Abstract
Using random chemical mutagenesis we obtained the mutant of Cupriavidus necator H16 which was capable of improved (about 35 %) production of poly(3-hydroxybuytrate) (PHB) compared to the wild-type strain. The mutant exhibited significantly enhanced specific activities of enzymes involved in oxidative stress response such as malic enzyme, NADP-dependent isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase and glutamate dehydrogenase. Probably, due to the activation of these enzymes, we also observed an increase of NADPH/NADP+ ratio. It is likely that as a side effect of the increase of NADPH/NADP+ ratio the activity of PHB biosynthetic pathway was enhanced, which supported the accumulation of PHB. Furthermore, the mutant was also able to incorporate propionate into copolymer poly(3-hydroxybuytyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] more efficiently than the wild-type strain (Y3HV/prec = 0.17 and 0.29 for the wild-type strain and the mutant, respectively)). We assume that it may be caused by lower availability of oxaloacetate for the utilization of propionyl-CoA in 2-methylcitrate cycle due to increased action of malic enzyme. Therefore, propionyl-CoA was incorporated into copolymer rather than transformed to pyruvate via 2-methylcitrate cycle. Thus, the mutant was capable of the utilization of waste frying oils and the production of P(3HB-co-3HV) with better yields and improved content of 3HV resulting in better mechanical properties of copolymer than the wild-type strain. The results of this work may be used for the development of innovative fermentation strategies for the production of PHA and also it might help to define novel targets for the genetic manipulations of PHA producing bacteria.
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References
Adwitiya P, Ashwini P, Avinash AK, Badri R, Kajal D, Vomsi P, Sridividya S (2009) Mutagenesis of Bacillus thuringiensis IAM 12077 for increasing poly(-β-)hydroxybutyrate (PHB) production. Turk J Biol 33:225–230
Akiyama M, Taima Y, Doi Y (1992) Production of poly(3-hydroxyalkanoates) by bacterium of the genus Alcaligenes utilizing long-chain fatty acids. Appl Microbiol Biotehnol 37:698–701
Alias Z, Tan IKP (2005) Isolation of palm oil-utilazing, polyhydroxyalkanoate (PHA)-producing bacteria by an enrichement technique. Bioresour Technol 96:1229–1234
Annuar MSM, Tan IKP, Ibrahim S, Ramachandran KB (2007) Production of medium-chain-length poly(3-hydroxyalkanoates) from crude fatty acids mixture by Pseudomonas putida. Food Bioprod Process 85:104–119
Ayub N, Tribelli PM, Lopez NI (2009) Polyhydroxyalkanoates are essential for maintenance of redox state in Antarctic bacterium Pseudomonas sp. 14–3 during low temperature adaptation. Extremophiles 13:59–66
Bramer CO, Steinbuchel A (2001) The methylcitric acid pathway in Ralstonia eutropha: new genes identified and involved in propionate metabolism. Microbiology 147:2203–2214
Bramer CO, Steinbuchel A (2002) The malate dehydrogenase of Ralstonia eutropha and functionality of the C3/C4 metabolism in Tn5-induced mdh mutant. FEMS Microbiol Lett 212:159–164
Brandl H, Gross RA, Lenz RW, Fuller RC (1988) Pseudomonas oleovorans as a source of poly(beta-hydroxyalkanoates) for potential application as a biodegradable polyester. Appl Environ Microbiol 54:1977–1982
Bruland N, Voss I, Bramer C, Steinbuchel A (2010) Unravelling the C3/C4 carbon metabolism in Ralstonia eutropha H16. J Appl Microbiol 109:79–90
Budde CF, Riedel SL, Hubner F, Risch S, Popovic MK, Rha C, Sinskey AJ (2011) Growth and polyhydroxybutyrate production by Ralstonia eutropha in emulsified plant oil medium. Appl Microbiol Biotechnol 89:1611–1619
Calhoun LN, Kwon YM (2010) Structure, function and regulation of the DNA-binding protein Dps and its role in acid and oxidative stress resistance in Escherichia coli: a review. J Appl Microbiol 110:375–386
Chan PL, Yu V, Wai L, Yu HF (2006) Production of medium-chain-length polyhydroxyalkanoates by Pseudomonas aeruginosa with fatty acids and alternative carbon sources. Appl Biochem Biotechnol 129:933–941
