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35. MOESM1 of 5-Aminolevulinic acid fermentation using engineered Saccharomyces cerevisiae

Author

Hara, Kiyotaka, Saito, Masaru, Kato, Hiroko, Morikawa, Kana, Kikukawa, Hiroshi, Nomura, Hironari, Fujimoto, Takanori, Hirono-Hara, Yoko, Watanabe, Shigeyuki, Kanamaru, Kengo, and Kondo, Akihiko

Abstract

Additional file 1. Table S1. Effect of glycine addition on intracellular metabolites in HEM1-overexpressing S. cerevisiae. Figure S1. Relative mRNA levels in HEM1-, HEM1/ACO1- and HEM1/ACO2-overexpressing strains.

Published
2019
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38. Engineering of the Long-Main-Chain Monomer-Incorporating Polyhydroxyalkanoate Synthase PhaCARfor the Biosynthesis of Poly[(R)-3-hydroxybutyrate-co-6-hydroxyhexanoate]

Author

Hozumi, Yuka, Hachisuka, Shin-ichi, Tomita, Hiroya, Kikukawa, Hiroshi, and Matsumoto, Ken’ichiro

Abstract

Polyhydroxyalkanoate (PHA) synthases (PhaCs) are useful and versatile tools for the production of aliphatic polyesters. Here, the chimeric PHA synthase PhaCARwas engineered to increase its capacity to incorporate unusual 6-hydroxyhexanoate (6HHx) units. Mutations at positions 149 and 314 in PhaCARwere previously found to increase the incorporation of an analogous natural monomer, 3-hydroxyhexanoate (3HHx). We attempted to repurpose the mutations to produce 6HHx-containing polymers. Site-directed saturation mutants at these positions were applied for P(3HB-co-6HHx) synthesis in Escherichia coli. As a result, the N149D and F314Y mutants effectively increased the 6HHx fraction. Moreover, the pairwise NDFY mutation further increased the 6HHx fraction, which reached 22 mol %. This increase was presumably caused by altered enzyme activity rather than altered expression levels, as assessed based on immunoblot analysis. The glass transition temperature and crystallinity of P(3HB-co-6HHx) decreased as the 6HHx fraction increased.

Published
2024
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43. Asymmetric oxidation of 1,3-propanediols to chiral hydroxyalkanoic acids by Rhodococcussp. 2N

Author

Kikukawa, Hiroshi, Koyasu, Rena, Yasohara, Yoshihiko, Ito, Noriyuki, Mitsukura, Koichi, and Yoshida, Toyokazu

Abstract

ABSTRACTRhodococcussp. 2N was found as a 1,3-propanediols-oxidizing strain from soil samples through enrichment culture using 2,2-diethyl-1,3-propanediol (DEPD) as the sole carbon source. The culture condition of the strain 2N was optimized, and the highest activity was observed when 0.3% (w/v) DEPD was added in the culture medium as an inducer. Chiral HPLC analysis of the hydroxyalkanoic acid converted from 2-ethyl-2-methyl-1,3-propanediol (EMPD) revealed that the strain 2N catalyzed the (R)-selective oxidation of EMPD. The reaction products and intermediates from DEPD and EMPD were identified by nuclear magnetic resonance analyses, and the results suggested that only one hydroxymethyl group of the propanediols was converted to carboxy group via two oxidation steps. Under optimized conditions and after a 72-h reaction time, the strain 2N produced 28 mM (4.1 g/L) of 2-(hydroxymethyl)-2-methylbutanoic acid from EMPD with a molar conversion yield of 47% and 65% ee (R).

Published
2019
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50. Engineering of the Long-Main-Chain Monomer-Incorporating Polyhydroxyalkanoate Synthase PhaC AR for the Biosynthesis of Poly[( R )-3-hydroxybutyrate- co -6-hydroxyhexanoate].

Author

Hozumi Y, Hachisuka SI, Tomita H, Kikukawa H, and Matsumoto K

Subjects
Protein Engineering methods, Polyesters chemistry, Polyesters metabolism, Mutagenesis, Site-Directed, Polyhydroxyalkanoates chemistry, Polyhydroxyalkanoates biosynthesis, Bacterial Proteins genetics, Bacterial Proteins metabolism, Bacterial Proteins chemistry, Acyltransferases genetics, Acyltransferases metabolism, Escherichia coli genetics, Escherichia coli metabolism, Caproates chemistry, Caproates metabolism
Abstract

Polyhydroxyalkanoate (PHA) synthases (PhaCs) are useful and versatile tools for the production of aliphatic polyesters. Here, the chimeric PHA synthase PhaC AR was engineered to increase its capacity to incorporate unusual 6-hydroxyhexanoate (6HHx) units. Mutations at positions 149 and 314 in PhaC AR were previously found to increase the incorporation of an analogous natural monomer, 3-hydroxyhexanoate (3HHx). We attempted to repurpose the mutations to produce 6HHx-containing polymers. Site-directed saturation mutants at these positions were applied for P(3HB- co -6HHx) synthesis in Escherichia coli . As a result, the N149D and F314Y mutants effectively increased the 6HHx fraction. Moreover, the pairwise NDFY mutation further increased the 6HHx fraction, which reached 22 mol %. This increase was presumably caused by altered enzyme activity rather than altered expression levels, as assessed based on immunoblot analysis. The glass transition temperature and crystallinity of P(3HB- co -6HHx) decreased as the 6HHx fraction increased.

Published
2024
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