High-rate 3-methylcatechol production in Pseudomonas putida strains by means of a novel expression system
High-rate 3-methylcatechol production in Pseudomonas putida strains by means of a novel expression system
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DOI：
10.1007/s002530000566
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发表时间：
2001-05-01
影响因子：
5
通讯作者：
Wery, J
中科院分区：
文献类型：
--
作者：
Hüsken, LE;Beeftink, R;Wery, J
将甲苯生物转化为3-甲基儿茶酚作为生产有价值的3-取代邻苯二酚的模型体系。为此，获得了一种生产3-甲基儿茶酚的改良微生物体系。恶臭假单胞菌含有参与甲苯转化为3-甲基儿茶酚的rodC1C2BAD基因，通过一种新的整合表达系统将这些基因的额外拷贝导入恶臭假单胞菌。构建了一个含有todC1C2BAD基因的表达盒，该表达盒位于萘和菲降解可诱导调控区NAGR的控制下。将此结构引入野生型恶臭假单胞菌F1(通过3-甲基儿茶酚降解甲苯)或突变恶臭假单胞菌F107(积累3-甲基儿茶酚)，产生携带多个表达框拷贝的生物催化剂。结果表明，3-甲基儿茶酚的累积最高可达14 mm(1.74g L(-1))，比产率达到105mmolmin(-1)g(-1)细胞干重的水平，是其他邻苯二酚生产系统的4倍。结果表明，这些性质在生物催化剂中保持稳定，在生产过程中不需要抗生素。这是获得设计型生物催化剂的重要一步。
The bioconversion of toluene into 3-methylcatechol was studied as a model system for the production of valuable 3-substituted catechols in general. For this purpose, an improved microbial system for the production of 3-methylcatechol was obtained. Pseudomonas putida strains containing the rodC1C2BAD genes involved in the conversion of toluene into 3-methylcatechol were used as hosts for introducing extra copies of these genes by means of a novel integrative expression system. A construct was made containing an expression cassette with the todC1C2BAD genes cloned under the control of the inducible regulatory control region for naphthalene and phenanthrene degradation, nagR. Introducing this construct into wild-type P. putida Fl, which degrades toluene via 3-methylcatechol, or into mutant P. putida F107, which accumulates 3-methylcatechol, yielded biocatalysts carrying multiple copies of the expression cassette. As a result, up to 14 mM (1.74 g l(-1)) of 3-methylcatechol was accumulated and the specific production rate reached a level of 105 mu mol min(-1) g(-1) cell dry weight, which is four times higher than other catechol production systems. It was shown that these properties were kept stable in the biocatalysts without the need for antibiotics in the production process. This is an important step for obtaining designer biocatalysts.