CHAPTER 6 METABOLIC PATHWAY RECONSTRUCTION

Transcription

1 66 CHAPTER 6 METABOLIC PATHWAY RECONSTRUCTION 6.1 GENERAL A metabolic pathway is a series of biochemical reactions that occur within a cell and these chemical reactions are catalyzed by certain enzymes. The large number of unknown enzymes in A. terreus leads to pathway gaps in metabolic pathway construction and incomplete knowledge at molecular level. These unknown enzymes have to be reannotated with serious effort. To improve the productivity of lovastatin, the reactants in the biosynthesis pathway have to be altered so that production of lovastatin is augmented and co-metabolites that increase downstream processing cost are reduced. Hence a detailed understanding of metabolic pathways in A. terreus is crucial. The combined reannotated data from functional and comparative analysis of closely related species gives more information required for pathway reconstruction. Biological pathway analysis can also be performed in comparison with Aspergillus species of choice. The comparative analysis with model organism, A. niger and other closely related species A. oryzae and A. nidulans helps in identifying key enzymes involved in the metabolic process of A. terreus. Based on enzyme data the reactions are collected and assigned to corresponding pathways. Candidates for missing enzyme in A. terreus are predicted using the comparison with other Aspergillus species and used for filling pathway gaps. The reactions are grouped based on pathway information. From each reaction of a particular pathway reactant-product pairs have been generated and the reactants and products are linked based on their association to construct the pathway.

2 RESULTS The protein sequence with the reannotated functional information along with their reactions and pathways are given in Appendix 6. Previously in A. terreus, only 780 sequences are annotated as enzymes. Using integrated functional reannotation approach 2392 sequences are predicted as enzymes with high confidence, 1359 with medium confidence level, 545 with low level of confidence. Out of 10,406 sequences in A. terreus, based on comparative analysis, 1291 metabolic reactions are transferred from A. oryzae, 2125 from A. niger and 674 from A. nidulans. On combining functional and comparative analysis in A. terreus 2,561 sequences are assigned to metabolic data with high confidence. In which 1,892 protein sequences are linked to pathways and reactions and 669 sequences are assigned to reactions which in turn are used for reconstruction of metabolic pathways (Subazini et al 2011). The total number of proteins assigned to metabolic data is shown in Table 6.1. An accurate understanding of central carbon metabolism, energy metabolism, amino acid metabolism, carbohydrate metabolism, lipid metabolism, cofactor metabolism and secondary metabolism of A. terreus has been accomplished using comparative and functional data. The generated reactions and Pathway information are stored in ASPMP - Aspergillus terreus Metabolic Pathway database ( and the reconstruction of pathways is done dynamically from reaction set, using GraphViz (Zhao 2006) and Perl (

3 Construction of ASPMP Database ASPMP has been designed as a user friendly interface for the reconstructed metabolic pathway data implemented with Perl and Graphviz (Zhao 2006). The reactions in a pathway are used for generation of the pathway dynamically using Graphviz by network expansion method. It has options for searching pathways based on EC number. The user can also generate new pathways. Table 6.1 Proteins associated to metabolic pathways Metabolic pathways Proteins assigned Amino acid 369 Biodegradation of xenobiotics 104 Carbohydrate metabolism 334 Cofactor pathway 28 Complex lipid metabolism 6 Lipid metabolism 120 Energy metabolism 66 Metabolism of Cofactors and Vitamins 136 Metabolism of Complex Carbohydrates 164 Metabolism of Complex Lipids 131 Metabolism of Other Amino Acids 36 Nucleotide metabolism 184 Membrane transport 40 Replication and Repair 11 Secondary metabolism 35 Others 128 Total 1892

4 Querying by EC number It is possible to seek out the accession numbers and the pathways belonging to A. terreus corresponding to the specific EC number queried by the user. A typical output of such queries or instance shows that aldehyde dehydrogenase (NAD+) (EC ) is involved in at least five different pathways; Glycerolipid metabolism, Bile acid biosynthesis, Ascorbate and aldarate metabolism, Butanoate metabolism, Glycolysis or Gluconeogenesis. If the match for a particular EC number is not available then user gets Not available response from the database.the main advantage of querying the database by EC numbers provides users with information on pathways in which a particular enzyme is involved and also the accession number of protein sequences of A. terreus corresponding to the EC number Exploring pathways ASPMP metabolic pathways are generated by connecting the seed reactions that are clustered to a pathway. The pathways are listed on the web page ( and are grouped under 11 major categories. A user can click on the corresponding subcategory to visualize the metabolic pathway, as a GIF image. Figure 6.1 shows the image generated for Glycerol metabolism. The enzymes are shown in the middle of the reactions with violet color; reactants and products are shaded in green and linked with directed lines on either side of enzymes. The direction of the arrow represents the forward flow of metabolic reaction and reversible reactions are indicated with double headed arrows.

