Biosynthesis of polyhydroxyalkanoate copolymers from methanol by Methylobacterium extorquens AM1 and the engineered strains under cobalt-deficient conditions
Biosynthesis of polyhydroxyalkanoate copolymers from methanol by Methylobacterium extorquens AM1 and the engineered strains under cobalt-deficient conditions
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DOI：
10.1007/s00253-013-5490-9
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发表时间：
2014-04-01
影响因子：
5
通讯作者：
Fukui, Toshiaki
中科院分区：
文献类型：
--
作者：
Orita, Izumi;Nishikawa, Kouta;Fukui, Toshiaki
扭脱甲基杆菌AM 1已被证明在甲基营养生长过程中积累仅由（R）-3-羟基丁酸酯（3 HB）组成的聚羟基链烷酸酯（PHA）。本研究表明，野生型菌株AM 1生长在Co2+缺乏的条件下积累的共聚酯的3 HB和C-5-单体，（R）-3-羟基戊酸酯（3 HV），使用甲醇作为唯一的碳源。3 HV单位应该来源于丙酰辅酶A，通过Co2+限制受损的乙基丙二酰辅酶A途径合成。当缺乏丙酰辅酶A羧化酶的菌株与甲醇一起孵育时，3 HV单位向PHA中的显性掺入强烈支持了这一假设。进一步的基因工程改造M.使用extortquens AM 1进行PHA共聚物的甲基营养合成。重组菌株M.其中原始PHA合酶基因phaC（Me）已被phaC（Ac）取代的扭脱昆菌AM 1C（Ac），编码来自嗜水气单胞菌的具有广泛底物特异性的酶，从甲醇产生由3 HB、3 HV和C-6-单体（R）-3-羟基己酸酯组成的PHA酶。菌株AM 1C（Ac）中积累的PHA的细胞含量和分子量均高于野生型菌株。M.扭脱喹菌AM 1C（Ac）对生长和PHA合成特性无显著影响。过表达编码β-酮硫解酶和NADPH-乙酰乙酰辅酶A还原酶的基因增加了细胞PHA含量和PHA中的3 HV组成，尽管细胞在甲醇上的生长下降。这项研究开辟了生产实用的PHA共聚物的可能性与甲基营养型细菌使用甲醇作为原料。
Methylobacterium extorquens AM1 has been shown to accumulate polyhydroxyalkanoate (PHA) composed solely of (R)-3-hydroxybutyrate (3HB) during methylotrophic growth. The present study demonstrated that the wild-type strain AM1 grown under Co2+-deficient conditions accumulated copolyesters of 3HB and a C-5-monomer, (R)-3-hydroxyvalerate (3HV), using methanol as the sole carbon source. The 3HV unit was supposed to be derived from propionyl-CoA, synthesized via the ethylmalonyl-CoA pathway impaired by Co2+ limitation. This assumption was strongly supported by the dominant incorporation of the 3HV unit into PHA when a strain lacking propionyl-CoA carboxylase was incubated with methanol. Further genetic engineering of M. extorquens AM1 was employed for the methylotrophic synthesis of PHA copolymers. A recombinant strain of M. extorquens AM1C(Ac) in which the original PHA synthase gene phaC (Me) had been replaced by phaC (Ac) , encoding an enzyme with broad substrate specificity from Aeromonas caviae, produced a PHA terpolymer composed of 3HB, 3HV, and a C-6-monomer, (R)-3-hydroxyhexanoate, from methanol. The cellular content and molecular weight of the PHA accumulated in the strain AM1C(Ac) were higher than those of PHA in the wild-type strain. The triple deletion of three PHA depolymerase genes in M. extorquens AM1C(Ac) showed no significant effects on growth and PHA biosynthesis properties. Overexpression of the genes encoding beta-ketothiolase and NADPH-acetoacetyl-CoA reductase increased the cellular PHA content and 3HV composition in PHA, although the cell growth on methanol was decreased. This study opens up the possibility of producing practical PHA copolymers with methylotrophic bacteria using methanol as a feedstock.