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 argAATG Y19C to form an operon. These genes were introduced under different promoters; lacIp, lacIqp, lacIq1p, 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 YifEQ100TAG, 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.
Highlights
Putrescine is a polyamine and consists of two amino groups and four methylene groups
As arginine is a key substrate of putrescine synthesis through the arginine decarboxylase (ADC) pathway, to increase putrescine production, we constructed plasmids with the ArgA desensitized to the feedback inhibition by arginine
Efficient initiation codon of argAY19C improved the putrescine production- Arginine is a key substrate of putrescine synthesis through the ADC pathway
Summary
Putrescine is a polyamine and consists of two amino groups and four methylene groups. This compound is widely distributed in living organisms (Pegg 1986; Michael 2016; Keller et al 2019). Several studies have focused on putrescine as it regulates rapid cell proliferation and differentiation at the level of gene expression (Heby 1981; Michael 2018; Igarashi and Kashiwagi 2000, 2018, 2019). E. coli has two putrescine synthetic pathways (Fig. 1): the ornithine decarboxylase (ODC) pathway and the arginine decarboxylase (ADC) pathway, which use ornithine and arginine as starting compounds, respectively (Fig. 1). The other pathway, the ADC pathway, converts arginine to putrescine via agmatine. Haywood and Large (1985) reported that speG encodes a putative diamine acetyltransferase that converts putrescine to acetylputrescine in Candida boidinii. We recently reported that PotFGHI can import spermidine under biofilm-forming conditions (Thongbhubate et al 2021)
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