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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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Fermentation profile of (a) SJB001, (b) SJB003, (c) SJB004, (d) SJB015, (e) SJB006, (f) SJB007, (g) SJB009 and (h) SJB010. ● glucose, ▲ acetic acid, ■ arginine and ◆ DCW.
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Fig2: Fermentation profile of (a) SJB001, (b) SJB003, (c) SJB004, (d) SJB015, (e) SJB006, (f) SJB007, (g) SJB009 and (h) SJB010. ● glucose, ▲ acetic acid, ■ arginine and ◆ DCW.

Mentions: When cultivated in bioreactors, SJB015 displayed a slightly improved arginine production compared to the previous constructs (Table 2). In particular, YP/x was relatively high (0.50 g arg/g dcw). However, cell growth was seriously hampered for this strain and SJB015 had the lowest μ and YX/S and the final cell density was lower than that of the other strains (data not shown). Consequently the volumetric productivity of SJB015 was relatively low (0.08 g/L/h). In addition, SJB015 produced high levels of acetate. When a DCW of about 7 g/L was reached (Figure 2d), cell growth stopped, arginine production drastically decreased and the remaining sugar (approximately 30% of the initial glucose) was mainly used for acetate formation (up to 28 g/L).Figure 2


Metabolic engineering of Escherichia coli for enhanced arginine biosynthesis.

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

Fermentation profile of (a) SJB001, (b) SJB003, (c) SJB004, (d) SJB015, (e) SJB006, (f) SJB007, (g) SJB009 and (h) SJB010. ● glucose, ▲ acetic acid, ■ arginine and ◆ DCW.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Fermentation profile of (a) SJB001, (b) SJB003, (c) SJB004, (d) SJB015, (e) SJB006, (f) SJB007, (g) SJB009 and (h) SJB010. ● glucose, ▲ acetic acid, ■ arginine and ◆ DCW.
Mentions: When cultivated in bioreactors, SJB015 displayed a slightly improved arginine production compared to the previous constructs (Table 2). In particular, YP/x was relatively high (0.50 g arg/g dcw). However, cell growth was seriously hampered for this strain and SJB015 had the lowest μ and YX/S and the final cell density was lower than that of the other strains (data not shown). Consequently the volumetric productivity of SJB015 was relatively low (0.08 g/L/h). In addition, SJB015 produced high levels of acetate. When a DCW of about 7 g/L was reached (Figure 2d), cell growth stopped, arginine production drastically decreased and the remaining sugar (approximately 30% of the initial glucose) was mainly used for acetate formation (up to 28 g/L).Figure 2

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