Directed Multistep Biocatalysis for the Synthesis of the Polyketide Oxytetracycline in Permeabilized Cells of <i>Escherichia coli</i>

Krauser, Steffen; Hoffmann, Thomas; Heinzle, Elmar

doi:10.1021/cs501825u

Cited by 12 publications

(5 citation statements)

References 44 publications

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“…As PTDH endows microorganisms with the unique ability to grow on PT, the enzyme has found use as a component of biocontainment strategies. − The favorable thermodynamics of the reaction catalyzed by PTDH (Δ G ° = −15 kcal/mol) and the low cost of PT (∼$0.1 USD/g) have also attracted considerable attention to the enzyme as a cofactor regeneration system. − This potential utility spurned several engineering efforts that led to the production of PTDH variants with increased thermostability, increased activity, and a decreased specificity for NAD + over NADP + that allows for efficient regeneration of both NADH and NADPH . These enzymes have been employed in various biocatalytic systems, both as stand-alone cofactor regenerators and as fusions with flavin monooxygenases within self-sufficient biocatalysts. − …”

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confidence: 99%

Temperature-Independent Kinetic Isotope Effects as Evidence for a Marcus-like Model of Hydride Tunneling in Phosphite Dehydrogenase

Howe

Donk

2019

Biochemistry

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Phosphite dehydrogenase catalyzes the transfer of a hydride from phosphite to NAD+, producing phosphate and NADH. We have evaluated the role of hydride tunneling in a thermostable variant of this enzyme (17X-PTDH) by measuring the temperature dependence of the primary 2H kinetic isotope effects (KIEs) between 5 and 45 °C. Pre-steady-state kinetic measurements were used to demonstrate that the hydride transfer is rate-determining across this temperature range and that the observed KIEs are equal to the intrinsic isotope effect on the chemical step. The KIEs on the pre-exponential factor (A H/A D) and the activation energy (ΔE a) were 1.6 ± 0.1 and 0.21 ± 0.05 kcal/mol, respectively, suggesting that 17X-PTDH facilitates extensive tunneling of both isotopes via a Marcus-like model. Site-directed mutagenesis was used to evaluate the role of an active site threonine (Thr104) found on the back face of the nicotinamide in promoting the close packing of the substrates. In mutants with reduced steric bulk at this position, values of A H/A D and ΔE a fall within the range describing semiclassical “over the barrier” reactivity, suggesting that Thr104 acts as a steric backstop to promote tunneling in 17X-PTDH. Whereas hydrogen tunneling is now a widely appreciated feature of C–H activating enzymes, these observations with a P–H activating system are consistent with the proposal that tunneling is likely to be a common feature on all enzymes that catalyze hydrogen transfers.

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confidence: 99%

Temperature-Independent Kinetic Isotope Effects as Evidence for a Marcus-like Model of Hydride Tunneling in Phosphite Dehydrogenase

Howe

Donk

2019

Biochemistry

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“…Moreover, recently study provided a strategy of introducing a polyphosphate kinase-mediated ATP regeneration system in Streptomyces albulus to increase intracellular ATP levels by 71.56% [ 55 ]. The ATP regeneration system has also been applied in many other microorganisms, such as C. glutamicum [ 56 ] and E. coli [ 57 ] to increase intracellular ATP levels. The application of polyphosphate kinase (PPK) performs remarkably well to re-phosphorylate ADP for ATP production with polyP as phosphate donor, such as additional phosphate salts.…”

Section: Resultsmentioning

confidence: 99%

Metabolic engineering of an industrial bacterium Zymomonas mobilis for anaerobic l-serine production

Wang,

Yan

et al. 2024

Synthetic and Systems Biotechnology

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“…131,167,191 The biosynthesis of some SdNPs involves not only SAM but also other cofactors, such as NAD(P)H, ATP, and acetyl-CoA. 11,192 The SAM biosynthesis is also tightly connected with ATP and NAD(P)H, all of which are primary cofactors that have fundamental functions to the host cell. 44,49,129,137 This is another challenge when developing high-performing MCFs of SdNP.…”

Section: Discussionmentioning

confidence: 99%

Improving Microbial Cell Factory Performance by Engineering SAM Availability

Lv,

Chang,

Zhang

et al. 2024

J. Agric. Food Chem.

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Methylated natural products are widely spread in nature. S-Adenosyl-L-methionine (SAM) is the secondary abundant cofactor and the primary methyl donor, which confer natural products with structural and functional diversification. The increasing demand for SAM-dependent natural products (SdNPs) has motivated the development of microbial cell factories (MCFs) for sustainable and efficient SdNP production. Insufficient and unsustainable SAM availability hinders the improvement of SdNP MCF performance. From the perspective of developing MCF, this review summarized recent understanding of de novo SAM biosynthesis and its regulatory mechanism. SAM is just the methyl mediator but not the original methyl source. Effective and sustainable methyl source supply is critical for efficient SdNP production. We compared and discussed the innate and relatively less explored alternative methyl sources and identified the one involving cheap one-carbon compound as more promising. The SAM biosynthesis is synergistically regulated on multilevels and is tightly connected with ATP and NAD(P)H pools. We also covered the recent advancement of metabolic engineering in improving intracellular SAM availability and SdNP production. Dynamic regulation is a promising strategy to achieve accurate and dynamic fine-tuning of intracellular SAM pool size. Finally, we discussed the design and engineering constraints underlying construction of SAM-responsive genetic circuits and envisioned their future applications in developing SdNP MCFs.

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Directed Multistep Biocatalysis for the Synthesis of the Polyketide Oxytetracycline in Permeabilized Cells of Escherichia coli

Cited by 12 publications

References 44 publications

Temperature-Independent Kinetic Isotope Effects as Evidence for a Marcus-like Model of Hydride Tunneling in Phosphite Dehydrogenase

Temperature-Independent Kinetic Isotope Effects as Evidence for a Marcus-like Model of Hydride Tunneling in Phosphite Dehydrogenase

Metabolic engineering of an industrial bacterium Zymomonas mobilis for anaerobic l-serine production

Improving Microbial Cell Factory Performance by Engineering SAM Availability

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