Choi JI, Lee SY (1997) Process analysis and economical evaluation for poly(3-hydroxybutyrate) production by fermentation. Bioprocess Eng 17:335–342
Ewering C, Bramer C, Bruland N, Bethke A, Steinbuchel A (2006) Occurence and expression of tricarboxylate synthases in Ralstonia eutropha. Appl Microbiol Biotechnol 71:80–89
Faccin DJL, Correa MP, Rech R, Ayub MAZ, Secchi AR, Cardozo NSM (2012) Modeling P(3HB) production by Bacillus megaterium. J Chem Technol Biotechnol 87:325–333
Gao X, Yuan XX, Shi ZY, Guo YY, Shen XW, Chen JC, Wu Q, Chen GQ (2012) Production of copolyesters of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates by E-coli containing an optimized PHA synthase gene. Microb Cell Fact 11:130
Guzman H, Van-Thouac D, Martin J, Hatti-Kaul R, Quillaguaman J (2009) A process for the production of ectoine and poly(3-hydroxybutyrate) by Halomonas boliviensis. Appl Microbiol Biot 84:1069–1077
Ienczak JL, Schmidell W, de Aragao GMF (2013) High-cell-density culture strategies for polyhydroxyalkanoate production: a review. J Ind Microbiol Biotechnol 40:275–286. doi:10.1007/s10295-013-1236-z
Jung YM, Lee YH (2000) Utilization of oxidative pressure for enhanced production of poly-b-hydroxybutyrate and poly(3-hydroxybuytyrate-3-hydroxyvalerate) in Ralstonia eutropha. J Biosci Bioeng 90:266–270
Kacmar J, Carlson R, Balogh SJ, Srienc F (2005) Staining and quantification of poly-3-hydroxybuytyrate in Saccharomyces cerevisiae and Cupriavidus necator cell population using automated flow cytometry. Cytometry Part A 69A:27–35
Kartika IA, Yani M, Ariono D, Evon P, Rigal L (2013) Biodiesel production from jatropha seeds: solvent extraction and in situ transesterification in a single step. Fuel 106:111–117
Kessler B, Wilholt B (1999) Poly(3-hydroxyalkanoates. In: Flickinger MC, Drew SW (eds) Encyclopedia of bioprocess technology—fermentation, biocatalysis and bioseparation. Wiley, New York, pp 2024–2040
Kessler B, Wilholt B (2001) Factors involved in the regulatory of polyhydroxyalkanoate metabolism. J Biotechnol 86:97–104
Kimura H, Takahashi T, Hiraka H, Iwana M, Takeishi M (1999) Effective biosynthesis of poly(3-hydroxybutyrate) from plant oils by Chromobacterium sp. Polym J 31:210–212
Lee IY, Kim MK, Park YH, Lee SY (1996) Regulatory effect of cellular nicotinamide nucleotides and enzyme activities on poly(3-hydroxybutyrate) synthesis in recombinant Escherichia coli. Biotechnol Bioeng 52:702–712
Marles-Wright J, Lewis RJ (2007) Stress response of bacteria. Curr Opin Struc Biol 17:755–760
Marsudi S, Unno H, Hori K (2008) Palm oil utilization for the simultaneous production of polyhydroxyalkanoates and rhamnolipids by Pseudomonas aeruginosa. Appl Microbiol Biotechnol 78:955–961
Moen B, Janbu AO, Langsrud S, Langsrud O, Hobman JL, Constantinidou C, Kohler A, Knut R (2009) Global responses of Escherichia coli to adverse conditions determined by microarrays and FT-IR spectroscopy. Can J Microbiol 55:714–728
Mothes G, Ackermann JU (2005) Synthesis of poly(3-hydroxy-butyrate-co-4-hydroxybutyrate) with a target mole fraction of 4-hydroxybutyric acid units by two-stage continuous cultivation of Delftia acidovorans P4a. Eng Life Sci 5:58–62
Mozejko J, Wilke A, Przybylek G, Ciesielski S (2012) Mcl-PHAs Produced by Pseudomona ssp Gl01 using fed-batch cultivation with waste rapeseed oil as carbon source. J Microbiol Biotechnol 22:371–377
Murakami K, Tsubouchi R, Fukayama M, Ogawa T, Yoshino M (2006) Oxidative inactivation of reduced NADP-generating enzymes in E. coli: iron-dependent inactivation with affinity cleavage of NADP-isocitrate dehydrogenase. Arch Microbiol 186:385–392
Obruca S, Marova I, Snajdar O, Mravcova L, Svoboda Z (2010a) Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Cupriavidus necator from waste rapeseed oil using propanol as a precursor of 3-hydroxyvalerate. Biotechnol Lett 39:1925–1932
Obruca S, Marova I, Stankova M, Mravcova L, Svoboda Z (2010b) Effect of ethanol and hydrogen peroxide on poly(3-hydroxybutyrate) biosynthetic pathway in Cupriavidus necator H16. World J Microbiol Biotechnol 26:1261–1267
