Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, N13-W8, Kita-ku, Sapporo 060-8628, Japan.
Biomacromolecules. 2010 Mar 8;11(3):815-9. doi: 10.1021/bm901437z.
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.
Lactate (LA)-polymerizing enzyme (LPE) 是一种新建立的聚羟基烷酸酯 (PHA) 合酶类别，能够将 LA 单元纳入聚合物链中。我们之前使用来自 Pseudomonas sp. 61-3 的 PHA 合酶的 Ser325Thr/Glu481Lys 突变体（PhaC1(Ps)ST/QK）作为第一个 LPE，在重组大肠杆菌中合成了 P(LA-co-3-hydroxybutyrate)s [P(LA-co-3HB)s]。在这项研究中，我们通过饱和突变位置 392（F392X）精细调节共聚物中的 LA 分数，该位置对应于 Ralstonia eutropha 的 PHA 合酶位置 420 的增强活性突变。在 LPE 位置 392 的 19 个饱和突变体中，有 17 个突变体产生了具有各种 LA 分数（16-45 mol%）的 P(LA-co-3HB)s，而 PhaC1(Ps)ST/QK 在相同的培养条件下仅产生 26 mol%的 LA。特别是，F392S 突变体具有最高的 45 mol%的 LA 分数，并且与 PhaC1(Ps)ST/QK（44 wt%）相比，聚合物含量也增加了（62 wt%）。F392S 突变体与促进 LA 生产的厌氧培养条件的结合，使 LA 分数进一步增加到 62 mol%。具有各种 LA 分数的 P(LA-co-3HB)s 由于其单体组成而表现出不同的熔融温度和熔融焓。因此，LPE 位置 392 的突变极大地有助于调节共聚物中 LA 分数的微调，这对于调节 LA 分数依赖性的热性能很有用。
