Enhancement of the NAD(P)(H) Pool in <i>Escherichia coli</i> for Biotransformation

Heuser, Florian; Schroer, Kirsten; Lütz, Stephan; Bringer-Meyer, Stephanie; Sahm, Hermann

doi:10.1002/elsc.200720203

Cited by 44 publications

(15 citation statements)

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“…The quantification of intracellular cofactor pool sizes and cofactor dynamics during biotransformation processes are valuable information that provides better understanding of whole cell biocatalysts. Thus limitations can be detected and overcome, for example, by genetic engineering methods (Heuser et al, 2007) and optimization of process conditions. The LC-MS/ MS-technique is also an interesting tool for microbial cells that perform enzyme-coupled regeneration of the cofactors (Gröger et al, 2006(Gröger et al, , 2007Schroer et al, 2007a) for example, by coexpression of formate dehydrogenase or glucose dehydrogenase or carrying out cofactor regeneration by reactions of the central metabolism (Park et al, 2007).…”

Section: Discussionmentioning

confidence: 99%

“…Wubbolts et al (1990) reported the overexpression of nicotinic acid phosphoribosyltransferase from Escherichia coli which cause a fivefold increase in the intracellular concentration of NAD. Related strategies were described by Berrios-Rivera et al (2002b) and Heuser et al (2007). The rapid and reliable quantification of intracellular cofactor concentrations is essential for evaluating the efficiency of such strain engineering strategies.…”

Section: Introductionmentioning

confidence: 99%

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Metabolomics for biotransformations: Intracellular redox cofactor analysis and enzyme kinetics offer insight into whole cell processes

Schroer

Zelić

Oldiges

et al. 2009

Biotech & Bioengineering

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For redox reactions catalyzed by microbial cells the analysis of involved cofactors is of special interest since the availability of cofactors such as NADH or NADPH is often limiting and crucial for the biotransformation efficiency. The measurement of these cofactors has usually been carried out using spectrophotometric cycling assays. Today LC-MS/MS methods have become a valuable tool for the identification and quantification of intracellular metabolites. This technology has been adapted to measure all four nicotinamide cofactors (NAD, NADP, NADH, and NADPH) during a whole cell biotransformation process catalyzed by recombinant Escherichia coli cells. The cells overexpressing an alcohol dehydrogenase from Lactobacillus brevis were used for the reduction of methyl acetoacetate (MAA) with substrate-coupled cofactor regeneration by oxidation of 2-propanol. To test the reliability of the measurement the data were evaluated using a process model. This model was derived using the measured concentrations of reactants and cofactors for initiation as well as the kinetic constants from in vitro measurements of the isolated enzyme. This model proves to be highly effective in the process development for a whole cell redox biotransformation in predicting both the right concentrations of cofactors and reactants in a batch and in a CSTR process as well as the right in vivo expression level of the enzyme. Moreover, a sensitivity analysis identifies the cofactor regeneration reaction as the limiting step in case for the reduction of MAA to the corresponding product (R)-methyl 3-hydroxybutyrate. Using the combination of in vitro enzyme kinetic measurements, measurements of cofactors and reactants and an adequate model initiated by intracellular concentrations of all involved reactants and cofactors the whole cell biotransformation process can be understood quantitatively.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Metabolomics for biotransformations: Intracellular redox cofactor analysis and enzyme kinetics offer insight into whole cell processes

Schroer

Zelić

Oldiges

et al. 2009

Biotech & Bioengineering

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“…In several studies, the cellular cofactor concentration has been increased through manipulating the NAD ϩ biosynthetic pathway (12,16) or feeding permeabilized cells (18,36,37). The NAD(H) level was increased up to 6-fold previously (12). Yet, what is the maximal NAD(H) level that live cells can tolerate?…”

Section: Discussionmentioning

confidence: 99%

“…The pncB was also mutated in the operator region to protect against the regulatory effect of NadR and overexpressed under the lac promoter, resulting in a 2.2-fold increase of the NAD(H) level (22). Cooverexpression of the pncB and nadE gene led to a more dramatic 7-fold increase of the NAD(H) level (12). The NAD(H) level was also reduced with the nadD mutation, which encoded the NAMN adenylyltransferase (25).…”

