Enhanced Terpenoid Biosynthesis Pathway based on Spycatcher-Spytag Scaffold Protein
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Terpenoid belongs to the most diverse family of natural product. It contains more than 25,000 structures and can be found in all classes of living thing ,like plants, insects, microorganisms, etc. Tepenoid metabolics have many different application, such as drug, nutraceutical and jet fuel. The microbial terpenoid biosynthesis involves a complicated pathway that contain many different enzymes and intermediates. Simply putting these enzyme together in bacteria will only result in low production level of terpenoid. Based on the previous work done by our
labmate, in the simple menaquinol synthesis model system, enzyme assemblies through scaffold proteins showed higher enzyme activities than the simple enzyme solution mixture. Due to the shorter distance of the
intermediate transportation, the enzyme complex produce much more final product than the wild type enzyme mixtures. We want to apply this strategy in the terpenoid synthesis pathway. Fusing the enzyme with the scaffold protein to create complex enzyme assemblies in order to enhance the terpenoid overproduction. For the scaffold protein, spy and snoop system will be used. In the spy system, the interesting adhesion protein from bacteria contain a isopeptide bond between lysine and aspartic acid residue in its natural state. By splitting and engineering the protein into two parts, the larger part catcher and the smaller part tag can bind and form covalent isopeptide bond
when mixed in solution. Snoop system is very similar to the spy one. The larger part of the spy system contains a reactive lysine while the smaller part of snoop system contains the reactive lysine, so these two system would not have cross reaction and are orthogonal. The plasmids containing the genes of terpenoid biosynthetic pathways were constructed by Gibson assembly. The resultant plasmids were co-transformed into E.coli to permit
the production of terpenoid. The desired terpenoid were extracted from E.coli and analyzed either by HPLC or spectrophotometer.

Enzyme fused with spy and snoop scaffold protein can form large enzyme assemblies. (A) Menaquinol biosynthesis enzymes fused with spy and snoop system respectively. Fused enzyme formed complex enzyme assemblies when mixed in solution and show no cross reaction between these two system. (B) Spycatcher and Spytag are able to react efficiently in the cytosolic environment

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著者Jiale QU,Wei KANG,Jiang XIA
會議名稱The 13th International Symposium on the Genetics of Industrial Microorganisms

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