Preferences help
enabled [disable] Abstract
Number of results
2017 | 64 | 1 | 123-133
Article title

Group II intron-mediated deletion of lactate dehydrogenase gene in an isolated 1,3-propanediol producer Hafnia alvei AD27

Title variants
Languages of publication
Our previous studies showed that glycerol fermentation by Hafnia alvei AD27 strain was accompanied by formation of high quantities of lactate. The ultimate aim of this work was the elimination of excessive lactate production in the 1,3-propanediol producer cultures. Group II intron-mediated deletion of ldh (lactate dehydrogenase) gene in an environmental isolate of H. alvei AD27 strain was conducted. The effect of the Δldh genotype in H. alvei AD27 strain varied depending on the culture medium applied. Under lower initial glycerol concentration (20 gL-1), lactate and 1,3-propanediol production was fully abolished, and the main carbon flux was directed to ethanol synthesis. On the other hand, at higher initial glycerol concentrations (40 gL-1), 1,3-propanediol and lactate production was recovered in the recombinant strain. The final titers of 1,3-propanediol and ethanol were similar for the recombinant and the WT strains, while the Δldh genotype displayed significantly decreased lactate titer. The by-products profile was altered upon ldh gene deletion, while glycerol utilization and biomass accumulation remained unaltered. As indicated by flow-cytometry analyses, the internal pH was not different for the WT and the recombinant Δldh strains over the culture duration, however, the WT strain was characterized by higher redox potential.
Physical description
  • Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Poznań, Poland
  • Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Poznań, Poland
  • Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Poznań, Poland
  • Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Poznań, Poland
  • Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Poznań, Poland
  • Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Poznań, Poland
  • Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Poznań, Poland
  • Ahrens K, Menzel K, Zeng AP, Deckwer WD (1998) Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: III. Enzymes and fluxes of glycerol dissimilation and 1,3-propanediol formation. Biotechnol Bioeng 59: 544-552. doi: 10.1002/(SICI)1097-0290(19980905)59:5<544::AID-BIT3>3.0.CO;2-A.
  • Anand P, Saxena RK, Marwah RG (2011) A novel downstream process for 1,3-propanediol from glycerol-based fermentation. Appl Microbiol Biotechnol 90: 1267-1276. doi: 10.1007/s00253-011-3161-2.
  • Barbirato F, Camarasca-Claret C, Bories A, Grivet JP (1995) Description of the glycerol fermentation by a new 1,3-propanediol producing microorganism: Enterobacter agglomerans. Appl Microbiol Biotechnol 43: 786-793. doi: 10.1007/bf02431909
  • Biebl H, Zeng AP, Menzel K, Deckwer WD (1998) Fermentation of glycerol to 1,3-propanediol and 2,3-butanediol by Klebsiella pneumoniae Appl Microbiol Biotechnol 50: 24-29. doi: 10.1007/s002530051251.
  • Celińska E (2010) Debottlenecking the 1,3-propanediol pathway by metabolic engineering. Biotechnol Adv 28: 519-530. doi: 10.1016/j.biotechadv.2010.03.003.
  • Celińska E (2012) Klebsiella spp. as a 1,3-propanediol producer - the metabolic engineering approach. Crit Rev Biotechnol 32: 274–288. doi: 10.3109/07388551.2011.616859.
  • Celińska E (2015a) Fully glycerol-independent microbial production of 1, 3-propanediol via non-natural pathway: paving the way to success with synthetic tiles. Biotechnol J 10: 242-243. doi: 10.1002/biot.201400360.
  • Celińska E, Drożdżyńska A, Jankowska M, Białas W, Czaczyk K, Grajek W (2015b) Genetic engineering of Citrobacter freundii AD970 strain towards enhanced 1,3-propanediol production. Process Biochem 50: 48-60. doi: 10.1016/j.procbio.2014.09.001
  • Celińska E, Grajek W (2013) A novel multigene expression construct for modification of glycerol metabolism in Yarrowia lipolytica. Microbial Cell Factories 12: 102. doi: 10.1186/1475-2859-12-102.
  • Celinska E, Grajek W (2009) Biotechnological production of 2,3-butanediol - current state and prospects. Biotechnol Adv 27: 715–725. doi: 10.1016/j.biotechadv.2009.05.002.
  • Chen Y, McClane BA, Fisher DJ, Rood JI, Gupta P (2005a) Construction of an alpha toxin gene knockout mutant of Clostridium perfringens type A by use of a mobile group II intron. Appl Environ Microbiol 71: 7542-7547. doi: 10.1128/AEM.71.11.7542-7547.2005.
  • Chen Y, Klein JR, McKay LL, Dunny GM (2005b) Quantitative analysis of group II intron expression and splicing in Lactococcuslactis. Appl Environ Microbiol 71: 2576-2586. doi: 10.1128/AEM.71.5.2576-2586.2005.
