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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 foods such as oyster mushrooms and red yeast rice. In industrialized production of lovastatin, the natural producer Aspergillus terreus is used. Howere, toxins also occur during the production. Thus, a host with the potential to produce lovastatin more securely is needed. The perfect performance of Aspergillus niger in industrial production of organic acid producer makes it an inviting host for our project. Moreover, it seems to be a wise choice as there is a lot of industry experience about A. niger which has high similarity to A. terreus. In this project, the potential of A. niger to biosynthesis lovastatin are assessed by cloning and transferring the genes from A. terreus to the same species funges A. niger. Also the lovastatin resistance of A. niger is in consideration.

Rationale

Lovastatin biosynthesis needs the participation of polyketide synthases (PKS), enoyl reductase, esterase and cytochrome P450 oxygenase[2]. Among them, LovB (lovastatin nonaketide synthase), LovG (thioesterase) [3]. and LovC (enoyl reductase) together take charge of most of the production pathway by releasing an intermediate-dihydromonacolin L acid from nine malonyl-CoA units [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 were obtained from analysis of Aspergillus terreus and the functionality of some of these domains has been demonstrated by experiments [5]

Aim

The target of this project is to show that Aspergillus niger is an suitable candidate host for lovastatin production. By cloning the enzymes LovB (3038AA), LovG (256AA) and LovC (363AA), which play significant roles in the lovastatin biosynthesis pathway, they can be expressed in Aspergillus niger. Moreover, LovB was split into single domains during cloning, checking the possibilty to reassemble and fuse those domains is a new biosynthetic engineering strategy.

Approach

Domain separation

The minimal polyketide synthase domains of LovB as standalone proteins have never been clearly defined nor have their activities and substrate specicifities been systematically assayed. With the help of literature, Domcut and BLAST to define the boundaries of each domain, we are able to split enzymes into separate domains, while maintaining their individual functionality. By splitting enzymes into separate domains, it is possible to rearrange domains from different sources and to design purposive multidomain enzymes producing novel products.

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 isolated and digested before inserting into a vector of A. niger for expression(Figure 1).

Figure 1: Lov gene expression.

Design for modular fusion

A module with particular nucleic acid sequence in both ends of each domain is shown in Figure 2. 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 vector 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 2: 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.

Research Methods

gBlocks and codon optimized

The nucleic acid chains for most domains are relatively too big to be cloned using gBlocks, which are double-stranded, sequence-verified genomic blocks up to 750bp in length. Thus each domain should still be split into parts, making corresponding DNA fragments in a form of 500bp or 750bp which were produced by IDT (Integrated DNA Technologies). The design of those fragments also included restriction site attached to both ends of the domain and an overlap of 42bp for fragments belong to one domain. Considering that the codon preference of the same amino acid may differ from A. terreus to A.niger, modifications have been made during translation from amino acid sequence to nucleic acid sequence.

DNA assembly

Gibson assembly allows for successful assembly of multiple DNA fragments, so this method was used to fuse two or three DNA fragments together for a complete domain. A combination of 1µL CloneJET (pJET) from Thermo scientific with the concentration of 50ng/µL, 2µL of each gBlocks with the concentration of 40ng/µL, and adequate amount of MiliQ water (depends on the number of gBlocks inserted) to make 10µL in total, was incubated with 10µL 2X Gibson Assembly Master Mix in a final volume of 20µL. The samples were incubated at 50 °C temperature for one hour before transformed into competent cells.

Colony PCR

Competent cells after ligation were transferred onto LB medium agar plate containing ampicillin with the concentration of 0.1% to pick successful ligated samples. 8 single colonies for each assembled domain were chosen for further PCR analysis. 1% agarose gel was used to separate fragments of different size apart.

Preparation of plasmid

The reagents and protocol from a Miniprep Kit of Thermo was used to isolate plasmid. The corresponding culture medium of selected colonies would be used for plasmid isolation. After it, the concentration of plasmid was measured by Nano drop.

Digestion

Restriction enzymes from NEB (New England Biolabs) were used for digesting fragments. The digested fragments would be inserted into a vector for gene expression in fungi. According to the designed structure of gBlocks which would be mentioned later, NotI and NsiI were used for inserting single domain into vector, while AgeI and BspEI were used to form scar area while more than one domain would be fused together. The protocol was obtained from double digestion manual of NEB.

Purification of DNA fragment

1% agarose gel was used for DNA extraction. After DNA fragments with different size were separated in the gel, the band suits the expected size was cut out. The reagents and protocol from Gel extraction Kit of Thermo were used to purify the fragment, then the concentration of fragment was measured by Nano drop.

Results

The results of assembly could be observed by gel of colony PCR shown in Figure 3. Correct assembly of Gblocks of each domain could be obtained. According to the expected size of fragment and the density of culture medium, two samples for each domain were chosen for further plasmid preparation. The plasmid with higher concentration was used for following digestion.

Figure 3: Colony PCR results for the domains.

To check the reality of produce lovastain by A. niger, lovastatin resistance of A. niger was tested. The fungi was incubated on plates with lovastain of different concertrations.

Figure 4: Lovastatin plate.

Future perspective

The production medium of lovastatin in Aspergillus niger was designed with xylose as the carbon source. After LovB gene is inserted into A. niger, the expression level could be detected. Additionally, the combination of LovB, LovC, LovG and even other necessary lov enzymes together is feasible and result in a super lovastatin productive enzyme.

References

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.