Disruption of a Global Regulatory Gene to Enhance Central Carbon Flux into Phenylalanine Biosynthesis in Escherichia coli

Genetic engineering of microbes for commercial metabolite production traditionally has sought to alter the levels and/or intrinsic activities of key enzymes in relevant biosynthetic pathway(s). Microorganisms exploit similar strategies for flux control, but also coordinate flux through sets of related pathways by using global regulatory circuits. We have engineered a global regulatory system of Escherichia coli, Csr (carbon storage regulator), to increase precursor for aromatic amino acid biosynthesis. Disruption of csrA increases gluconeogenesis, decreases glycolysis, and thus elevates phosphoenolpyruvate, a limiting precursor of aromatics. A strain in which the aromatic (shikimate) pathway had been optimized produced twofold more phenylalanine when csrA was disrupted. Overexpression of tktA (transketolase) to increase the other precursor, erythrose-4-phosphate, yielded ∼1.4-fold enhancement, while both changes were additive. These effects of csrA were not mediated by increasing the regulatory enzymes of phenylalanine biosynthesis. This study introduces the concept of “global metabolic engineering” for second-generation strain improvement. Received: 25 October 2000 / Accepted: 8 December 2000     

Regulation of Aconitase Synthesis in Bacillus subtilis: Induction, Feedback Repression, and Catabolite Repression

The synthesis of aconitase in Bacillus subtilis wild-type and different citric acid cycle mutants has been studied and the influence of various growth conditions examined. Aconitase is induced by citrate and precursors of citrate and repressed by glutamate. Induction and repression counteract each other, and at equimolar concentrations of citrate and glutamate, aconitase synthesis is unaffected. Induction by citrate can partly overcome catabolite repression of aconitase. Isocitrate dehydrogenase show endogenous induction of aconitase due to citrate accumulation. Leaky mutants defective in citrate synthase and aconitase cannot be induced by citrate, which indicates that they carry a regulatory mutation. The complex regulation of aconitase is discussed with reference to the participation of this enzyme in glutamate biosynthesis and energy metabolism.     

Metabolic regulation of citrate and iron by aconitases: role of iron–sulfur cluster biogenesis

Iron and citrate are essential for the metabolism of most organisms, and regulation of iron and citrate biology at both the cellular and systemic levels is critical for normal physiology and survival. Mitochondrial and cytosolic aconitases catalyze the interconversion of citrate and isocitrate, and aconitase activities are affected by iron levels, oxidative stress and by the status of the Fe–S cluster biogenesis apparatus. Assembly and disassembly of Fe–S clusters is a key process not only in regulating the enzymatic activity of mitochondrial aconitase in the citric acid cycle, but also in controlling the iron sensing and RNA binding activities of cytosolic aconitase (also known as iron regulatory protein IRP1). This review discusses the central role of aconitases in intermediary metabolism and explores how iron homeostasis and Fe–S cluster biogenesis regulate the Fe–S cluster switch and modulate intracellular citrate flux.     

A novel metabolic network leads to enhanced citrate biogenesis in <Emphasis Type="Italic">Pseudomonas </Emphasis><Emphasis Type="Italic">fluorescens</Emphasis> exposed to aluminum toxicity

Aluminum (Al), an environmental toxin, is known to have a negative impact on various biological systems. However, some microbes have devised intricate mechanisms to combat the toxic influence of this trivalent metal. In this study, Pseudomonas fluorescens grown in malate invoked a unique metabolic shift to promote the synthesis of citrate, a metabolite involved in the sequestration of Al. Electrophoretic and spectrophotometric assays revealed several malate-metabolizing enzymes including malate dehydrogenase (MDH) and malic enzyme (ME) displayed increases in activity and expression in the Al-treated cells. Whereas pyruvate dehydrogenase (PDH) also showed increased activity and expression in the Al-stressed cultures, phosphoenolpyruvate carboxykinase (PEPCK) displayed a marked diminution in the Al-treated cells. The upregulation of citrate synthase (CS) coupled with the diminished activities of aconitase (ACN) and NAD-isocitrate dehydrogenase (NAD-ICDH) appeared to be instrumental in the accumulation of citrate. HPLC experiments revealed high levels of citrate in the Al-stressed cultures. Thus, an Al-enriched environment provoked a metabolic shift in P. fluorescens dedicated to the conversion of malate to citrate.     

Iron-shortage-induced increase in citric acid content and reduction of cytosolic aconitase activity in Citrus fruit vesicles and calli

Aconitase, which catalyses the conversion of citrate into isocitrate, requires Fe for its activity. The yeast and animal enzyme loses its enzymatic activity under Fe shortage and binds to RNA of genes involved in Fe homeostasis, altering their expression. Thus, the enzyme provides a regulatory link between organic acid metabolism and Fe cellular status. Roots and leaves of Fe-deficient plants show induction in organic acids, especially citrate. Although no RNA-binding activity has been so far demonstrated for the plant aconitase, whether alternations in enzyme activity by Fe could play a role in this induction remain unanswered. This question was investigated in lemon fruit [ Citrus limon (L.) Burm var Eureka ], characterized by the accumulation of citrate to about 0.3 M in the juice vesicles cells (pulp). Calli and isolated juice vesicles showed two- to three-fold induction in citrate level when subjected to Fe shortage. The mRNA level of aconitase exhibited no changes under reduced Fe concentrations. Analysis of aconitase isozymes demonstrated that out of two aconitase isozymes, typically detected in citrus fruit, only the cytosolic form displayed a reduced activity under low Fe concentrations. Our data support the notion of a limited Fe-availability-induced reduction in cytosolic aconitase, resulting in a slower rate of citrate breakdown and a concomitant increase in citrate levels.     

