Dataset Information

Construction of Streptomyces coelicolor A3(2) mutants that exclusively produce NA4/NA6 intermediates of agarose metabolism through mutation induction.

ABSTRACT: NA4/NA6, an intermediate degradation product of β-agarase, is a high value-added product with anticancer, anti-obesity, and anti-diabetic effects. Therefore, a method that enables the efficient production of NA4/NA6 would be useful from economic and medical perspectives. In this study, we aimed to generate a Streptomyces coelicolor A3(2) mutant M22-2C43 that produces NA4/NA6 as a final product; this method serves as a more efficient alternative to the enzymatic conversion of β-agarase for the generation of these products. The M22-2C43 strain was generated through two rounds of mutagenesis and screening for increased β-agarase activity and effective production of NA4/NA6. We assembled the complete genomes of two mutants, M22 and M22-2C43, which were identified following a two-round screening. Large and small genetic changes were found in these two mutants, including the loss of two plasmids present in wild-type S. coelicolor A3(2) and chromosome circularization of mutant M22-2C43. These findings suggest that mutant M22-2C43 can produce NA4/NA6 as a degradation product due to functional inactivation of the dagB gene through a point mutation (G474A), ultimately preventing further degradation of NA4/NA6 to NA2. To our knowledge, this is the first report of a microbial strain that can effectively produce NA4/NA6 as the main degradation product of β-agarase, opening the door for the use of this species for the large-scale production of this valuable product.

SUBMITTER: Kim J

PROVIDER: S-EPMC10624881 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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Construction of Streptomyces coelicolor A3(2) mutants that exclusively produce NA4/NA6 intermediates of agarose metabolism through mutation induction.

Kim Jina J Kim Eun Joo EJ Ko Hye-Jeong HJ Lee Yeon-Hee YH Hong Soon-Kwang SK Shin Miyoung M Lee Je Hyeon JH Kwak Woori W

Scientific reports 20231103 1

NA4/NA6, an intermediate degradation product of β-agarase, is a high value-added product with anticancer, anti-obesity, and anti-diabetic effects. Therefore, a method that enables the efficient production of NA4/NA6 would be useful from economic and medical perspectives. In this study, we aimed to generate a Streptomyces coelicolor A3(2) mutant M22-2C43 that produces NA4/NA6 as a final product; this method serves as a more efficient alternative to the enzymatic conversion of β-agarase for the ge ...[more]

PMID: 37923760

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Project description:4-Coumarate:coenzyme A ligase (4CL) plays a key role in phenylpropanoid metabolism, providing precursors for a large variety of important plant secondary metabolites, such as lignin, flavonoids, and phytoalexins. Although 4CLs have been believed to be specific to plants, a gene encoding a 4CL-like enzyme which shows more than 40% identity in amino acid sequence to plant 4CLs was found in the genome of the gram-positive, filamentous bacterium Streptomyces coelicolor A3(2). The recombinant enzyme, produced in Escherichia coli with a histidine tag at its N-terminal end, showed distinct 4CL activity. The optimum pH and temperature of the reaction were pH 8.0 and 30 degrees C, respectively. The K(m) value for 4-coumarate and k(cat) were determined as 131 +/- 4 micro M and 0.202 +/- 0.007 s(-1), respectively. The K(m) value was comparable to those of plant 4CLs. The substrate specificity of this enzyme was, however, distinctly different from those of plant 4CLs. The enzyme efficiently converted cinnamate (K(m), 190 +/- 2 micro M; k(cat), 0.475 +/- 0.012 s(-1)), which is a very poor substrate for plant 4CLs. Furthermore, the enzyme showed only low activity toward caffeate and no activity toward ferulate, both of which are generally good substrates for plant 4CLs. The enzyme was therefore named ScCCL for S. coelicolor A3(2) cinnamate CoA ligase. To determine the amino acid residues providing the unique substrate specificity of ScCCL, eight ScCCL mutant enzymes having a mutation(s) at amino acid residues that probably line up along the substrate-binding pocket were generated. Mutant A294G used caffeate as a substrate more efficiently than ScCCL, and mutant A294G/A318G used ferulate, which ScCCL could not use as a substrate, suggesting that Ala(294) and Ala(318) are involved in substrate recognition. Furthermore, the catalytic activities of A294G and A294G/A318G toward cinnamate and 4-coumarate were greatly enhanced compared with those of the wild-type enzyme.

Dataset Information

Construction of Streptomyces coelicolor A3(2) mutants that exclusively produce NA4/NA6 intermediates of agarose metabolism through mutation induction.

Publications

Construction of Streptomyces coelicolor A3(2) mutants that exclusively produce NA4/NA6 intermediates of agarose metabolism through mutation induction.

Similar Datasets

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