Metabolic engineering of Escherichia coli for enhanced arginine biosynthesis

Abstract

BackgroundArginine 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.ResultsFirstly, 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.ConclusionsIn 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.

Tuning of the Carbon-to-Nitrogen Ratio for the Production of l-Arginine by Escherichia coli

“…Looking more closely into the acetate production is interesting since acetate is the main by-product during L-arginine production [11]. Conditions that minimise its formation are desirable.…”

“…Prior to the fermentation, the pH was adjusted to 7 using 2 M HCl or 5 M NaOH when necessary. The temperature was set to 32 • C and the stirring speed to 500 rpm [11]. Throughout the fermentation, the pH was maintained at 7 with automatic addition of 5 M NaOH and the dissolved oxygen level at 50% with automatic addition of 5 vvm of air.…”

“…The strain E. coli SJB009 previously engineered to have an enhanced L-arginine production ability was used [11]. This strain is derived from E. coli K-12 C600.…”

“…Acetic acid, the main by-product formed during fermentation [11], was quantified by HPLC using refractive index detection. The cation exchange column (Aminex HPX87-H, BioRad, Hercules, CA, USA) and guard column (Micro-Guard IG Cation H Cartridge, BioRad) were maintained at 65 • C. The mobile phase was 5 mM H 2 SO 4 at a flow rate of 0.6 mL/min.…”

“…Combined with its fast growth, its robustness and the availability of molecular tools for its genetic engineering, E. coli is also a candidate for L-arginine production. An E. coli strain able to produce nearly 12 g/L of L-arginine with a yield of 0.17 g arginine /g glucose was recently engineered [11].…”