Section: Manipulating the Nadh/nadmentioning

confidence: 99%

Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD ⁺ -Auxotrophic Mutant

Zhou

Wang

Yang

et al. 2011

Appl Environ Microbiol

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NAD (NAD؉ ) and its reduced form (NADH) are omnipresent cofactors in biological systems. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD ؉ level is tightly controlled by a biosynthesis regulation mechanism. Here, we developed a strategy to determine the extreme NAD(H) levels in Escherichia coli cells that were genetically engineered to be NAD ؉ auxotrophic. First, we expressed the ntt4 gene encoding the NAD(H) transporter in the E. coli mutant YJE001, which had a deletion of the nadC gene responsible for NAD ؉ de novo biosynthesis, and we showed NTT4 conferred on the mutant strain better growth in the presence of exogenous NAD ؉ . We then constructed the NAD ؉ -auxotrophic mutant YJE003 by disrupting the essential gene nadE, which is responsible for the last step of NAD ؉ biosynthesis in cells harboring the ntt4 gene. The minimal NAD ؉ level was determined in M9 medium in proliferating YJE003 cells that were preloaded with NAD ؉ , while the maximal NAD(H) level was determined by exposing the cells to high concentrations of exogenous NAD(H). Compared with supplementation of NADH, cells grew faster and had a higher intracellular NAD(H) level when NAD ؉ was fed. The intracellular NAD(H) level increased with the increase of exogenous NAD ؉ concentration, until it reached a plateau. Thus, a minimal NAD(H) level of 0.039 mM and a maximum of 8.49 mM were determined, which were 0.044؋ and 9.6؋ those of wild-type cells, respectively. Finally, the potential application of this strategy in biotechnology is briefly discussed.

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“…NAD(P)(H), is a useful tool for metabolic engineering [78,79]. Such engineering requires knowledge of the NAD biosynthesis pathway genes, to which comparative genomics has contributed significantly for the early pathway steps leading to quinolinate, the universal de novo precursor of the pyridine ring of NAD [80,81].…”

Section: Synergy Of Prokaryote–eukaryote Integrationsmentioning

confidence: 99%

‘Unknown’ proteins and ‘orphan’ enzymes: the missing half of the engineering parts list – and how to find it

Hanson

Pribat

Waller

et al. 2009

Biochemical Journal

178

161

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Like other forms of engineering, metabolic engineering requires knowledge of the components (the ‘parts list’) of the target system. Lack of such knowledge impairs both rational engineering design and diagnosis of the reasons for failures; it also poses problems for the related field of metabolic reconstruction, which uses a cell’s parts list to recreate its metabolic activities in silico. Despite spectacular progress in genome sequencing, the parts lists for most organisms that we seek to manipulate remain highly incomplete, due to the dual problem of ‘unknown’ proteins and ‘orphan’ enzymes. The former are all the proteins deduced from genome sequence that have no known function, and the latter are all the enzymes described in the literature (and often catalogued in the EC database) for which no corresponding gene has been reported. Unknown proteins constitute up to about half of the proteins in prokaryotic genomes, and much more than this in higher plants and animals. Orphan enzymes make up more than a third of the EC database. Attacking the ‘missing parts list’ problem is accordingly one of the great challenges for post-genomic biology, and a tremendous opportunity to discover new facets of life’s machinery. Success will require a co-ordinated community-wide attack, sustained over years. In this attack, comparative genomics is probably the single most effective strategy, for it can reliably predict functions for unknown proteins and genes for orphan enzymes. Furthermore, it is cost-efficient and increasingly straightforward to deploy owing to a proliferation of databases and associated tools.

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Enhancement of the NAD(P)(H) Pool in Escherichia coli for Biotransformation

Cited by 44 publications

References 48 publications

Metabolomics for biotransformations: Intracellular redox cofactor analysis and enzyme kinetics offer insight into whole cell processes

Metabolomics for biotransformations: Intracellular redox cofactor analysis and enzyme kinetics offer insight into whole cell processes

Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD ⁺ -Auxotrophic Mutant

‘Unknown’ proteins and ‘orphan’ enzymes: the missing half of the engineering parts list – and how to find it

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Enhancement of the NAD(P)(H) Pool in Escherichia coli for Biotransformation

Cited by 44 publications

References 48 publications

Metabolomics for biotransformations: Intracellular redox cofactor analysis and enzyme kinetics offer insight into whole cell processes

Metabolomics for biotransformations: Intracellular redox cofactor analysis and enzyme kinetics offer insight into whole cell processes

Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD + -Auxotrophic Mutant

‘Unknown’ proteins and ‘orphan’ enzymes: the missing half of the engineering parts list – and how to find it

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Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD ⁺ -Auxotrophic Mutant