A Toolkit for Metabolic Engineering of Bacteria

Keasling, Jay D.; Benemann, John R.; Pramanik, J.; Carrier, Trent A.; Jones, Kristala L.; Dien, Stephen J. Van

doi:10.1007/978-0-585-35132-2_11

Cited by 15 publications

(10 citation statements)

References 21 publications

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“…Since the Gibbs free energy of formation of maltose limits the amount of H 2 that can be produced, exceeding this level necessitated changes in energy management. Reduced biomass formation and decreased growth rate both offer this option (17,18,20). Typically, glucose oxidation leading to the production of 4 mol of H 2 /mol of glucose represents 33% of the stoichiometric maximum.…”

Section: Discussionmentioning

confidence: 99%

Uncoupling Fermentative Synthesis of Molecular Hydrogen from Biomass Formation in Thermotoga maritima

Singh

White

Demi̇rel

et al. 2018

Appl Environ Microbiol

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When carbohydrates are fermented by the hyperthermophilic anaerobeThermotoga maritima, molecular hydrogen (H2) is formed in strict proportion to substrate availability. Excretion of the organic acids acetate and lactate provide an additional sink for removal of excess reductant. However, mechanisms controlling energy management of these metabolic pathways are largely unexplored. To investigate this topic, transient gene inactivation was used to block lactate production as a strategy to produce spontaneous mutant cell lines that overproduced H2through mutation of unpredicted genetic targets. Single-crossover homologous chromosomal recombination was used to disrupt lactate dehydrogenase (encoded byldh) with a truncatedldhfused to a kanamycin resistance cassette expressed from a native PgroESLpromoter. Passage of the unstable recombinant resulted in loss of the genetic marker and recovery of evolved cell lines, including strain Tma200. Relative to the wild type, and considering the mass balance of fermentation substrate and products, Tma200 grew more slowly, produced H2at levels above the physiologic limit, and simultaneously consumed less maltose while oxidizing it more efficiently. Whole-genome resequencing indicated that the ABC maltose transporter subunit, encoded bymalK3, had undergone repeated mutation, and high-temperature anaerobic [14C]maltose transport assays demonstrated that the rate of maltose transport was reduced. Transfer of themalK3mutation into a clean genetic background also conferred increased H2production, confirming that the mutant allele was sufficient for increased H2synthesis. These data indicate that a reduced rate of maltose uptake was accompanied by an increase in H2production, changing fermentation efficiency and shifting energy management.IMPORTANCEBiorenewable energy sources are of growing interest to mitigate climate change, but like other commodities with nominal value, require innovation to maximize yields. Energetic considerations constrain production of many biofuels, such as molecular hydrogen (H2) because of the competing needs for cell mass synthesis and metabolite formation. Here we describe cell lines of the extremophileThermotoga maritimathat exceed the physiologic limits for H2formation arising from genetic changes in fermentative metabolism. These cell lines were produced using a novel method called transient gene inactivation combined with adaptive laboratory evolution. Genome resequencing revealed unexpected changes in a maltose transport protein. Reduced rates of sugar uptake were accompanied by lower rates of growth and enhanced productivity of H2.

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Section: Discussionmentioning

confidence: 99%

Uncoupling Fermentative Synthesis of Molecular Hydrogen from Biomass Formation in Thermotoga maritima

Singh

White

Demi̇rel

et al. 2018

Appl Environ Microbiol

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“…Pada masing-masing perlakuan ini bakteri B. cereus A66 ditumbuhkan terlebih dahulu (prakultur) dalam 70 ml medium TGY (1% bacto tryptone, 0,1% glucose, 0,5% yeast extract) [16] [20]. Dalam hal ini sel bakteri dapat merilis fosfat karena dalam kondisi tekanan lingkungan tanpa fosfat ini telah menginduksi PPX sehingga terjadi hidrolisis cadangan polifosfat [21]. Dengan demikian maka terjadi penurunan konsentrasi P dalam sel secara signifikan.…”

Section: Perlakuan Kontrol (Tiga Perlakuan)unclassified

Efek Pelaparan dan Akumulasi Polifosfat terhadap Biopresipitasi Uranium pada Bacillus cereus A66

