New strategy to improve efficiency for gene replacement in <i>Klebsiella pneumoniae</i>

Dong, Wei; Sun, Junsong; Shi, Jiping; Liu, Pengfu; Hao, Jian

doi:10.1007/s10295-013-1250-1

Cited by 8 publications

(4 citation statements)

References 12 publications

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“…Documented culture temperatures for 2-ketogluconic-acidproducing strains, excluding Klebsiella pneumoniae, are between 28°C and 30°C, whereas Klebsiella pneumoniae NCT418 (Klebsiella aerogenes NCT418) is cultured at 35°C [4,9,10,12,13,17]. Commonly, K. pneumoniae CGMCC 1.6636 and the budA mutated strain are cultured at 37°C [18,19].…”

Section: Effect Of Culture Temperature On 2-ketogluconic Acid Productionmentioning

confidence: 99%

“…pneumoniae CGMCC 1.6366 (TUAC01) is a strain isolated for the production of 1,3-propanediol [5]. Efficient gene replacement in K. pneumoniae has been exploited, and several mutants derived from K. pneumoniae CGMCC 1.6366 have been constructed [18,19]. The budA mutant that has disrupted the gene encoding alpha-acetolactate decarboxylase, a key enzyme in the 2,3-butanediol synthesis pathway, was found to accumulate 2-ketogluconic acid in cultures exposed to acidic conditions [20].…”

Section: Introductionmentioning

confidence: 99%

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Two-Stage Fermentation for 2-Ketogluconic Acid Production by Klebsiella pneumoniae

Sun¹,

Dong²,

Shi³

et al. 2014

Journal of Microbiology and Biotechnology

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2-Ketogluconic acid production by Klebsiella pneumoniae is a pH-dependent process, strictly proceeding under acidic conditions. Unfortunately, cell growth is inhibited by acidic conditions, resulting in low productivity of 2-ketogluconic acid. To overcome this deficiency, a two-stage fermentation strategy was exploited in the current study. During the first stage, the culture was maintained at neutral pH, favoring cell growth. During the second stage, the culture pH was switched to acidic conditions favoring 2-ketogluconic acid accumulation. Culture parameters, including switching time, dissolved oxygen levels, pH, and temperature were optimized for the fed-batch fermentation. Characteristics of glucose dehydrogenase and gluconate dehydrogenase were revealed in vitro, and the optimal pHs of the two enzymes coincided with the optimum culture pH. Under optimum conditions, a total of 186 g/l 2- ketogluconic acid was produced at 26 h, and the conversion ratio was 0.98 mol/mol. This fermentation strategy has successfully overcome the mismatch between optimum parameters required for cell growth and 2-ketogluconic acid accumulation, and this result has the highest productivity and conversion ratio of 2-ketogluconic and produced by microorganism.

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Section: Effect Of Culture Temperature On 2-ketogluconic Acid Productionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Two-Stage Fermentation for 2-Ketogluconic Acid Production by Klebsiella pneumoniae

Sun¹,

Dong²,

Shi³

et al. 2014

Journal of Microbiology and Biotechnology

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“…R-acetoin can be further reduced to 2,3-butanediol when catalyzed by butanediol dehydrogenase. An efficient gene replacement method suitable for K. pneumoniae has been explored, and many mutants have been constructed 5,6,7 . A budC mutant, which lost its 2,3-butanediol dehydrogenase activity, accumulates high levels of acetoin in culture broth.…”

Section: Introductionmentioning

confidence: 99%

Production of Chemicals by <em>Klebsiella pneumoniae</em> Using Bamboo Hydrolysate as Feedstock

Dong

Zhang

et al. 2017

JoVE

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Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was pretreated with NaOH and washed to a neutral pH. Cellulase was added to the pretreated bamboo powder to generate the hydrolysate, which contained 30 g/L glucose and 15 g/L xylose and was used as the carbon source to prepare a medium for chemical production. When cultured in microaerobic conditions, 12.7 g/L 2,3-butanediol was produced by wildtype K. pneumoniae. In aerobic conditions, 13.0 g/L R-acetoin was produced by the budC mutant of K. pneumoniae. A mixture of 25.5 g/L 2-ketogluconic acid and 13.6 g/L xylonic acid was produced by the budA mutant of K. pneumoniae in a two-stage, pH-controlled fermentation with high air supplementation. In the first stage of fermentation, the culture was maintained at a neutral pH; after cell growth, the fermentation proceeded to the second stage, during which the culture was allowed to become acidic.

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“…In other microorganisms, this inhibition might vary, and linear DNA that reaches the host cytoplasm may be digested by host RecBCD exonuclease, meaning that longer homologous extensions are required. K. pneumoniae CGMCC 1.6366 is a strain used for production of 1,3‐propanediol , and ways to improve the efficiency of Red recombinase‐mediated gene replacement in this strain have been explored . In the current study, we investigated the efficiency of Gam inhibition of RecBCD in K. pneumoniae CGMCC 1.6366, and developed a method for improving the efficiency of gene replacement.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of RecBCD in Klebsiella pneumoniae by Gam and its effect on the efficiency of gene replacement

Chen

Dong

Liu

et al. 2015

J. Basic Microbiol.

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Gam protein is an inhibitor of the host RecBCD exonuclease, and this inhibition is essential to the proficiency of Red recombinase-mediated gene replacement. In Klebsiella pneumoniae, the efficiency of this gene replacement was lower than that in Escherichia coli, and the minimum length of homologous extensions required was longer. Thus, it was supposed that the inhibitory effect of Gam against RecBCD was weak in K. pneumoniae. To test this hypothesis, a Gam-deficient Red recombinase expression plasmid and a ΔrecB K. pneumoniae mutant were constructed. The Gam-deficient Red recombinase showed a reduced capacity for gene replacement compared with that of the complete Red recombinase. The efficiency of gene replacement in the ΔrecB mutant was 6-8 times higher than the wild-type strain, and the minimum length for the homologous extensions was reduced to 100 bp. These results indicate that Gam does inhibit the RecBCD exonuclease in K. pneumoniae, but that this inhibition is not stringent. Furthermore, mutation of recB presents a convenient and efficient method to enhance the Red recombinase assisted gene replacement in K. pneumoniae.

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New strategy to improve efficiency for gene replacement in Klebsiella pneumoniae

Cited by 8 publications

References 12 publications

Two-Stage Fermentation for 2-Ketogluconic Acid Production by Klebsiella pneumoniae

Two-Stage Fermentation for 2-Ketogluconic Acid Production by Klebsiella pneumoniae

Production of Chemicals by <em>Klebsiella pneumoniae</em> Using Bamboo Hydrolysate as Feedstock

Inhibition of RecBCD in Klebsiella pneumoniae by Gam and its effect on the efficiency of gene replacement

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