Enzymatic process optimization for the in vitro production of isoprene from mevalonate.
Enzymatic process optimization for the in vitro production of isoprene from mevalonate.
复制标题
从甲羟戊酸体外生产异戊二烯的酶法优化
DOI：
10.1186/s12934-016-0622-4
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发表时间：
2017-01-09
影响因子：
6.4
通讯作者：
Xian M
中科院分区：
文献类型：
--
作者：
Cheng T;Liu H;Zou H;Chen N;Shi M;Xie C;Zhao G;Xian M
背景异戊二烯作为合成橡胶的重要主体化学品，可由微生物生物合成。但由于细胞代谢的复杂性，为了使体内过程可行，往往需要合理的工程和优化。在体外合成异戊二烯或类异戊二烯的生物化学方法发展很快。结果本研究建立了一种体外酶化学合成方法，利用甲氧戊酸低端途径中的5种酶来合成异戊二烯。我们发现，单个酶的水平和比例会显著影响整个系统的效率。优化后的工艺采用10种平衡酶单位(MVK、PMK、MVD5.0µM，IDI 10.0µM，ISPS 80.0µM)，在2 mg/L的体外体系中，可产生6323.5µm ol/L/h(430 mg/L/h)的异戊二烯。在50 mg/L的放大过程中，仅用1个平衡的酶单位(MVK、PMK、MVD为0.5µM，IDI为1.0µM，ISPS为8.0µM)，系统可在40 h内生产302 mg/L异戊二烯，与体内对照相比，具有较高的产率和较长的反应时间。结论通过优化较低MVA途径的酶水平，可以建立合成生化方法用于甲戊酸酶促合成异戊二烯或类异戊二烯。
BackgroundAs an important bulk chemical for synthetic rubber, isoprene can be biosynthesized by robust microbes. But rational engineering and optimization are often demanded to make the in vivo process feasible due to the complexities of cellular metabolism. Alternative synthetic biochemistry strategies are in fast development to produce isoprene or isoprenoids in vitro.ResultsThis study set up an in vitro enzyme synthetic chemistry process using 5 enzymes in the lower mevalonate pathway to produce isoprene from mevalonate. We found the level and ratio of individual enzymes would significantly affect the efficiency of the whole system. The optimized process using 10 balanced enzyme unites (5.0 µM of MVK, PMK, MVD; 10.0 µM of IDI, 80.0 µM of ISPS) could produce 6323.5 µmol/L/h (430 mg/L/h) isoprene in a 2 ml in vitro system. In a scale up process (50 ml) only using 1 balanced enzyme unit (0.5 µM of MVK, PMK, MVD; 1.0 µM of IDI, 8.0 µM of ISPS), the system could produce 302 mg/L isoprene in 40 h, which showed higher production rate and longer reaction phase with comparison of the in vivo control.ConclusionsBy optimizing the enzyme levels of lower MVA pathway, synthetic biochemistry methods could be set up for the enzymatic production of isoprene or isoprenoids from mevalonate.