5 70 Figure 6.1 Glycerol metabolism generated by network expansion method using Graphviz Enzymes in the middle of the reactions are shaded with violet color; Reactants and products are shaded in green Construction of new subnetworks The database facilitates users to construct new pathways using the reaction and enzymes list. Reactions and their catalyzing enzymes should be separated by TAB space and each reaction-enzyme pair should be given in the new line. The reactions and enzymes can also be directly given in the text area available in the web page or it can be browsed via notepad or any text editor to generate the network. Figure 6.2 shows a simple network generated from the sample set of reaction and enzyme given via text area.

6 71 Figure 6.2 Subnetwork generated by network expansion method for user entered reactions using Graphviz Enzymes in the middle of the reactions are shaded with violet color; Reactants and Products are shaded in green Central carbon metabolism of A. terreus Acetyl CoA, an important intracellular metabolite in central carbon metabolism serves as a precursor for the polyketide biosynthesis. Polyketide synthesis starts with the generation of malonyl CoA from acetyl CoA by acetylcoa carboxylase (ATEG_09702). Wattanachaisaereekul et al (2008) has engineered yeast for increased precursor supply of 6-MSA a polyketide. Thus increasing acetyl CoA flux (amount of acetyl CoA at a given time) helps in increasing the production of lovastatin. The study of central carbon metabolism of A. terreus with respect to acetyl CoA helps in determining the possibilities of redirecting the carbon fluxes, and over expression of enzymes to improve lovastatin biosynthesis. The central carbon metabolism constructed for A. terreus is shown in Figure 6.3.

7 72 Figure 6.3 The central carbon metabolism constructed for A. terreus The catalyzing enzymes are shown in Yellow color, Reactants and Products in green color and Hydrogen ion is shaded in blue color

8 DISCUSSION The availability of whole-genome sequences gives valuable information for generation of metabolic networks using reannotated data, which can be further utilized for in silico flux balance analysis. The quality of annotation affects the metabolic model (Seo and Lewin 2009), which further affects the study of flux. According to Francke et al (2005) a gap or hole is a reaction in a pathway that is not coupled to a gene-product because the appropriate enzymes were present but could not be recovered by the annotation procedure. Thus in this study, metabolic-network reconstructions of fully sequenced A. terreus strains are performed carefully via the relevant information obtained from both integrated functional and comparative genomics approaches, so that the combined analysis improves the reannotation. The comparative analysis with closely related organisms helps in overcoming missing of low similarity enzymes in diverse organisms (Kurtz et al 2004) coding for same enzymatic function. The selection of enzymes encoding function relies on sequence similarity between the proteins in closely related Aspergillus species. That is comparison of A. terreus with Aspergillus species helps in discovering the functions of certain enzymes that are not identified by comparison with diverse species. The addition of reactions for filling holes is done after systematic verification to prevent inconsistencies. The addition of new reactions for filling holes will affect the amount of product from a substrate on simulation of pathway to obtain the maximum theoretical yield. Thus while adding new reactions to the pathway careful investigation is done with the literature study of physiology of an organism. Glucose phosphatase involved in the conversion of Glucose to Glucose-6-phosphate in Embden-Meyerhoff-Parnas pathway. There is a contradiction identified in annotation of ATEG_06116 by comparative and functional annotation of A. terreus. ATEG_06116 (conserved hypothetical protein) is reannotated as Sphingoid base-phosphate phosphatase by

9 74 integrated functional annotation approach. glucose phosphatase is missing in the reannotated data. Osterman and Overbeek (2003) states that the use of metabolic profile of closely related Aspergillus species as references allow identification of common enzymatic role in A. terreus with phylogenetically related organisms. A. niger, A. nidulans, A. oryzae are phylogenetically related organism with A. terreus so the comparative analysis with these organisms are used here to predict the function of ATEG_06116 as glucose phosphatase with good E-Value and identity percentage. Further verification of the function of ATEG_06116 is done by protein interaction approach and assigned the function glucose phosphatase. To maintain the reliability of metabolic pathway reconstruction only enzymes that are predicted with high and medium confidence level through integrated functional annotation approaches are utilized, those with low confidence levels are ignored through manual curation (Radrich et al 2010). ATEG_07882 codes for citrate synthase involved in TCA cycle and is identified using both comparative and functional analysis with high confidence. ATEG_07196, previously annotated as mutase is updated as phosphoglycerate mutase present in Embden-Meyerhoff-Parnas (EMP) Pathway by both integrated functional annotation and comparative analysis. Functional annotation of A. terreus and comparative analysis with A. oryzae identifies gluconokinase of Pentose Phosphate pathway (PPP) which phosphorylates D-gluconate to 6-phospho D-gluconate utilizing ATP. Acetate kinase of TCA cycle is also identified using both functional annotation and comparative analysis. Thus the data generated from the integration of functional annotation and comparative analysis provides a comprehensive set of reactions that can be used for the reconstruction of central carbon metabolism. This reconstructed pathway is further used for metabolic flux analysis.