Determining the extremes of the cellular NAD(H) level by using an Escherichia coli NAD(+)-auxotrophic mutant

Abstract

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.

Fig. 1.

Overview of NAD⁺ metabolism. (A) NAD⁺ biosynthetic and salvage pathways in E. coli. (B) Schematic representation of the linkage between NAD⁺ biosynthesis and the transport pathway. Dashed lines show the NAD⁺ transport model in chloroplasts and mitochondria, discovered in eukaryotes S. cerevisiae and A. thaliana; solid lines refer to the NAD⁺ metabolic pathway and transport pathway in prokaryotes E. coli and Chlamydia, respectively. Abbreviations: ASP, asparate; DHAP, dihydroxyacetone phosphate; ImASP, imino asparate; QA, quinolinate; NA, nicotinate; NM, nicotinamide; NMN, nicotinamide mononucleotide; NmR, nicotinamide ribonucleoside; NaR, nicotinate ribonucleoside; NAMN, nicotinic acid mononucleotide; dNAD, deamino NAD⁺; Ade, adenosine. Enzymes: NadB, l-aspartate oxidase; NadA, quinolinate synthetase; NadC, quinolinate phosphoribosyltransferase; NadD, NAMN adenyltransferase; NadE, NAD⁺ synthetase; UshA, NMN glycohydrolase; DeoD, purine-nucleoside phosphorylase PncA, Nm deamidase; PncB, NA phosphoribosyltransferase; NudC, NAD⁺ pyrophosphatase; NadK, NAD⁺ kinase. ScNDT1 and ScNDT2, NAD⁺ carriers NDT1 and NDT2 from S. cerevisiae; AtNDT1 and AtNDT2, NAD⁺ carriers NDT1 and NDT2 from A. thaliana.

Fig. 2.

Verification of the function of NTT4 in the ΔnadC mutant. (A) NTT4 function was verified by expressing it in E. coli JW0105, which lacks the de novo NAD⁺ biosynthesis pathway due to nadC disruption. (B) NTT4-expressing strain YJE001 had better growth than the control strain when cultivated in M9 medium containing 100 μM NAD⁺.

Fig. 3.

Construction and characterization of the NAD⁺-auxotrophic mutant YJE003. (A) NAD⁺-auxotrophic mutants were constructed by disrupting the nadE gene in an NTT4 expression background. (B) Phenotype of the NAD⁺-auxotrophic mutant YJE003. About 10⁹ overnight cultured cells were suspended in 1 ml double-distilled H₂O (OD₆₀₀, 0.1), and 10-μl aliquots of dilutions from 10⁻¹ to 10⁻⁶ were spotted on the corresponding plates (from left to right).

Fig. 4.

Determination of the minimal cofactor level for cell growth, based on the NAD⁺ distribution strategy with the NAD⁺-auxotrophic mutant YJE003. (A) Schematic of the NAD⁺ distribution strategy. The auxotrophic mutant YJE003 was preloaded with a certain amount of NAD⁺ after cultivating in M9 medium containing 100 μM NAD⁺ to early log phase (OD₆₀₀, ≈0.8), washed twice with M9 medium, and transferred into the M9 medium without NAD⁺. The cells proliferated, and the cofactor was distributed into daughter cells until the cellular cofactor limited cell division. (B) Growth curve of YJE003 in M9 medium without NAD⁺ with different inoculation densities (0.1 to 0.3). (C) Intracellular cofactor concentrations were measured when the cells reached the stationary phase. The data represent the averages ± standard deviations for at least three independent samples.

Fig. 5.

Manipulation of the intracellular NAD(H) level with different exogenous NAD⁺ feeding levels. (A) Growth curve of YJE003 cultivated in M9 medium containing different concentrations of NAD⁺. μ is the growth rate (h⁻¹), which was calculated from the linear slopes of the logarithmic plots of growth curves. (B) Intracellular NAD(H) concentrations measured at 24 h when the cells reached stationary phase. The data represent the averages ± standard deviations for at least three independent samples.