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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 [1]. 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[2]. Among them, LovB (3038AA), LovG (256AA) [3]. and LovC (363AA) together take charge of most of the production pathway by releasing an intermediate-dihydromonacolin L acid [3] 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 [2][4]. 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 [5]

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.

Design for modular fusion

A module with particular nucleic acid sequence in both ends of each domain is shown in Figure 1. A structure of AgeI site–NsiI site-Inserted gene–BspEI site–TAATAG–NotI site was formed to meet the criteria: a). make the fusion of single or multiple domains feasible b). stop codon only exists when the domain is the last or single one c). compatible with Funbrick system. When a single domain was inserted into a vector, NotI and NsiI were used to digest the fragment for sticky ends (the domain remains from shadow area of B to shadow area of E) and the vector to make a gap, before fusing them together. When more than one domains were planned to be inserted, the first one should be digested by NsiI and BspEI (from B to C) and the last one should be digested by AgeI and NotI (from A to E). The restriction enzymes AgeI and BspEI were used in pair when fusing domains together. A scar area of (a combination of unshadow area of A and C) was formed, which cannot be digested by AgeI or BspEI anymore. During expression, the nucleotides of the connection part (scar+ site B) would be translated into amino acid sequence of Ser-Gly-Met-His, whose structure is relatively simple thus won’t change the structure of original domain severely as an additional part.

Figure 1: Module of gBlocks. Shadow area of sequence A was restriction site for AgeI while B for NsiI, C for BspEI and E for NotI. Sequence D consists of two stop codons which ensure the inserted domain be expressed precisely.

Reference

1.Tobert, J. A. (2003). Lovastatin and beyond: The history of the HMG-CoA reductase inhibitors. Nature Reviews Drug Discovery, 2(7), 517-526.
2.Campbell, C. D., & Vederas, J. C. (2010). Biosynthesis of Lovastatin and Related Metabolites Formed by Fungal Iterative PKS Enzymes. Biopolymers, 93(9), 755-763.
3.Xu, W., Chooi, Y. H., Choi, J. W., Li, S., Vederas, J. C., Da Silva, N. A., & Tang, Y. (2013). LovG: The Thioesterase Required for DihydromonacolinL Release and Lovastatin Nonaketide Synthase Turnover in Lovastatin Biosynthesis. Angewandte Chemie-International Edition, 52(25), 6472-6475.
4.Ames, B. D., Nguyen, C., Bruegger, J., Smith, P., Xu, W., Ma, S., . . . Tsai, S. C. (2012). Crystal structure and biochemical studies of the trans-acting polyketide enoyl reductase LovC from lovastatin biosynthesis. Proceedings of the National Academy of Sciences of the United States of America, 109(28), 11144-11149.
5.Ma, S. M., & Tang, Y. (2007). Biochemical characterization of the minimal polyketide synthase domains in the lovastatin nonaketide synthase LovB. Febs Journal, 274(11), 2854-2864.