  • Chen Z, Geng F, Zeng AP (2015) Protein design and engineering of a de novo pathway for microbial production of 1,3-propanediol from glucose. Biotechnol J 10: 284-289. doi: 10.1002/biot.201400235.
  • Cheng KK, Liu HJ, Liu DH (2005) Multiple growth inhibition of Klebsiella pneumoniae in 1,3-propanediol fermentation. Biotechnol Lett 27: 19-22. doi: 10.1007/s10529-004-6308-8.
  • Cui YL, Zhou JJ, Gao LR, Zhu CQ, Jiang X, Fu SL, Gong H (2014) Utilization of excess NADH in 2,3-butanediol-deficient Klebsiella pneumoniae for 1,3-propanediol production. J Appl Microbiol 117: 690-698. doi: 10.1111/jam.12588.
  • Dietz D, Zeng AP (2014) Efficient production of 1,3-propanediol from fermentation of crude glycerol with mixed cultures in a simple medium. Bioprocess Biosyst Eng 37: 225-233. doi: 10.1007/s00449-013-0989-0.
  • Drożdżyńska A, Pawlicka J, Kubiak P, Kośmider A, Pranke D, Olejnik-Schmidt A, Czaczyk K (2014) Conversion of glycerol to 1,3-propanediol by Citrobacterfreundii and Hafnia alvei - newly isolated strains from the Enterobacteriaceae. N Biotechnol 31: 402–410. doi: 10.1016/j.nbt.2014.04.002.
  • Frazier CL, San Filippo J, Lambowitz AM, Mills DA (2003) Genetic manipulation of Lactococcus lactis by using targeted group II introns: generation of stable insertions without selection. Appl Environ Microbiol 69: 1121-1128. doi: 10.1128/AEM.69.2.1121-1128.2003.
  • Guo X, Fang H, Zhuge B, Zong H, Song J, Zhuge J, Du X (2013) budC knockout in Klebsiella pneumoniae for bioconversion from glycerol to 1,3-propanediol. Biotechnol Appl Biochem 60: 557-563. doi: 10.1002/bab.1114.
  • Hao J, Xu F, Liu HJ, Liu DH (2006) Downstream processing of 1,3-propanediol fermentation broth. J Chem Technol Biotechnol 81: 102-108. doi: 10.1002/jctb.1369
  • Heap JT, Pennington OJ, Cartman ST, Carter GP, Minton NP (2007) The ClosTron: a universal gene knock-out system for the genus Clostridium. J Microbiol Methods 70: 452-464. doi: 10.1016/j.mimet.2007.05.021.
  • Homann T, Tag C, Biebl H, Deckwer WD, Schink B (1990) Fermentation of glycerol to 1,3-propanediol by Klebsiella and Citrobacter strains. Appl Microbiol Biotechnol 33: 121-126. doi: 10.1007/BF00176511
  • Hong WK, Kim CH, Heo SY, Luo LH, Oh BR, Rairakhwada D, Seo JW (2011) 1,3-Propandiol production by engineered Hansenula polymorpha expressing dhagenes from Klebsiella pneumoniae. Bioprocess Biosyst Eng 34: 231-236. doi: 10.1007/s00449-010-0465-z.
  • Horng YT, Chang KC, Chou TC, Yu CJ, Chien CC, Wei YH, Soo PC (2010) Inactivation of dhaD and dhaK abolishes by-product accumulation during 1,3-propanediol production in Klebsiella pneumoniae. J Ind Microbiol Biotechnol 37: 707-716. doi: 10.1007/s10295-010-0714-9.
  • Ji XJ, Huang H, Zhu JG, Hu N, Li S (2009) Efficient 1,3-propanediol production by fed-batch cultureof Klebsiella Pneumoniae: the role of pH fluctuation. Appl Biochem Biotechnol 159: 605-613. doi: 10.1007/s12010-008-8492-9.
  • Kaeding T, DaLuz J, Kube J, Zeng AP (2015) Integrated study of fermentation and downstream processing in a miniplant significantly improved the microbial 1,3-propanediol production from raw glycerol. Bioprocess Biosyst Eng 38: 575-586. doi: 10.1007/s00449-014-1297-z.
  • Kang TS, Korber DR, Tanaka T (2014) Bioconversion of glycerol to 1,3-propanediol in thin stillage-based media by engineered Lactobacillus panis PM1. J Ind Microbiol Biotechnol 41: 629-635. doi: 10.1007/s10295-014-1403-x.
  • Kaur G, Srivastava AK, Chand S (2013) Bioconversion of glycerol to 1,3-propanediol: a mathematical model-based nutrient feeding approach for high production using Clostridium diolis. Bioresour Technol 142: 82-87. doi: 10.1016/j.biortech.2013.05.040.
