Enhanced incorporation of 3-hydroxy-4-methylvalerate unit into biosynthetic polyhydroxyalkanoate using leucine as a precursor.
Enhanced incorporation of 3-hydroxy-4-methylvalerate unit into biosynthetic polyhydroxyalkanoate using leucine as a precursor.
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DOI：
10.1186/2191-0855-1-6
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发表时间：
2011-05-18
期刊：
影响因子：
3.7
通讯作者：
Tsuge T
中科院分区：
文献类型：
--
作者：
Saika A;Watanabe Y;Sudesh K;Abe H;Tsuge T
表达假单胞菌61-3聚羟基链烷酸酯（PHA）合酶1（PhaC 1 Ps）的真养罗尔斯通氏菌（Ralstonia eutropha）PHB-4从作为碳源的果糖合成含有3-羟基丁酸酯（3 HB）和少量（0.5摩尔%）3-羟基-4-甲基戊酸酯（3 H4 MV）的PHA共聚物。在这项研究中，使用支链氨基酸亮氨酸作为3 H4 MV的前体，研究了3 H4 MV与PHA的增强掺入。亮氨酸具有与3 H4 MV相同的碳骨架，并且预期是天然的和可自我产生的前体。我们发现，在培养基中补充过量的亮氨酸（10 g/L）可增强3 H4 MV的掺入。这一发现表明3 H4 MV可以来源于亮氨酸。为了通过消除反馈抑制来增加宿主菌株中亮氨酸生物合成的代谢通量，对细胞进行N-甲基-N '-硝基-N-亚硝基胍（NTG）诱变并产生亮氨酸类似物抗性突变体。突变株的3 H4 MV分数在统计学上高于未补充亮氨酸的亲本菌株。另外，通过提供过量的亮氨酸，突变体合成了含有3.1摩尔%的3 H4 MV和1.2摩尔%的3-羟基戊酸酯（3 HV）作为次要成分的基于3 HB的PHA共聚物，这显著影响了共聚物的热性质。本研究表明，它是可能的，以提高单体供应的3 H4 MV到PHA通过操纵亮氨酸代谢。
Ralstonia eutropha PHB-4 expressing Pseudomonas sp. 61-3 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Ps) synthesizes PHA copolymer containing 3-hydroxybutyrate (3HB) and a small amount (0.5 mol%) of 3-hydroxy-4-methylvalerate (3H4MV) from fructose as a carbon source. In this study, enhanced incorporation of 3H4MV into PHA was investigated using branched amino acid leucine as a precursor of 3H4MV. Leucine has the same carbon backbone as 3H4MV and is expected to be a natural and self-producible precursor. We found that the incorporation of 3H4MV was enhanced by the supplementation of excess amount (10 g/L) of leucine in the culture medium. This finding indicates that 3H4MV can be derived from leucine. To increase metabolic flux to leucine biosynthesis in the host strain by eliminating the feedback inhibition, the cells were subjected to N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis and leucine analog resistant mutants were generated. The mutants showed statistically higher 3H4MV fraction than the parent strain without supplementing leucine. Additionally, by supplying excess amount of leucine, the mutants synthesized 3HB-based PHA copolymer containing 3.1 mol% 3H4MV and 1.2 mol% 3-hydroxyvalerate (3HV) as minor constituents, which significantly affected the thermal properties of the copolymer. This study demonstrates that it is possible to enhance the monomer supply of 3H4MV into PHA by manipulating leucine metabolism.
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影响因子：
5
作者：
Kato, M;Bao, HJ;Doi, Y
通讯作者：
Doi, Y
DOI：
10.1016/0922-338x(96)82217-3
发表时间：
1996-01-01
期刊：
JOURNAL OF FERMENTATION AND BIOENGINEERING
影响因子：
--
作者：
Lee, IY;Kim, GJ;Park, YH
通讯作者：
Park, YH
影响因子：
5.5
作者：
SHIMAMURA, E;KASUYA, K;DOI, Y
通讯作者：
DOI, Y
影响因子：
6.2
作者：
Matsusaki, H;Abe, H;Doi, Y
通讯作者：
Doi, Y
影响因子：
2.7
作者：
Choi, GG;Kim, MW;Rhee, YH
通讯作者：
Rhee, YH