Metabolic regulation of Escherichia coli and its gdhA, glnL, gltB, D mutants under different carbon and nitrogen limitations in the continuous culture

Abstract

Background: It is quite important to understand how the central metabolism is regulated under nitrogen (N)- limitation as well as carbon (C)- limitation. In particular, the effect of C/N ratio on the metabolism is of practical interest for the heterologous protein production, PHB production, etc. Although the carbon and nitrogen metabolisms are interconnected and the overall mechanism is complicated, it is strongly desirable to clarify the effects of culture environment on the metabolism from the practical application point of view.

Results: The effect of C/N ratio on the metabolism in Escherichia coli was investigated in the aerobic continuous culture at the dilution rate of 0.2 h-1 based on fermentation data, transcriptional RNA level, and enzyme activity data. The glucose concentration was kept at 10 g/l, while ammonium sulfate concentration was varied from 5.94 to 0.594 g/l. The resultant C/N ratios were 1.68 (100%), 2.81(60%), 4.21(40%), 8.42(20%), and 16.84(10%), where the percentage values in brackets indicate the ratio of N- concentration as compared to the case of 5.94 g/l of ammonium sulfate. The mRNA levels of crp and mlc decreased, which caused ptsG transcript expression to be up-regulated as C/N ratio increased. As C/N ratio increased cra transcript expression decreased, which caused ptsH, pfkA, and pykF to be up-regulated. At high C/N ratio, transcriptional mRNA level of soxR/S increased, which may be due to the activated respiratory chain as indicated by up-regulations of such genes as cyoA, cydB, ndh as well as the increase in the specific CO2 production rate. The rpoN transcript expression increased with the increase in C/N ratio, which led glnA, L, G and gltD transcript expression to change in similar fashion. The nac transcript expression showed similar trend as rpoN, while gdhA transcript expression changed in reverse direction. The transcriptional mRNA level of glnB, which codes for PII, glnD and glnK increased as C/N ratio increases. It was shown that GS-GOGAT pathway was activated for gdhA mutant under N- rich condition. In the case of glnL mutant, GOGAT enzyme activity was reduced as compared to the wild type under N- limitation. In the case of gltB, D mutants, GDH and GS enzymes were utilized under both N- rich and N- limited conditions. In this case, the transcriptional mRNA level of gdhA and corresponding GDH enzyme activity was higher under N- limitation as compared to N- rich condition.

Conclusion: The metabolic regulation of E.coli was clarified under both carbon (C)- limitation and nitrogen (N)- limitation based on fermentation, transcriptional mRNA level and enzyme activities. The overall regulation mechanism was proposed. The effects of knocking out N- assimilation pathway genes were also clarified.

Figure 1

Central metabolic pathways of E.coli concerned with C- metabolism and N- assimilation.

Figure 2

Comparison of fermentor characteristics of the wild type E.coli at various C/N ratios: (a) biomass, glucose and acetate concentration (g/l), cell yield (g/g); (b) specific rates of glucose consumption, acetate and CO₂ production (mmol/g DCW. h).

Figure 3

Comparison of the transcriptional mRNA levels of the wild type E.coli genes cultivated at 100% (C/N = 1.68), 40% (C/N = 4.21), 20% (C/N = 8.42) and 10% (C/N = 1.68) N- concentration: (a) global regulatory, (b) N- regulatory, (c) metabolic pathway, (d) respiratory chain.

Figure 4

Comparison of fermentor characteristics of the wild type E.coli and nitrogen assimilation related mutants, gdhA, glnL, gltB, and gltD at 100% N- concentration (C/N ratio = 1.68): (a) biomass, glucose and acetate concentration (g/l), cell yield (g/g); (b) specific rates of glucose consumption, acetate and CO₂ production (mmol/g DCW. h).

Figure 5

Comparison of fermentor characteristics of the wild type E.coli and nitrogen assimilation related mutants, gdhA, glnL, gltB, and gltD at 20% N- concentration (C/N ratio = 8.42): (a) biomass, glucose and acetate concentration (g/l), cell yield (g/g); (b) specific rates of glucose consumption, acetate and CO₂ production (mmol/g DCW. h).

Figure 6

Enzyme Activities of N- assimilation pathway such as GDH, GS, and GOGAT in the wild type E.coli and its mutants in Units*/mg protein.

Figure 7

Proposed overall mechanism of N- assimilation in E.coli under C- limited (N- rich) and N- limited conditions.

Figure 8

Proposed schematic illustration of N-assimilation pathways for the wild type E.coli and its mutants under C- limiting (N-rich) and N-limiting conditions.

Figure 9

Proposed scheme of N- assimilation in E.coli in response to the changes in C- and N-concentrations.