Studies of the production of fungal polyketides in Aspergillus nidulans by using systems biology tools

Abstract

Many filamentous fungi produce polyketide molecules with great significance as human pharmaceuticals; these molecules include the cholesterol-lowering compound lovastatin, which was originally isolated from Aspergillus terreus. The chemical diversity and potential uses of these compounds are virtually unlimited, and it is thus of great interest to develop a well-described microbial production platform for polyketides. Using genetic engineering tools available for the model organism Aspergillus nidulans, we constructed two recombinant strains, one expressing the Penicillium griseofulvum 6-methylsalicylic acid (6-MSA) synthase gene and one expressing the 6-MSA synthase gene and overexpressing the native xylulose-5-phosphate phosphoketolase gene (xpkA) for increasing the pool of polyketide precursor levels. The physiology of the recombinant strains and that of a reference wild-type strain were characterized on glucose, xylose, glycerol, and ethanol media in controlled bioreactors. Glucose was found to be the preferred carbon source for 6-MSA production, and 6-MSA concentrations up to 455 mg/liter were obtained for the recombinant strain harboring the 6-MSA gene. Our findings indicate that overexpression of xpkA does not directly improve 6-MSA production on glucose, but it is possible, if the metabolic flux through the lower part of glycolysis is reduced, to obtain quite high yields for conversion of sugar to 6-MSA. Systems biology tools were employed for in-depth analysis of the metabolic processes. Transcriptome analysis of 6-MSA-producing strains grown on glucose and xylose in the presence and absence of xpkA overexpression, combined with flux and physiology data, enabled us to propose an xpkA-msaS interaction model describing the competition between biomass formation and 6-MSA production for the available acetyl coenzyme A.

FIG. 1.

(A) Principles of metabolic network analysis with [1-¹³C]glucose. The ¹³C labeling pattern of pyruvate is dependent on the active metabolic pathways. For instance, pyruvate formed in the Embden-Meyerhof-Parnas (EMP) pathway contains ¹³C at position 3, while activity of the PP pathway results in loss of labeled carbon. Pyruvate is the precursor of valine, which is incorporated into biomass. Based on the labeling pattern of valine and other proteinogenic amino acids, the fluxes in central carbon metabolism can be resolved. (B) Major metabolic fluxes of the A. nidulans strains investigated, including strains A4 (wt), AR16msaGP74 (msaS), and AR1phk6msaGP74 (msaS+xpkA). All fluxes are relative to a glucose uptake rate of 100 mol (arbitrary value). The dashed arrows indicate that the metabolite is a precursor drained for biomass formation. See File S2 in the supplemental material for abbreviations used for metabolites. TCA, tricarboxylic acid; PPP, pentose phosphate pathway.

FIG. 2.

Summary of the significant effects of PHK overexpression (PHK) and 6-MSAS insertion (MSA) in A. nidulans on gene transcription levels. Each Venn diagram summarizes the transcriptome analysis performed with glucose or xylose medium. In each of the three circles are the numbers of genes affected by either PHK overexpression, 6-MSAS insertion, or the additive effect. The additive effect can be a synergetic effect (either positive or negative) of PHK overexpression and 6-MSAS. In each subdivision of the Venn diagrams the total number of significant genes is indicated (larger number), and these genes are divided into minor groups (smaller numbers); the boxes indicate the directions of significant responses to the PHK overexpression, 6-MSAS insertion, or additive effects. Superscripts indicate the numbers of genes responding in the same patterns on both carbon sources. For instance, the sixth box of the central subdivision of the glucose Venn diagram shows that there are three genes that are induced by both the PHK overexpression and 6-MSAS insertion effects, but these effects are downregulated (lessened) by the additive effect. Furthermore, these three genes are the same for both carbon sources.

FIG. 3.

Detail from an overview of the additive effects of 6-MSAS expression and PHK overexpression. ▾, additive effect causes lower-level expression of one or more isoenzymes of the process; ▴, higher-level expression; , the level of expression of some genes with the function is higher while that of other genes is lower; , a gene could not be assigned to the function. Adapted from a map of A. niger metabolism published by Andersen et al. (3).

FIG. 4.

Model of the effects of PHK overexpression and 6-MSAS insertion in A. nidulans. A tilted oval indicates a hypothetical maximum flux above which a large growth-inhibiting and flux-decreasing response is induced.