Improvement of putrescine production through the arginine decarboxylase pathway in Escherichia coli K-12

Abstract

In the bio-based polymer industry, putrescine is in the spotlight for use as a material. We constructed strains of Escherichia coli to assess its putrescine production capabilities through the arginine decarboxylase pathway in batch fermentation. N-Acetylglutamate (ArgA) synthase is subjected to feedback inhibition by arginine. Therefore, the 19th amino acid residue, Tyr, of argA was substituted with Cys to desensitize the feedback inhibition of arginine, resulting in improved putrescine production. The inefficient initiation codon GTG of argA was substituted with the effective ATG codon, but its replacement did not affect putrescine production. The essential genes for the putrescine production pathway, speA and speB, were cloned into the same plasmid with argA^{ATG Y19C} to form an operon. These genes were introduced under different promoters; lacIp, lacI^qp, lacI^q1p, and T5p. Among these, the T5 promoter demonstrated the best putrescine production. In addition, disruption of the puuA gene encoding enzyme of the first step of putrescine degradation pathway increased the putrescine production. Of note, putrescine production was not affected by the disruption of patA, which encodes putrescine aminotransferase, the initial enzyme of another putrescine utilization pathway. We also report that the strain KT160, which has a genomic mutation of YifE^Q100TAG, had the greatest putrescine production. At 48 h of batch fermentation, strain KT160 grown in terrific broth with 0.01 mM IPTG produced 19.8 mM of putrescine.

Keywords: Glutamate-putrescine ligase; Macrodomain Ori protein; N-acetylglutamate synthase; Terrific broth.

Fig. 1

The metabolic map of putrescine in E. coli K-12. The Xs indicate the knocked out genes. Thick arrows indicate the overexpression of genes. Dash arrows indicate the increased expression of genes. Blunt end arrows indicate inhibition of the expression of genes. GdhA glutamate dehydrogenase, HdfR DNA-binding transcriptional dual regulator HdfR, GltBD glutamate synthase, GlnA glutamine synthetase, GlnE glutamine synthetase adenylyltransferase, CarAB carbamoyl phosphate synthetase, ArgA N-acetylglutamate synthase, ArgB acetylglutamate kinase, ArgC N-acetylglutamylphosphate reductase, ArgD N-acetylornithine aminotransferase, ArgE acetylornithine deacetylase, ArgF ornithine carbamoyltransferase, ArgI ornithine carbamoyltransferase, ArgG argininosuccinate synthetase, ArgH argininosuccinate lyase, ArgR DNA-binding transcriptional dual regulator ArgR, SpeA biosynthetic arginine decarboxylase, SpeB agmatinase, SpeC constitutive ornithine decarboxylase, SpeD S-adenosylmethionine decarboxylase, SpeE spermidine synthase, SpeF inducible ornithine decarboxylase, SpeG spermidine N-acetyltransferase, PatA putrescine aminotransferase, PatD γ-aminobutyraldehyde dehydrogenase, GabT 4-aminobutyrate aminotransferase, GabD succinate-semialdehyde dehydrogenase GabD, Sad succinate-semialdehyde dehydrogenase Sad, PuuA glutamate-putrescine ligase, PuuB γ-glutamylputrescine oxidase, PuuC γ-glutamyl-γ-aminobutyraldehyde dehydrogenase, PuuD γ-glutamyl-γ-aminobutyrate hydrolase, PuuP putrescine importer, YdcSTUV putrescine importer, PotFGHI putrescine importer, PlaP putrescine importer, PotE putrescine-ornithine antiporter/putrescine importer, SapBCDF putrescine exporter, TCA cycle tricarboxylic acid cycle

