Subtoxic product levels limit the epoxidation capacity of recombinant E. coli by increasing microbial energy demands

Kuhn, Daniel; Fritzsch, Frederik S. O.; Zhang, Xiumei; Wendisch, Volker F.; Blank, Lars M.; Bühler, Bruno; Schmid, Andreas

doi:10.1016/j.jbiotec.2012.07.194

Cited by 25 publications

(24 citation statements)

References 55 publications

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“…Otto et al (68) observed product inhibition of isolated StyAB at styrene oxide concentrations above 0.5 mM. On the other hand, it was shown that styrene oxide concentrations below the toxicity limit already affected the metabolic and biocatalytic performance of recombinant E. coli JM101 (pSPZ10) (9). However, as only half the amount of styrene oxide was formed in the induction setups leading to low specific activities compared to that in the induction setups resulting in maximal specific epoxidation activities (see Fig.…”

Section: Discussionmentioning

confidence: 99%

“…This includes the regeneration of required cofactors, increased enzyme stability, the possibility to combine multiple enzyme reactions, and the deactivation of reactive oxygen species (1)(2)(3)(4). However, the efficiency of whole cells as biocatalysts is often limited by the toxicity of apolar substrates and products (5)(6)(7)(8)(9), which typically involves detrimental effects on cellular membranes (10). Solvents having a log partition coefficient in an equimolar mixture of octanol and water (P OW ) between 1 and 4 are regarded to be extremely toxic, as they readily intercalate into the membrane (11,12).…”

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confidence: 99%

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Engineering of Pseudomonas taiwanensis VLB120 for Constitutive Solvent Tolerance and Increased Specific Styrene Epoxidation Activity

Volmer

Neumann

Bühler

et al. 2014

Appl Environ Microbiol

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The application of whole cells as biocatalysts is often limited by the toxicity of organic solvents, which constitute interesting substrates/products or can be used as a second phase for in situ product removal and as tools to control multistep biocatalysis. Solvent-tolerant bacteria, especially Pseudomonas strains, are proposed as promising hosts to overcome such limitations due to their inherent solvent tolerance mechanisms. However, potential industrial applications suffer from tedious, unproductive adaptation processes, phenotypic variability, and instable solvent-tolerant phenotypes. In this study, genes described to be involved in solvent tolerance were identified in Pseudomonas taiwanensis VLB120, and adaptive solvent tolerance was proven by cultivation in the presence of 1% (vol/vol) toluene. Deletion of ttgV, coding for the specific transcriptional repressor of solvent efflux pump TtgGHI gene expression, led to constitutively solvent-tolerant mutants of P. taiwanensis VLB120 and VLB120⌬C. Interestingly, the increased amount of solvent efflux pumps enhanced not only growth in the presence of toluene and styrene but also the biocatalytic performance in terms of stereospecific styrene epoxidation, although proton-driven solvent efflux is expected to compete with the styrene monooxygenase for metabolic energy. Compared to that of the P. taiwanensis VLB120⌬C parent strain, the maximum specific epoxidation activity of P. taiwanensis VLB120⌬C⌬ttgV doubled to 67 U/g of cells (dry weight). This study shows that solvent tolerance mechanisms, e.g., the solvent efflux pump TtgGHI, not only allow for growth in the presence of organic compounds but can also be used as tools to improve redox biocatalysis involving organic solvents.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Engineering of Pseudomonas taiwanensis VLB120 for Constitutive Solvent Tolerance and Increased Specific Styrene Epoxidation Activity

Volmer

Neumann

Bühler

et al. 2014

Appl Environ Microbiol

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“…They are particularly coenzyme. SMOs catalyze the transformation of styrene into (S)-styrene oxide in the upper catabolic pathway of styrene degradation [52,53]. Most two-component SMOs are isolated from the genera of Pseudomonas and Rhodococcus and share high sequence similarities [50,51,[54][55][56][57][58].…”