Octavia

Yuwono²,

Taftazani³

2018

Biotropika

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Pelaparan dan akumulasi polifosfat pada bakteri diduga dapat meningkatkan biopresipitasi uranium. Tujuan penelitian ini adalah untuk mengetahui efek pelaparan dan akumulasi polifosfat terhadap peningkatan biopresipitasi uranium pada Bacillus cereus A66. Pada penelitian ini B. cereus A66 ditumbuhkan terlebih dahulu (prakultur) dalam Tryptone Glucose Yeast Extract (TGY), pada suhu ruang (± 28ºC) hingga fase logaritmik (± 16 jam). Pada perlakuan pertama, prakultur B. cereus A66 diberi perlakuan pelaparan fosfat dalam medium P-free, selanjutnya dipindahkan ke medium P-uptake untuk akumulasi fosfat. Untuk mengamati biopresipitasi uranium, sel bakteri dipindahkan ke dalam larutan uranium 1 mM. Pada perlakuan kedua, prakultur B. cereus A66 langsung dipindahkan ke dalam larutan uranium tanpa fase pelaparan dan akumulasi polifosfat. Sedangkan pada perlakuan ketiga, B. cereus A66 tanpa fase pelaparan dikultur dalam medium P-uptake kemudian dipindahkan ke larutan uranium. Pada perlakuan keempat, B. cereus A66 dikondisikan dengan pelaparan fosfat dalam medium P-free, diikuti dengan pemindahan ke dalam larutan uranium. Perlakuan kedua, ketiga dan keempat dirancang untuk mengkonfirmasi efek perlakuan pertama dalam penelitian ini. Hasil penelitian menunjukkan bahwa B. cereus A66 yang mengalami pelaparan fosfat dapat mengakumulasi fosfat delapan kali lebih banyak ketika dipindahkan ke medium P-uptake, dibandingkan dengan B. cereus A66 yang tidak mengalami pelaparan fosfat. Selain itu, B. cereus A66 yang mengakumulasi lebih banyak fosfat juga menunjukkan peningkatan biopresipitasi uranium sebesar 1,5 kali lebih banyak dibandingkan dengan B. cereus A66 yang tidak mengalami pelaparan fosfat. Fenomena ini diyakini digerakkan oleh metabolisme polifosfat yang dikontrol oleh aktivitas gen PPK dan PPX. Secara keseluruhan hasil penelitian ini menunjukkan bahwa semakin banyak polifosfat terakumulasi dalam sel, semakin meningkat respon biopresipitasi uranium dalam hal jumlah uranium yang diambil dari larutan dan efisiensi waktu pengambilannya. Strategi ini dapat dikembangkan lebih lanjut untuk bioremediasi air dan tanah yang terkontaminasi uranium.

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“…Although growth de-coupled production strategies could increase the biohydrogen efficiency [4,5], the coupled synthesis of the target chemical to biomass yield is usually of interest for metabolic engineering. This allows for easy selection of overproducing strains by selecting the strains with the fastest growth.…”

Section: Design Of Knockoutsmentioning

confidence: 99%

“…It is expected that such theoretical high-yielding pathways would compete with the growth yield. Growth de-coupled production approaches have therefore been suggested for increasing the efficiency in biohydrogen production [4,5].…”

Section: Introductionmentioning

confidence: 99%

An in silico re-design of the metabolism in Thermotoga maritima for increased biohydrogen production

Nogales

Guđmundsson

Thiele

2012

International Journal of Hydrogen Energy

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A Toolkit for Metabolic Engineering of Bacteria

Cited by 15 publications

References 21 publications

Uncoupling Fermentative Synthesis of Molecular Hydrogen from Biomass Formation in Thermotoga maritima

Uncoupling Fermentative Synthesis of Molecular Hydrogen from Biomass Formation in Thermotoga maritima

Efek Pelaparan dan Akumulasi Polifosfat terhadap Biopresipitasi Uranium pada Bacillus cereus A66

An in silico re-design of the metabolism in Thermotoga maritima for increased biohydrogen production

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