  • Karberg M, Guo H, Zhong J, Coon R, Perutka J, Lambowitz AM (2001) Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria. Nat Biotechnol 19: 1162-1167. doi: 10.1038/nbt1201-1162.
  • Kumar V, Sankaranarayanan M, Durgapal M, Zhou S, Ko Y, Ashok S, Sarkar R, Park S (2013) Simultaneous production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol using resting cells of the lactate dehydrogenase-deficient recombinant Klebsiella pneumoniae overexpressing an aldehyde dehydrogenase. Bioresour Technol 135: 555-563. doi: 10.1016/j.biortech.2012.11.018.
  • Kumar V, Sankaranarayanan M, Jae KE, Durgapal M, Ashok S, Ko Y, Sarkar R, Park S (2012) Co-production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol using resting cells of recombinant Klebsiella pneumoniae J2B strain overexpressing aldehyde dehydrogenase. Appl Microbiol Biotechnol 96: 373-383. doi: 10.1007/s00253-012-4187-9.
  • Liang Q, Zhang H, Li S, Qi Q (2011) Construction of stress-induced metabolic pathway from glucose to 1,3-propanediol in Escherichia coli. Appl Microbiol Biotechnol 89: 57-62. doi: 10.1007/s00253-010-2853-3.
  • Lin EC (1976) Glycerol dissimilation and its regulation in bacteria. Annu Rev Microbiol 30: 535-578. doi: 10.1146/annurev.mi.30.100176.002535.
  • Ma Z, Bian Y, Shentu X, Yu X (2013) Development of a novel recombinant strain Zygosacharomyces rouxii JL2011 for 1,3-propanediol production from glucose. Appl Microbiol Biotechnol 97: 4055-4064. doi: 10.1007/s00253-012-4501-6.
  • Maervoet VE, De Maeseneire SL, Avci FG, Beauprez J, Soetaert WK, De Mey M (2014) 1,3-propanediol production with Citrobacter werkmanii DSM17579: effect of a dhaD knock-out. Microb Cell Fact 13: 70. doi: 10.1186/1475-2859-13-70.
  • Metsoviti M, Zeng AP, Koutinas AA, Papanikolaou S (2013) Enhanced 1,3-propanediol production by a newly isolated Citrobacterfreundii strain cultivated on biodiesel-derived waste glycerol through sterile and non-sterile bioprocesses. J Biotechnol 163: 408-418. doi: 10.1016/j.jbiotec.2012.11.018.
  • Nakamura C, Whited G (2003) Metabolic engineering for the microbial production of 1,3-propanediol. Curr Opin Biotechnol 14: 454-459. doi: 10.1016/j.copbio.2003.08.005.
  • Oh BR, Seo JW, Heo SY, Luo LH, Hong WK, Park DH, Kim CH (2013) Efficient production of 1,3-propanediol from glycerol upon constitutive expression of the 1,3-propanediol oxidoreductase gene in engineered Klebsiella pneumoniae with elimination of by-product formation. Bioprocess Biosyst Eng 36: 757-763. doi: 10.1007/s00449-013-0901-y.
  • Pflügl S, Marx H, Mattanovich D, Sauer M (2014) Heading for an economic industrial upgrading of crude glycerol from biodiesel production to 1,3-propanediol by Lactobacillus diolivorans. Bioresour Technol 152: 499-504. doi: 10.1016/j.biortech.2013.11.041.
  • Rao Z, Ma Z, Shen W, Fang H, Zhuge J, Wang X (2008) Engineered Saccharomyces cerevisiae that produces 1,3-propanediol from D-glucose. J Appl Microbiol 105: 1768-1776. doi: 10.1111/j.1365-2672.2008.03868.x.
  • Ricci MA, Russo A, Pisano I, Palmieri L, de Angelis M, Agrimi G (2015) Improved 1,3-Propanediol Synthesis from Glycerol by the Robust Lactobacillus reuteri Strain DSM 20016. J Microbiol Biotechnol 25: 893-902. doi: 10.4014/jmb.1411.11078.
  • Rodriguez A, Wojtusik M, Ripoll V, Santos VE, Garcia-Ochoa F (2016) 1,3-Propanediol production from glycerol with a novel biocatalyst Shimwelliablattae ATCC 33430: Operational conditions and kinetics in batch cultivations. Bioresour Technol 200: 830-837. doi: 10.1016/j.biortech.2015.10.061.
  • Sabra W, Groeger C, Zeng AP (2016) Microbial Cell Factories for Diol Production. Adv Biochem Eng Biotechnol 155: 165-197. doi: 10.1007/10_2015_330.
  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual 2nd edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
  • Saxena RK, Anand P, Saran S, Isar J (2009) Microbial production of 1,3-propanediol: Recent developments and emerging opportunities. Biotechnol Adv 27: 895-913. doi: 10.1016/j.biotechadv.2009.07.003.