Aconitase and citric acid fermentation by Aspergillus niger.

In view of the often-cited theory that citric acid accumulation is caused by an inhibition of aconitase activity, the equilibrium of the reaction of aconitase was investigated by comparing in vivo steady-state concentrations of citrate and isocitrate in Aspergillus niger grown under various citric acid-producing conditions. With the equilibrium catalyzed by the A. niger enzyme in vitro, similar values were obtained. The validity of our in vivo measurements was verified by the addition of the aconitase inhibitor fluorocitrate, which appreciably elevated the citrate:isocitrate ratio. The results strongly argue against an inhibition of aconitase during citric acid fermentation.     

Aconitase and citric acid fermentation by Aspergillus niger

In view of the often-cited theory that citric acid accumulation is caused by an inhibition of aconitase activity, the equilibrium of the reaction of aconitase was investigated by comparing in vivo steady-state concentrations of citrate and isocitrate in Aspergillus niger grown under various citric acid-producing conditions. With the equilibrium catalyzed by the A. niger enzyme in vitro, similar values were obtained. The validity of our in vivo measurements was verified by the addition of the aconitase inhibitor fluorocitrate, which appreciably elevated the citrate:isocitrate ratio. The results strongly argue against an inhibition of aconitase during citric acid fermentation.     

Inhibition of aconitase in citrus fruit callus results in a metabolic shift towards amino acid biosynthesis

Citrate, a major determinant of citrus fruit quality, accumulates early in fruit development and declines towards maturation. The isomerization of citrate to isocitrate, catalyzed by aconitase is a key step in acid metabolism. Inhibition of mitochondrial aconitase activity early in fruit development contributes to acid accumulation, whereas increased cytosolic activity of aconitase causes citrate decline. It was previously hypothesized that the block in mitochondrial aconitase activity, inducing acid accumulation, is caused by citramalate. Here, we investigated the effect of citramalate and of another aconitase inhibitor, oxalomalate, on aconitase activity and regulation in callus originated from juice sacs. These compounds significantly increased citrate content and reduced the enzyme’s activity, while slightly inducing its protein level. Citramalate inhibited the mitochondrial, but not cytosolic form of the enzyme. Its external application to mandarin fruits resulted in inhibition of aconitase activity, with a transient increase in fruit acidity detected a few weeks later. The endogenous level of citramalate was analyzed in five citrus varieties: its pattern of accumulation challenged the notion of its action as an endogenous inhibitor of mitochondrial aconitase. Metabolite profiling of oxalomalate-treated cells showed significant increases in a few amino acids and organic acids. The activities of alanine transaminase, aspartate transaminase and aspartate kinase, as well as these of two γ-aminobutyrate (GABA)-shunt enzymes, succinic semialdehyde reductase (SSAR) and succinic semialdehyde dehydrogenase (SSAD) were significantly induced in oxalomalate-treated cells. It is suggested that the increase in citrate, caused by aconitase inhibition, induces amino acid synthesis and the GABA shunt, in accordance with the suggested fate of citrate during the acid decline stage in citrus fruit.     

Inhibition of aconitase by nitric oxide leads to induction of the alternative oxidase and to a shift of metabolism towards biosynthesis of amino acids

Nitric oxide (NO) is a free radical molecule involved in signalling and in hypoxic metabolism. This work used the nitrate reductase double mutant of Arabidopsis thaliana (nia) and studied metabolic profiles, aconitase activity, and alternative oxidase (AOX) capacity and expression under normoxia and hypoxia (1% oxygen) in wild-type and nia plants. The roots of nia plants accumulated very little NO as compared to wild-type plants which exhibited ～20-fold increase in NO emission under low oxygen conditions. These data suggest that nitrate reductase is involved in NO production either directly or by supplying nitrite to other sites of NO production (e.g. mitochondria). Various studies revealed that NO can induce AOX in mitochondria, but the mechanism has not been established yet. This study demonstrates that the NO produced in roots of wild-type plants inhibits aconitase which in turn leads to a marked increase in citrate levels. The accumulating citrate enhances AOX capacity, expression, and protein abundance. In contrast to wild-type plants, the nia double mutant failed to show AOX induction. The overall induction of AOX in wild-type roots correlated with accumulation of glycine, serine, leucine, lysine, and other amino acids. The findings show that NO inhibits aconitase under hypoxia which results in accumulation of citrate, the latter in turn inducing AOX and causing a shift of metabolism towards amino acid biosynthesis.     

The Escherichia coli CsrB and CsrC small RNAs are strongly induced during growth in nutrient-poor medium

The carbon storage regulatory (Csr) system is a complex network controlling various phenotypes in many eubacteria. So far, the external conditions by which the system is regulated are poorly understood. Here we show that the expression of the two noncoding small RNAs CsrB and CsrC in Escherichia coli is strongly increased in cultures grown in minimal medium. Addition of tryptone, casamino acids or a mixture of amino acids to a culture grown in minimal medium led to a rapid reduction in the levels of CsrB. Based on this we propose that the expression of the Csr sRNAs is controlled by the amino acid availability in the growth medium.