Heterologous expression and characterization of bacterial 2-C-methyl-D-erythritol-4-phosphate pathway in Saccharomyces cerevisiae.

Transfer of a biosynthetic pathway between evolutionary distant organisms can create a metabolic shunt capable of bypassing the native regulation of the host organism, hereby improving the production of secondary metabolite precursor molecules for important natural products. Here, we report the engineering of Escherichia coli genes encoding the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway into the genome of Saccharomyces cerevisiae and the characterization of intermediate metabolites synthesized by the MEP pathway in yeast. Our UPLC-MS analysis of the MEP pathway metabolites from engineered yeast showed that the pathway is active until the synthesis of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate, but appears to lack functionality of the last two steps of the MEP pathway, catalyzed by the [4Fe-4S] iron sulfur cluster proteins encoded by ispG and ispH. In order to functionalize the last two steps of the MEP pathway, we co-expressed the genes for the E. coli iron sulfur cluster (ISC) assembly machinery. By deleting ERG13, thereby incapacitating the mevalonate pathway, in conjunction with labeling experiments with U-¹³C6 glucose and growth experiments, we found that the ISC assembly machinery was unable to functionalize ispG and ispH. However, we have found that leuC and leuD, encoding the heterodimeric iron-sulfur cluster protein, isopropylmalate isomerase, can complement the S. cerevisiae leu1 auxotrophy. To our knowledge, this is the first time a bacterial iron-sulfur cluster protein has been functionally expressed in the cytosol of S. cerevisiae under aerobic conditions and shows that S. cerevisiae has the capability to functionally express at least some bacterial iron-sulfur cluster proteins in its cytosol.

Gene deletion of cytosolic ATP: citrate lyase leads to altered organic acid production in Aspergillus niger.

With the availability of the genome sequence of the filamentous fungus Aspergillus niger, the use of targeted genetic modifications has become feasible. This, together with the fact that A. niger is well established industrially, makes this fungus an attractive micro-organism for creating a cell factory platform for production of chemicals. Using molecular biology techniques, this study focused on metabolic engineering of A. niger to manipulate its organic acid production in the direction of succinic acid. The gene target for complete gene deletion was cytosolic ATP: citrate lyase (acl), which had previously been identified by using genome-scale stoichiometric metabolic model simulations. The acl gene was deleted using the bipartite gene-targeting method, and the mutant was characterized in batch cultivation. It was found that the succinic acid yield was increased threefold by deleting the acl gene. Additionally, the total amount of organic acids produced in the deletion strain was significantly increased. Genome-scale stoichiometric metabolic model predictions can be used for identifying gene targets. Deletion of the acl led to increased succinic acid production by A. niger.

Production of the polyketide 6-MSA in yeast engineered for increased malonyl-CoA supply.

The heterologous production of fungal polyketides was investigated using 6-methylsalicylic acid synthase (6-MSAS) as a model polyketide synthase and Saccharomyces cerevisiae as a host. In order to improve the production of 6-MSA by enhancing the supply of precursors, the promoter of the gene (ACC1) encoding acetyl-CoA carboxylase, which catalyzes the conversion of acetyl-CoA to malonyl-CoA, was replaced with a strong, constitutive promoter (TEF1p) in a strain harboring two plasmids carrying the genes encoding 6-MSAS from Penicillium patulum and PPTase from Aspergillus nidulans, respectively. The strain was characterized in batch cultivations with a glucose minimal media (20 g/L), and a 60% increase in 6-MSA titer was observed compared to a strain having the native promoter in front of ACC1. The production of 6-MSA was scaled up by the cultivation in minimal media containing 50 g/L of glucose, and hereby a final titer of 554+/-26 mg/L of 6-MSA was obtained.

Metabolic model integration of the bibliome, genome, metabolome and reactome of Aspergillus niger.

The release of the genome sequences of two strains of Aspergillus niger has allowed systems-level investigations of this important microbial cell factory. To this end, tools for doing data integration of multi-ome data are necessary, and especially interesting in the context of metabolism. On the basis of an A. niger bibliome survey, we present the largest model reconstruction of a metabolic network reported for a fungal species. The reconstructed gapless metabolic network is based on the reportings of 371 articles and comprises 1190 biochemically unique reactions and 871 ORFs. Inclusion of isoenzymes increases the total number of reactions to 2240. A graphical map of the metabolic network is presented. All levels of the reconstruction process were based on manual curation. From the reconstructed metabolic network, a mathematical model was constructed and validated with data on yields, fluxes and transcription. The presented metabolic network and map are useful tools for examining systemwide data in a metabolic context. Results from the validated model show a great potential for expanding the use of A. niger as a high-yield production platform.

Optimization of heterologous production of the polyketide 6-MSA in Saccharomyces cerevisiae.

Polyketides are a group of natural products that have gained much interest due to their use as antibiotics, cholesterol lowering agents, immunosuppressors, and as other drugs. Many organisms that naturally produce polyketides are difficult to cultivate and only produce these metabolites in small amounts. It is therefore of general interest to transfer polyketide synthase (PKS) genes from their natural sources into heterologous hosts that can over-produce the corresponding polyketides. In this study we demonstrate the heterologous expression of 6-methylsalicylic acid synthase (6-MSAS), naturally produced by Penicillium patulum, in the yeast Saccharomyces cerevisiae. In order to activate the PKS a 4'-phosphopantetheinyl transferase (PPTase) is required. We therefore co-expressed PPTases encoded by either sfp from Bacillus subtilis or by npgA from Aspergillus nidulans. The different strains were grown in batch cultures. Growth and product concentration were measured and kinetic parameters were calculated. It was shown that both PPTases could be efficiently used for activation of PKS's in yeast as good yields of 6-MSA were obtained with both enzymes.