Acetylacetone is widely used as fuel additive and dyeing intermediate. It is also applied in the fields of metal extraction, metal plating, and resin modification. Traditionally, acetylacetone is manufactured through chemical routes using acetone and ketene.
However, the chemical routes suffer from drawbacks such as multiple steps, harsh conditions, low yield, and environmental problems, which hamper further applications of petrochemical-based acetylacetone.
"Biosynthetic methods have the advantages of eco-friendliness, mild conditions and high selectivity. So, we expect to get acetylacetone through biological methods," said Dr. FENG Xinjun from the Qingdao Institute of Bioenergy and Bioprocess Technology (QIBEBT) of the Chinese Academy of Sciences.
Inspired by the biodegradation pathway, the QIBEBT researchers established a biosynthetic pathway of acetylacetone from fermentable sugars. And the acetylacetone was produced by engineered Escherichia coli by key enzyme screening and cultivation condition regulation. The acetylacetone is produced biologically for the first time.
To improve the production, the researchers increased the acetylacetone-cleaving enzyme (Dke1) activity based on sequence alignment and structure analysis. A double mutant was obtained with high catalytic activity.
The double-mutant strain presented the highest acetylacetone-producing capacity, 3.6-fold higher than that of the wild-type protein. And the production was achieved at 556 mg/L in fed-batch fermentation.
Fig. 1 Biodegradation-inspired biosynthetic pathway of acetylacetone. (Image by FENG Xinjun)
Meanwhile, the molecular basis for increased enzymatic activity was also revealed by structural simulation and molecular docking. "From the 3D structure, we can see that the channel, through which substrates enter the enzyme reactive center, was widened," Dr. FENG Xinjun said. "That's why the enzyme activity was enhanced."
Fig. 2 Analysis of the Dke1 structure with molecular docking. (a) Wild-type, (b) K15Q/A60D mutant. (Image by FENG Xinjun)
Dke1 is not absolutely specific for acetylacetone. Many related β-dicarbonyl compounds such as 3,5-heptanedione, 2,4-octanedione, and ethylacetoacetate can be accepted as substrate by Dke1. Thus, the results of this study also provide a possible biosynthesis method for other β-dicarbonyl compounds.
The study was published in Biotechnology for Biofuels on May 15. It was supported by Youth Innovation Promotion Association CAS, and the National Natural Science Foundation of China.
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