Section: Introductionmentioning

confidence: 99%

Biocatalytic Epoxidation for Green Synthesis

Lin

Liu

et al. 2016

Green Biocatalysis

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“…The benefits of metabolic engineering have especially become evident in fermentation processes, as exemplified by the development of strains for the biotechnological production of indigo (8) and propanediol (9). Metabolic engineering can rely on a set of powerful tools, such as flux balance analysis (10) and metabolic flux analysis (MFA) (11-13), used, inter alia, for the optimization of the production of various amino acids in Corynebacterium glutamicum (14,15).The efficiency of whole-cell redox biocatalysts often depends on microbial physiology (16, 17), for example, because the provision of (co)substrates/cofactors becomes limiting (18, 19), or because a toxic or inhibiting product has accumulated (20,21). With respect to cell physiology, 2-oxoacid-dependent nonheme Fe(II)-dependent dioxygenases (22, 23), such as proline-4-hydroxylases (P4Hs), are highly interesting.…”

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confidence: 99%

“…The efficiency of whole-cell redox biocatalysts often depends on microbial physiology (16,17), for example, because the provision of (co)substrates/cofactors becomes limiting (18,19), or because a toxic or inhibiting product has accumulated (20,21). With respect to cell physiology, 2-oxoacid-dependent nonheme Fe(II)-dependent dioxygenases (22,23), such as proline-4-hydroxylases (P4Hs), are highly interesting.…”

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confidence: 99%

Proline Availability Regulates Proline-4-Hydroxylase Synthesis and Substrate Uptake in Proline-Hydroxylating Recombinant Escherichia coli

Falcioni

Blank

Frick

et al. 2013

Appl Environ Microbiol

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Microbial physiology plays a crucial role in whole-cell biotransformation, especially for redox reactions that depend on carbon and energy metabolism. In this study, regio-and enantio-selective proline hydroxylation with recombinant Escherichia coli expressing proline-4-hydroxylase (P4H) was investigated with respect to its interconnectivity to microbial physiology and metabolism. P4H production was found to depend on extracellular proline availability and on codon usage. Medium supplementation with proline did not alter p4h mRNA levels, indicating that P4H production depends on the availability of charged prolyl-tRNAs. Increasing the intracellular levels of soluble P4H did not result in an increase in resting cell activities above a certain threshold (depending on growth and assay temperature). Activities up to 5-fold higher were reached with permeabilized cells, confirming that host physiology and not the intracellular level of active P4H determines the achievable whole-cell proline hydroxylation activity. Metabolic flux analysis revealed that tricarboxylic acid cycle fluxes in growing biocatalytically active cells were significantly higher than proline hydroxylation rates. Remarkably, a catalysis-induced reduction of substrate uptake was observed, which correlated with reduced transcription of putA and putP, encoding proline dehydrogenase and the major proline transporter, respectively. These results provide evidence for a strong interference of catalytic activity with the regulation of proline uptake and metabolism. In terms of whole-cell biocatalyst efficiency, proline uptake and competition of P4H with proline catabolism are considered the most critical factors. I n redox biocatalysis, especially when activation of molecular oxygen is involved, the whole cell still remains the preferred catalyst. Besides the capability to continuously regenerate redox cofactors via central carbon metabolism, the whole-cell approach provides advantages with respect to operational catalyst stability, e.g., via continuous (multicomponent) enzyme synthesis, assembly, and stabilization, as well as degradation of reactive oxygen species (1-3).Oxygenases are of special interest for industrial applications, since they catalyze the highly specific oxyfunctionalization of unactivated C-H bonds under mild conditions, with molecular oxygen as the oxygen donor (4). The efficiency of an oxygenase-containing whole-cell biocatalyst depends on both enzyme and host cell performance. Hence, biocatalyst engineering must encompass both factors and be integrated with biochemical process engineering (5, 6). The technological progress in enzyme engineering of the recent decades now allows fast generation of enzyme variants with improved properties (7). For in vivo applications, intracellular conditions have to be considered, and host cell engineering often is necessary to allow the exploitation of the catalytic capability of (engineered) enzymes. The benefits of metabolic engineering have especially become evident in fermentation processes, as ex...

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Subtoxic product levels limit the epoxidation capacity of recombinant E. coli by increasing microbial energy demands

Cited by 25 publications

References 55 publications

Engineering of Pseudomonas taiwanensis VLB120 for Constitutive Solvent Tolerance and Increased Specific Styrene Epoxidation Activity

Engineering of Pseudomonas taiwanensis VLB120 for Constitutive Solvent Tolerance and Increased Specific Styrene Epoxidation Activity

Biocatalytic Epoxidation for Green Synthesis

Proline Availability Regulates Proline-4-Hydroxylase Synthesis and Substrate Uptake in Proline-Hydroxylating Recombinant Escherichia coli

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