Adjustable mutations in lactate (LA)-polymerizing enzyme for the microbial production of LA-based polyesters with tailor-made monomer composition.
Lactate (LA)-polymerizing enzyme (LPE) is a newly established class of polyhydroxyalkanoate (PHA) synthase, which can incorporate LA units into a polymer chain. We previously synthesized P(LA-co-3-hydroxybutyrate)s [P(LA-co-3HB)s] in recombinant Escherichia coli using the first LPE, which is the Ser325Thr/Glu481Lys mutant of PHA synthase from Pseudomonas sp. 61-3 [PhaC1(Ps)ST/QK]. In this study, we finely regulated LA fraction in the copolymer by saturated mutations at position 392 (F392X), which corresponds to the activity-enhancing mutations at position 420 of PHA synthase from Ralstonia eutropha. Among the 19 saturated mutants of LPE at position 392, 17 mutants produced P(LA-co-3HB)s with various LA fractions (16-45 mol %), whereas PhaC1(Ps)ST/QK produced P(LA-co-3HB) with 26 mol % LA under the same culture condition. In particular, the F392S mutation exhibited the highest LA fraction of 45 mol %, and also increased polymer content (62 wt %) compared with PhaC1(Ps)ST/QK (44 wt %). Combination of the F392S mutant and anaerobic culture conditions, which promote LA production, led to a further increase in LA fraction up to 62 mol %. The P(LA-co-3HB)s with various LA fractions exhibited altered melting temperatures and melting enthalpy depending on their monomer composition. Accordingly, the mutations at position 392 in LPE greatly contributed to fine-tuning of the LA fraction in the copolymers that is useful for regulating LA fraction-dependent thermal properties.
Yamada
Miwa
M
Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, N13-W8, Kita-ku, Sapporo 060-8628, Japan.
Matsumoto
Ken'ichiro
K
Shimizu
Kotaro
K
Uramoto
Shu
S
Nakai
Takanori
T
Shozui
Fumi
F
Taguchi
Seiichi
S
D000693:Anaerobiosis; D001483:Base Sequence; D017931:DNA Primers; D004798:Enzymes / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D019344:Lactic Acid / Q000378:metabolism*; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011091:Polyesters / Q000378:metabolism*
D017931:DNA Primers; D004798:Enzymes; D011091:Polyesters; D019344:Lactic Acid