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Metabolic engineering of Escherichia coli for enhanced arginine biosynthesis.

Ginesy M, Belotserkovsky J, Enman J, Isaksson L, Rova U - Microb. Cell Fact. (2015)

Bottom Line: The V216A mutation in argP (transcriptional regulator of argO, which encodes for an arginine exporter) was identified as a potential candidate for improved arginine production.The combination of multicopy of argP216 or argO and argA214 led to nearly 2-fold and 3-fold increase in arginine production, respectively, and a reduction of acetate formation.In this study, E. coli was successfully engineered for enhanced arginine production.

View Article: PubMed Central - PubMed

Affiliation: Biochemical Process Engineering, Division of Chemical Engineering, Department of Civil, Environmental and Natural Resources Engineering, Luleå University of Technology, SE-971 87, Luleå, Sweden. mireille.ginesy@ltu.se.

ABSTRACT

Background: Arginine is a high-value product, especially for the pharmaceutical industry. Growing demand for environmental-friendly and traceable products have stressed the need for microbial production of this amino acid. Therefore, the aim of this study was to improve arginine production in Escherichia coli by metabolic engineering and to establish a fermentation process in 1-L bioreactor scale to evaluate the different mutants.

Results: Firstly, argR (encoding an arginine responsive repressor protein), speC, speF (encoding ornithine decarboxylases) and adiA (encoding an arginine decarboxylase) were knocked out and the feedback-resistant argA214 or argA215 were introduced into the strain. Three glutamate independent mutants were assessed in bioreactors. Unlike the parent strain, which did not excrete any arginine during glucose fermentation, the constructs produced between 1.94 and 3.03 g/L arginine. Next, wild type argA was deleted and the gene copy number of argA214 was raised, resulting in a slight increase in arginine production (4.11 g/L) but causing most of the carbon flow to be redirected toward acetate. The V216A mutation in argP (transcriptional regulator of argO, which encodes for an arginine exporter) was identified as a potential candidate for improved arginine production. The combination of multicopy of argP216 or argO and argA214 led to nearly 2-fold and 3-fold increase in arginine production, respectively, and a reduction of acetate formation.

Conclusions: In this study, E. coli was successfully engineered for enhanced arginine production. The ∆adiA, ∆speC, ∆speF, ∆argR, ∆argA mutant with high gene copy number of argA214 and argO produced 11.64 g/L of arginine in batch fermentation, thereby demonstrating the potential of E. coli as an industrial producer of arginine.

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Arginine biosynthesis pathway inEscherichia coli. NAGS: N-acetylglutamate synthase, −: inhibition/negative regulation, [c]: cytoplasm, [p]: periplasm, [e]: extracellular. Targeted genes are indicated in bold.
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Fig1: Arginine biosynthesis pathway inEscherichia coli. NAGS: N-acetylglutamate synthase, −: inhibition/negative regulation, [c]: cytoplasm, [p]: periplasm, [e]: extracellular. Targeted genes are indicated in bold.

Mentions: In E. coli, arginine biosynthesis follows a linear pathway starting from the precursors glutamate and acetyl-CoA (Figure 1). The first enzyme in the biosynthetic pathway, N-acetylglutamate synthase (NAGS) encoded by the argA gene, is inhibited by arginine through feedback inhibition [11]. In addition, the arginine responsive repressor protein ArgR, encoded by the argR gene, negatively regulates transcription of arginine biosynthesis genes [12]. E. coli also possesses machineries for the export of some amino acids, including arginine. The arginine export pump ArgO, encoded by the argO gene, is transcriptionally regulated by ArgP [13-15]. The latter is responsive to intracellular arginine levels and activates the transcription of argO accordingly [15-17]. In addition, E. coli has degradative pathways for both L-arginine and its precursor L-ornithine. Two ornithine decarboxylases (encoded by the speC and speF genes) are responsible for the conversion of ornithine to putrescine [18,19], whereas arginine is first degraded into agmatine by an arginine decarboxylase (encoded by adiA), which is subsequently converted to putrescine and urea [18]. In summary, the biosynthesis pathway of arginine is constrained by several layers of metabolic and transcriptional regulations resulting in a complex network to engineer for arginine overproduction. Arginine overproducing E. coli strains have been classically obtained by selection of canavanine-resistant mutants [20]. Canavanine, an arginine analogue, inhibits growth by competing for arginine in protein synthesis [21]. The rationale for using this selection system is that mutants resistant to canavanine are likely to be derepressed for arginine synthesis, as over-production of arginine will release the inhibition caused by canavanine. When characterized, these mutants have subsequently been found to carry mutations in argA, argR and in some instances argP [12,22,23]. Not surprisingly, mutations in argA commonly resulted in an ArgA feedback resistant to arginine, which led some workers to derive further mutants by directed selection [24]. Similarly, mutations in argP resulted in ArgP acting in a constitutive manner, independent of the presence of arginine [14,17].Figure 1


Metabolic engineering of Escherichia coli for enhanced arginine biosynthesis.

