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Introduction

As an inhibitor of the enzyme (3-hydroxy-3-methylglutaryl CoA reductase), which plays a significant role in cholesterol biosynthesis, lovastatin is a medicinal compound used against cardiovascular diseases (Tobert, 2003). It is a naturally occurring drug which is found in food such as oyster mushrooms and red yeast rice. During the production of lovastatin in Aspergillus terreus, toxins could be produced at the same time. However, Aspergillus niger is a mass producer of organic acids with the potential to produce lovastatin more securely. By this project, a more suitable host for lovastatin biosynthesis is likely to be found by cloning and transferring the gene from Aspergillus terreus to the same species fungi Aspergillus niger.

Strategy and Approach

Biosynthesis gene

Lovastatin biosynthesis approach needs the participation of polyketide synthases (PKS), enoyl reductase, esterase and cytochrome P450 oxygenase (Campbell & Vederas, 2010). Among them, LovB (3038AA), LovG (256AA) (Xu et al., 2013)and LovC (363AA) together take charge of most of the production pathway by releasing an intermediate-dihydromonacolin L acid (Xu et al., 2013) after 35 reactions.

LovB contains KS (β-ketosynthase), MAT (acyl transferase), DH (dehydratase), MT (methyl transferase), KR (ketoreductase), ACP (acyl carrier protein), CON (nonribosomal-peptide synthase condensation) domains and an inactive ER domain, which is active in LovC(Ames et al., 2012; Campbell & Vederas, 2010). The amino acid sequences of LovB and LovC are obtained from analysis of Aspergillus terreus and the functionality of some domains has been demonstrated by experiments (Ma & Tang, 2007).

Domain separation

The minimal polyketide synthase domains of LovB as standalone proteins and assay for their activities and substrate specificities have never been clearly defined or systematically accomplished. With the help of literature,Domcut and BLAST to define the boundaries of each domain,it is able to split enzyme when the structure of separate domains remain functionally. By splitting enzyme into separate domains and expressing them in a vector, it can be possible to rearrange domains from different sources and design an enzyme for the function of particular purposes.

gBlocks and Gibson assembly

gBlocks gene fragments, a new tool for synthetic biology were ordered for cloning each domain with the consideration of codon optimization for Aspergillus niger. After the gene was assembled by Gibson assembly in E.coli,it would be isloated and digested before inserting into a vector of A. niger for expression.

Reference

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