Fig. 2

Base substitution on argA increased putrescine production. Putrescine concentration in the supernatant of strains harboring plasmids with different mutant argA: KT207 (argA^{ATG Y19C}), KT208 (argA^{GTG Y19C}), KT209 (argA^{ATG Y19}), and KT210 (argA^{GTG Y19}). The strains were grown in LB medium supplemented with 100 μg/mL of ampicillin at 37 °C for 48 h with reciprocal shaking at 120 rpm. When the OD₆₀₀ reached 0.4, 0.02 mM IPTG was added. **, p < 0.01; ***, p < 0.001. Data shown are averages ± standard deviations, and culture experiments were performed in triplicate

Fig. 3

Effect of SpeA, SpeB, and SpeC on putrescine production. A Bacterial cell growth and B putrescine concentration in the supernatant of strains FS123 (speC⁺, open circle), KN20 (speABC⁺, closed triangle), and KN24 (speAB⁺, closed square) grown in LB medium supplemented with 100 μg/mL of ampicillin. When the OD₆₀₀ reached 0.4, 0.02 mM IPTG was added. Data shown are averages ± standard deviations, and culture experiments were performed in triplicate

Fig. 4

Effects of the wild-type ArgA expressed from the genome on putrescine production. A Bacterial cell growth and B putrescine concentration in the supernatant of strains KT39 (ΔargA, open circle) and KT207 (argA⁺, closed triangle) grown in LB medium supplemented with 100 μg/mL of ampicillin. When the OD₆₀₀ reached 0.4, 0.02 mM IPTG was added. Data shown are averages ± standard deviations, and culture experiments were performed in triplicate

Fig. 5

Effects of promoters of speAB and desensitized argA on putrescine production. A Bacterial cell growth and B putrescine concentration in the supernatant of strains KT37 (lacI promoter, closed triangle), KT38 (lacI^q promoter, open circle), KT39 (lacI^q1 promoter, closed square), and KT148 (T5 promoter, open diamond) grown in LB medium supplemented with 100 μg/mL of ampicillin. When the OD₆₀₀ reached 0.4, 0.02 mM IPTG was added. Data shown are averages ± standard deviations, and culture experiments were performed in triplicate

Fig. 6

Effects of patA, puuA, and puuP genes on the putrescine production. The amount of putrescine in the culture supernatant of the strains KT148, KT152, KT153, KT154, and KT162 grown in LB medium supplemented with 100 μg/mL of ampicillin for 48 h. When the OD₆₀₀ reached 0.4, 0.03 mM IPTG was added. ***, p < 0.001; ns, not significantly different. Data shown are averages ± standard deviations, and culture experiments were performed in triplicate

Fig. 7

Effects of culture media on putrescine production by strain KT152. A Bacterial cell growth and B putrescine concentration in the supernatant of the strain KT152, whose putrescine utilization pathway (ΔpuuAP ΔpatA) was disrupted. Open triangles represent the strain KT152 grown in the LB medium supplemented with 100 μg/mL of ampicillin; closed circles represent the strain KT152 grown in terrific broth supplemented with 100 μg/mL of ampicillin. When the OD₆₀₀ reached 0.4, 0.03 mM IPTG was added. Data shown are averages ± standard deviations, and culture experiments were performed in triplicate

Fig. 8

Effects of the yifE mutant and putrescine transporter PotFGHI on putrescine production. a The putrescine concentration in the supernatant of the culture media of strains KT152, KT160, and KT161 grown in terrific broth supplemented with 100 μg/mL of ampicillin for 48 h. When the OD₆₀₀ reached 0.4, 0.02 mM IPTG was added. b The putrescine concentration in the supernatant of the culture media of strains KT160 grown in terrific broth supplemented with 100 μg/mL of ampicillin. When the OD₆₀₀ reached 0.4, 0.01 mM IPTG was added. * p < 0.001; ns, not significantly different. yifE: WT, yifE^wt; Q100TAG, yifE^Q100TAG gene. potFGHI: + , presence of potFGHI operon; -, absence of potFGHI operon. [IPTG]: 0.01 and 0.02 is the optimum concentration of IPTG in terms of mM. Data shown are averages ± standard deviations, and culture experiments were performed in triplicate