  • Seo MY, Seo JW, Heo SY, Baek JO, Rairakhwada D, Oh BR, Seo PS, Choi MH, Kim CH (2009) Elimination of by-product formation during production of 1,3-propanediol in Klebsiella pneumoniae by inactivation of glycerol oxidative pathway. Appl Microbiol Biotechnol 84: 527-534. doi: 10.1007/s00253-009-1980-1.
  • Tarmy EM, Kaplan NO (1968a) Chemical characterization of D-lactate dehydrogenase from Escherichia coli B. J Biol Chem 243: 2579-2586.
  • Tarmy EM, Kaplan NO (1968b) Kinetics of Escherichia coli B D-lactate dehydrogenase and evidence for pyruvate-controlled change in conformation. J Biol Chem 243: 2587-2596.
  • Wu Z, Wang Z, Wang G, Tan T (2013) Improved 1,3-propanediol production by engineering the 2,3-butanediol and formic acid pathways in integrative recombinant Klebsiella pneumoniae. J Biotechnol 168: 194-200. doi: 10.1016/j.jbiotec.2013.04.022.
  • Xiu ZL, Zeng AP (2008) Present state and perspective of downstream processing of biologically produced 1,3-propanediol and 2,3-butanediol. Appl Microbiol Biotechnol 78: 917-926. doi: 10.1007/s00253-008-1387-4.
  • Xu YZ, Guo NN, Zheng ZM, Ou XJ, Liu HJ, Liu DH (2009) Metabolism in 1,3-Propanediol Fed-Batch Fermentation by a D-Lactate Deficient Mutant of Klebsiella pneumoniae. Biotechnol Bioeng 104: 965-972. doi: 10.1002/bit.22455.
  • Yang G, Tian J, Li J (2007) Fermentation of 1,3-propanediol by a lactate deficient mutant of Klebsiellaoxytoca under microaerobic conditions. Appl Microbiol Biotechnol 73: 1017-1024. doi: 10.1007/s00253-006-0563-7.
  • Yao J, Zhong J, Fang Y, Geisinger E, Novick RP, Lambowitz AM (2006) Use of targetrons to disrupt essential and nonessential genes in Staphylococcus aureus reveals temperature sensitivity of Ll.LtrB group II intron splicing. RNA 12: 1271-1281. doi: 10.1261/rna.68706.
  • Zeng AP, Biebl H (2002) Bulk chemicals from biotechnology: the case of 1,3-propanediol production and the new trends. Adv Biochem Eng Biotechnol 74: 239-259. doi: 10.1007/3-540-45736-4.
  • Zeng AP, Biebl H, Schlieker H, Deckwer WD (1993) Pathway analysis of glycerol fermentation by Klebsiella pneumoniae: Regulation of reducing equivalent balance and product formation. Enzyme Microb Technol 15: 770-779. doi: 10.1016/0141-0229(93)90008-P
  • Zeng AP, Sabra W (2011) Microbial production of diols as platform chemicals: recent progresses. Curr Opin Biotechnol 22: 749-757. doi: 10.1016/j.copbio.2011.05.005.
  • Zhang Y, Li Y, Du C, Liu M, Cao Z (2006) Inactivation of aldehyde dehydrogenase: a key factor for engineering 1,3-propanediol production by Klebsiella pneumoniae. Metab Eng 8: 578-586. doi: 10.1016/j.ymben.2006.05.008.
  • Zhang Q, Teng H, Sun Y, Xiu Z, Zeng AP (2008) Metabolic flux and robustness analysis of glycerol metabolism in Klebsiella pneumoniae. Bioprocess Biosyst Eng 31: 127-135. doi: 10.1007/3-540-45736-4.
  • Zheng P, Wereath K, Sun J, Van den Heuve IJ, Zeng AP (2006) Overexpression of genes of the dha regulon and its effects on cell growth, glycerol fermentation to 1,3-propanediol and plasmid stability in Klebsiella pneumoniae. Process Biochem 41: 2160-2169. doi: 10.1016/j.procbio.2006.06.012
  • Zheng ZM, Xu YZ, Liu HJ, Guo NN, Cai ZZ, Liu DH (2008) Physiologic mechanisms of sequential products synthesis in 1,3-propanediol fed-batch fermentation by Klebsiella pneumoniae. Biotechnol Bioeng 100: 923-932. doi: 10.1002/bit.21830.
  • Zhong J, Karberg M, Lambowitz AM (2003) Targeted and random bacterial gene disruption using a group II intron (targetron) vector containing a retrotransposition-activated selectable marker. Nucleic Acids Res 31: 1656-1664. doi: 10.1093/nar/gkg248.
Document Type
Publication order reference
YADDA identifier
JavaScript is turned off in your web browser. Turn it on to take full advantage of this site, then refresh the page.