Obruca S, Marova I, Svoboda Z, Mikulikova R (2010c) Use of controlled exogenous stress for improvement of poly(3-hydroxybutyrate) production in Cupriavidus necator. Folia Microbiol 55:17–22
Pradella JGC, Taciro MK, Pataquiva AY (2010) High-cell-den-sity poly (3-hydroxybutyrate) production from sucrose using Burkholderia sacchari culture in airlift bioreactor. Bioresour Technol 101:8355–8360
Rangel DEN (2011) Stress induced cross-protection against environmental challenges on prokaryotic and eukarytotic microbe. World J Microbiol Biotechnol 27:1281–1296
Reinecke F, Steinbuchel A (2009) Ralstonia eutropha Strain H16 as model organism for PHA metabolism and for biotechnological production of technically interesting biopolymers. J Mol Microbiol Biotechnol 16:91–108
Ruiz JA, Lopez NI, Fernandez R, Mendez BS (2001) Polyhydroxyalkanoates degradation is associated with nucleotide accumulation and enhances stress resistance and survival of Pseudomonas oleovorans in natural water microcosms. Appl Environ Microbiol 67:225–230
Saito T, Fukui T, Ikeda F, Tanaka Y, Tomita K (1977) An NADP-linked acetoacetyl-CoA reductase from Zooglear amigera. Arch Microbiol 114:211–217
Serafim LS, Lemos PC, Albuquerque MGE, Reis MAM (2008) Strategies for PHA production by mixed cultures and renewable waste materials. Appl Microbiol Biotechnol 81:615–628
Singh R, Beriault R, Middaugh J, Hamel R, Chenier D, Appanna VD, Kalyuzhnyi S (2005) Aluminum-tolerant Pseudomonas fluorescens: rOS toxicity and enhanced NADPH production. Extremophiles 9:367–373
Singh R, Mailloux RJ, Puiseux-Dao S, Apanna V (2007) Oxidative stress evokes a metabolic adaptation that favors increased NADPH synthesis and decreased NADH production in Pseudomonas fluorescens. J Bacteriol 189:6665–6675
Slater S, Houmiel KL, Tran M, Mitsky TA, Taylor NB, Padgette SR, Gruys KJ (1998) Multiple beta-ketothiolases mediate poly(beta-hydroxyalkanoate) copolymer synthesis in Ralstonia eutropha. J Bacteriol 180:1979–1987
Spierkermann P, Rehm BHA, Kalscheuer R, Baumeister D, Steinbuchel A (1999) A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Arch Microbiol 171:73–80
Sudesh K, Abe H, Doi Y (2000) Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. Prog Polym Sci 25:1503–1555
Taniguchi I, Kagotani K, Kimura Y (2003) Microbial production of poly(hydroxyalkanoates)s from waste edible oils. Green Chem 5:545–548
Vidal-Mas J, Resina-Pelfort O, Haba E, Comas J, Maresa A, Vives-Rego J (2001) Rapid flow cytometry—Nile red assssment of PHA cellular content bad heterogenitity in cultures of Pseudomonas aeruginosa 4ZT2 (NCIB 40044) grown in waste frying oil. Antonie Van Leeuwenhoek 80:57–63
Wang ZX, Bramer C, Steinbuchel A (2003) Two phenotypypically compensating isocitrate dehydrogenase in Ralstonia eutropha. FEMS Microbiol Lett 227:9–16
Yamane T, Fukunaga M, Lee YW (1996) Increased PHB production by high-cell-density fed-batch culture of Alcaligenes latus, a growth associated PHB producer. Biotechnol Bioeng 50:197–202
Yu J, Si YT (2004) Metabolic carbon fluxes and biosynthesis of polyhydroxyalkanoates in Ralstonia eutropha on short fatty acids. Biotechnol Prog 20:1015–1024
Zhang Z, Yu J, Stanton RC (2000) A method for determination of pyridine nucleotides using a single extract. Anal Biochem 285:163–167
Zhang Y, Adams IP, Ratledge C (2007) Malic enzyme: the controlling activity for lipid production? Overexpression of malic enzyme in Mucor circinelloides leads to a 2.5-fold increase in lipid accumulation. Microbiol-SGM 153:2013–2025
Acknowledgments
This work was supported by project “Centre for Materials Research at FCH BUT” No. CZ.1.05/2.1.00/01.0012 from ERDF and by the project “Excellent young researcher at BUT” No. CZ.1.07./2.3.00/30.0039.
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Obruca, S., Snajdar, O., Svoboda, Z. et al. Application of random mutagenesis to enhance the production of polyhydroxyalkanoates by Cupriavidus necator H16 on waste frying oil. World J Microbiol Biotechnol 29, 2417–2428 (2013). https://doi.org/10.1007/s11274-013-1410-5
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DOI: https://doi.org/10.1007/s11274-013-1410-5