Central carbon metabolite profiling reveals vector-associated differences in the recombinant protein production host Escherichia coli BL21

García-Calvo, Laura; Rane, Divyata Vilas; Everson, Nikalet; Humlebrekk, Sigurd Tømmerberg; Mathiassen, Lise Femanger; Mæhlum, Astfrid Helene Morka; Malmo, Jostein; Bruheim, Per

doi:10.3389/fceng.2023.1142226

Cited by 3 publications

(2 citation statements)

References 104 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The biological significance of the two pathways depends on the intracellular concentration of NADPH in Priestia megaterium . Estimates of intracellular NADPH concentrations in bacterial cells fall in the 20- to 300-μM range ( 43 – 47 ). A similarly wide variation has been reported for eukaryotic cells ( 48 – 51 ).…”

Section: Discussionmentioning

confidence: 99%

Tryptophan extends the life of cytochrome P450

Ravanfar,

Sheng,

Gray

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Powerfully oxidizing enzymes need protective mechanisms to prevent self-destruction. The flavocytochrome P450 BM3 from Priestia megaterium (P450 BM3 ) is a self-sufficient monooxygenase that hydroxylates fatty acid substrates using O 2 and NADPH as co-substrates. Hydroxylation of long-chain fatty acids (≥C 14 ) is well coupled to O 2 and NADPH consumption, but shorter chains (≤C 12 ) are more poorly coupled. Hydroxylation of p -nitrophenoxydodecanoic acid by P450 BM3 produces a spectrophotometrically detectable product wherein the coupling of NADPH consumption to product formation is just 10%. Moreover, the rate of NADPH consumption is 1.8 times that of O 2 consumption, indicating that an oxidase uncoupling pathway is operative. Measurements of the total number of enzyme turnovers before inactivation (TTN) indicate that higher NADPH concentrations increase TTN. At lower NADPH levels, added ascorbate increases TTN, while a W96H mutation leads to a decrease. The W96 residue is about 7 Å from the P450 BM3 heme and serves as a gateway residue in a tryptophan/tyrosine (W/Y) hole transport chain from the heme to a surface tyrosine residue. The data indicate that two oxidase pathways protect the enzyme from damage by intercepting the powerfully oxidizing enzyme intermediate (Compound I) and returning it to its resting state. At high NADPH concentrations, reducing equivalents from the flavoprotein are delivered to Compound I by the usual reductase pathway. When NADPH is not abundant, however, oxidizing equivalents from Compound I can traverse a W/Y chain, arriving at the enzyme surface where they are scavenged by reductants. Ubiquitous tryptophan/tyrosine chains in highly oxidizing enzymes likely perform similar protective functions.

show abstract

Section: Discussionmentioning

confidence: 99%

Tryptophan extends the life of cytochrome P450

Ravanfar,

Sheng,

Gray

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…The reconstructed strain co-expressing NAMPT from Chitinophaga pinensis (CpNAMPT) and the artificial operon achieved 2.5 times higher NMN production (189 mg/L) than the parental strain [47]. Although the construction of artificial operons improved PRPP production, vectors expressing the operons and CpNAMPT constitute a double plasmid system that may cause metabolic burdens on cells [63,64]. Alternatively, PRPP can also be produced from ribose via a short pathway, and many bacteria such as E. coli and B. subtilis can use ribose as a carbon source [65,66].…”

Section: Improving the Supply Of Essential Precursorsmentioning

confidence: 99%

Biosynthesis of Nicotinamide Mononucleotide: Current Metabolic Engineering Strategies, Challenges, and Prospects

et al. 2023

View full text Add to dashboard Cite

Nicotinamide mononucleotide (NMN) is an essential precursor of nicotinamide adenine dinucleotide (NAD+), which is widely applied in the pharmaceutical and biotech industries. The biosynthesis of NMN is currently attracting much attention because it has non-toxic reaction conditions and low amounts of isomers, whereas chemical synthesis has low yields and is not environmentally friendly. This review systematically describes the two biosynthetic pathways of NMN in detail for the first time and introduces the latest studies on NMN production through different pathways using metabolic engineering strategies. NMN accumulation can be improved by optimizing the activity of key enzymes, enhancing the supply of precursors and co-factors, inhibiting the synthesis of byproducts, and promoting product export. Finally, we also discuss the current challenges of producing NMN and possible solutions for the future.

show abstract

Zic-HILIC MS/MS Method for NADomics Provides Novel Insights into Redox Homeostasis in Escherichia coli BL21 Under Microaerobic and Anaerobic Conditions

Rane,

García-Calvo,

Kristiansen

et al. 2024

Metabolites

View full text Add to dashboard Cite

Background: Nicotinamide adenine dinucleotide (NAD+), its precursors, and its derivatives (collectively NADome) play a crucial role in cellular processes and maintain redox homeostasis. Understanding the dynamics of these metabolic pools and redox reactions can provide valuable insights into metabolic functions, especially cellular regulation and stress response mechanisms. The accurate quantification of these metabolites is challenging due to the interconversion between the redox forms. Methods: Our laboratory previously developed a zwitterionic hydrophilic interaction liquid chromatography (zic-HILIC)–tandem mass spectrometry method for the quantification of five essential pyridine nucleotides, including NAD+ derivatives and it’s reduced forms, with 13C isotope dilution and matrix-matched calibration. In this study, we have improved the performance of the chromatographic method and expanded its scope to twelve analytes for a comprehensive view of NAD+ biosynthesis and utilization. The analytical method was validated and applied to investigate Escherichia coli BL21 under varying oxygen supplies including aerobic, microaerobic, and anaerobic conditions. Conclusions: The intracellular absolute metabolite concentrations ranged over four orders of magnitude with NAD+ as the highest abundant, while its precursors were much less abundant. The composition of the NADome at oxygen-limited conditions aligned more with that in the anaerobic conditions rather than in the aerobic phase. Overall, the NADome was quite homeostatic and E. coli rapidly, but in a minor way, adapted the metabolic activity to the challenging shift in the growth conditions and achieved redox balance. Our findings demonstrate that the zic-HILIC-MS/MS method is sensitive, accurate, robust, and high-throughput, providing valuable insights into NAD+ metabolism and the potential significance of these metabolites in various biological contexts.

show abstract

Central carbon metabolite profiling reveals vector-associated differences in the recombinant protein production host Escherichia coli BL21

Cited by 3 publications

References 104 publications

Tryptophan extends the life of cytochrome P450

Tryptophan extends the life of cytochrome P450

Biosynthesis of Nicotinamide Mononucleotide: Current Metabolic Engineering Strategies, Challenges, and Prospects

Zic-HILIC MS/MS Method for NADomics Provides Novel Insights into Redox Homeostasis in Escherichia coli BL21 Under Microaerobic and Anaerobic Conditions

Contact Info

Product

Resources

About