Biotransformation of glycerol to dihydroxyacetone by recombinant Gluconobacter oxydans DSM 2343. Appl. Microbiol. Biotechnol. 76: 553-559.
Guo T, Tang Y, Xi YL, He AY, Sun BJ, Wu H, et al. 2011. Clostridium beijerinckii mutant obtained by atmospheric pressure glow discharge producing high proportions of butanol and solvent yields. Biotechnol. Lett. 33: 2379-2383.
Hekmat D, Bauer R, Fricke J. 2003. Optimization of the microbial synthesis of dihydroxyacetone from glycerol with Gluconobacter oxydans. Bioprocess Biosyst. Eng. 26: 109-116.
Hekmat D, Bauer R, Neff V. 2007. Optimization of the microbial synthesis of dihydroxyacetone in a semi-continuous repeated-fed-batch process by in situ immobilization of Gluconobacter oxydans. Process Biochem. 42: 71-76.
Hoshino T, Sugisawa T, Shinjoh M, Tomiyama N, Miyazaki T. 2003. Membrane-bound D-sorbitol dehydrogenase of Gluconobacter suboxydans IFO 3255 - enzymatic and genetic characterization. Biochim. Biophys. Acta 1647: 278-288.
Hu ZC, Liu ZQ, Xu JM, Zheng YG, Shen YC. 2012. Improvement of 1,3-dihydroxyacetone production from Gluconobacter oxydans by ion beam implantation. Prep. Biochem. Biotechnol. 42: 15-28.
Hu ZC, Zheng YG. 2009. A high throughput screening method for 1,3-dihydroxyacetone-producing bacterium by cultivation in a 96-well microtiter plate. J. Rapid Methods Autom. Microbiol. 17: 233-241.
Hu ZC, Zheng YG. 2011. Enhancement of 1,3-dihydroxyacetone production by a UV-induced mutant of Gluconobacter oxydans with DO control strategy. Appl. Biochem. Biotechnol. 165: 11521160.
Lapenaite I, Kurtinaitiene B, Razumiene J, Laurinavicius V, Marcinkeviciene L, Bachmatova I, et al. 2005. Properties and analytical application of PQQ-dependent glycerol dehydrogenase from Gluconobacter sp. 33. Anal. Chim. Acta 549: 140-150.
Li G, Li HP, Wang LY, Wang S, Zhao HX, Sun WT, et al. 2008. Genetic effects of radio-frequency, atmospheric-pressure glow discharges with helium. Appl. Phys. Lett. 92: 221504.
Liu RM, Liang LY, Ma JF, Ren XY, Jiang M, Chen KQ, et al. 2013. An engineering Escherichia coli mutant with high succinic acid production in the defined medium obtained by the atmospheric and room temperature plasma. Process Biochem. 48: 1603-1609.
Liu YP, Sun Y, Tan C, Li H, Zheng XJ, Jin KQ, Wang G. 2013. Efficient production of dihydroxyacetone from biodieselderived crude glycerol by newly isolated Gluconobacter frateurii. Bioresour. Technol. 142: 384-389.
Ma L, Lu W, Xia Z, Wen J. 2010. Enhancement of dihydroxyacetone production by a mutant of Gluconobacter oxydans. Biochem. Eng. J. 49: 61-67.
Miyazaki T, Tomiyama N, Shinjoh M, Hoshino T. 2002. Molecular cloning and functional expression of D-sorbitol dehydrogenase from Gluconobacter suboxydans IFO3255, which requires pyrroloquinoline quinone and hydrophobic protein SldB for activity development in E. coli. Biosci. Biotechnol. Biochem. 66: 262-270.
Nabe K, Izuo N, Yamada S, Chibata I. 1979. Conversion of glycerol to dihydroxyacetone by immobilized whole cells of Acetobacter xylinum. Appl. Environ. Microbiol. 38: 1056-1060.
Nie GJ, Yang XR, Liu H, Wang Li, Gong GH, Jin W, Zheng ZM. 2013. N+ ion beam implantation of tannase-producing and Aspergillus niger and optimization of its process parameters under submerged fermentation. Ann. Microbiol. 63: 279-287.
Raška J, Skopal F, Komers K, Machek J. 2007. Kinetics of glycerol biotransformation to dihydroxyacetone by immobilized Gluconobacter oxydans and effect of reaction conditions. Collect. Czech. Chem. Commun. 72: 1269-1283.
Roy A, Kucukural A, Zhang Y. 2010. I-TASSER: a unified platform for automated protein structure and function prediction. Nat. Protoc. 5: 725-738.
Ruch FE, Lin EC. 1975. Independent constitutive expression of the aerobic and anaerobic pathways of glycerol catabolism in Klebsiella aerogenes. J. Bacteriol. 124: 348-352.
Silva GPD, Mack M, Contiero J. 2009. Glycerol: a promising and abundant carbon source for industrial microbiology. Biotechnol. Adv. 27: 30-39.
Toyama H, Chen ZW, Fukumoto M, Adachi O, Matsushita K, Mathews FS. 2005. Molecular cloning and structural analysis of quinohemoprotein alcohol dehydrogenase ADHIIG from Pseudomonas putida HK5. J. Mol. Biol. 352: 91-104.
Wang LY, Huang ZL, Li G, Zhao HX, Xing XH, Sun WT, et al. 2009. Novel mutation breeding method for Streptomyces avermitilis using an atmospheric pressure glow discharge plasma. J. Appl. Microbiol. 108: 851-858.
Wang Q, Feng LR, Wei L, Li HG, Wang L, Zhou Y. 2014. Mutation breeding of lycopene-producing strain Blakeslea trispora by a novel atmospheric and room temperature plasma (ARTP). Appl. Biochem. Biotechnol. 174: 452-460.
Xu S, Wang X, Du G, Zhou J, Chen J. 2014. Enhanced production of L-sorbose from D-sorbitol by improving the mRNA abundance of sorbitol dehydrogenase in Gluconobacter oxydans WSH-003. Microb. Cell Fact. 13: 146.
Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y. 2014. The I-TASSER Suite: protein structure and function prediction. Nat. Methods 12: 7-8.
Yang J, Zhang Y. 2015. I-TASSER server: new development for protein structure and function predictions. Nucleic Acids Res. 43: 174-181.
Yang W, Zhou Y, Zhao ZK. 2013. Production of dihydroxyacetone from glycerol by engineered Escherichia coli cells co-expressing gldA and nox genes. Afr. J. Biotechnol. 12: 4387-4392.
Zong H, Zhan Y, Li X, Peng L, Feng F, Li D. 2012. A new mutation breeding method for Streptomyces albulus by an atmospheric and room temperature plasma. Afr. J. Microbiol. Res. 6: 3154-3158.