Ginesy M, Belotserkovsky J, Enman J, Isaksson L, Rova U - Microb. Cell Fact. (2015)

Arginine biosynthesis pathway inEscherichia coli. NAGS: N-acetylglutamate synthase, −: inhibition/negative regulation, [c]: cytoplasm, [p]: periplasm, [e]: extracellular. Targeted genes are indicated in bold.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4358701&req=5

Fig1: Arginine biosynthesis pathway inEscherichia coli. NAGS: N-acetylglutamate synthase, −: inhibition/negative regulation, [c]: cytoplasm, [p]: periplasm, [e]: extracellular. Targeted genes are indicated in bold.
Mentions: In E. coli, arginine biosynthesis follows a linear pathway starting from the precursors glutamate and acetyl-CoA (Figure 1). The first enzyme in the biosynthetic pathway, N-acetylglutamate synthase (NAGS) encoded by the argA gene, is inhibited by arginine through feedback inhibition [11]. In addition, the arginine responsive repressor protein ArgR, encoded by the argR gene, negatively regulates transcription of arginine biosynthesis genes [12]. E. coli also possesses machineries for the export of some amino acids, including arginine. The arginine export pump ArgO, encoded by the argO gene, is transcriptionally regulated by ArgP [13-15]. The latter is responsive to intracellular arginine levels and activates the transcription of argO accordingly [15-17]. In addition, E. coli has degradative pathways for both L-arginine and its precursor L-ornithine. Two ornithine decarboxylases (encoded by the speC and speF genes) are responsible for the conversion of ornithine to putrescine [18,19], whereas arginine is first degraded into agmatine by an arginine decarboxylase (encoded by adiA), which is subsequently converted to putrescine and urea [18]. In summary, the biosynthesis pathway of arginine is constrained by several layers of metabolic and transcriptional regulations resulting in a complex network to engineer for arginine overproduction. Arginine overproducing E. coli strains have been classically obtained by selection of canavanine-resistant mutants [20]. Canavanine, an arginine analogue, inhibits growth by competing for arginine in protein synthesis [21]. The rationale for using this selection system is that mutants resistant to canavanine are likely to be derepressed for arginine synthesis, as over-production of arginine will release the inhibition caused by canavanine. When characterized, these mutants have subsequently been found to carry mutations in argA, argR and in some instances argP [12,22,23]. Not surprisingly, mutations in argA commonly resulted in an ArgA feedback resistant to arginine, which led some workers to derive further mutants by directed selection [24]. Similarly, mutations in argP resulted in ArgP acting in a constitutive manner, independent of the presence of arginine [14,17].Figure 1

Bottom Line: The V216A mutation in argP (transcriptional regulator of argO, which encodes for an arginine exporter) was identified as a potential candidate for improved arginine production.The combination of multicopy of argP216 or argO and argA214 led to nearly 2-fold and 3-fold increase in arginine production, respectively, and a reduction of acetate formation.In this study, E. coli was successfully engineered for enhanced arginine production.

View Article: PubMed Central - PubMed

Affiliation: Biochemical Process Engineering, Division of Chemical Engineering, Department of Civil, Environmental and Natural Resources Engineering, Luleå University of Technology, SE-971 87, Luleå, Sweden. mireille.ginesy@ltu.se.

ABSTRACT

Background: Arginine is a high-value product, especially for the pharmaceutical industry. Growing demand for environmental-friendly and traceable products have stressed the need for microbial production of this amino acid. Therefore, the aim of this study was to improve arginine production in Escherichia coli by metabolic engineering and to establish a fermentation process in 1-L bioreactor scale to evaluate the different mutants.

Results: Firstly, argR (encoding an arginine responsive repressor protein), speC, speF (encoding ornithine decarboxylases) and adiA (encoding an arginine decarboxylase) were knocked out and the feedback-resistant argA214 or argA215 were introduced into the strain. Three glutamate independent mutants were assessed in bioreactors. Unlike the parent strain, which did not excrete any arginine during glucose fermentation, the constructs produced between 1.94 and 3.03 g/L arginine. Next, wild type argA was deleted and the gene copy number of argA214 was raised, resulting in a slight increase in arginine production (4.11 g/L) but causing most of the carbon flow to be redirected toward acetate. The V216A mutation in argP (transcriptional regulator of argO, which encodes for an arginine exporter) was identified as a potential candidate for improved arginine production. The combination of multicopy of argP216 or argO and argA214 led to nearly 2-fold and 3-fold increase in arginine production, respectively, and a reduction of acetate formation.

Conclusions: In this study, E. coli was successfully engineered for enhanced arginine production. The ∆adiA, ∆speC, ∆speF, ∆argR, ∆argA mutant with high gene copy number of argA214 and argO produced 11.64 g/L of arginine in batch fermentation, thereby demonstrating the potential of E. coli as an industrial producer of arginine.

Show MeSH
Related in: MedlinePlus