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X-omics data for in depth understanding of polyketide production in Aspergillus nidulans
Journal of Biotechnology 131S (2007) S196–S210
Industrial Biotechnology
METABOLIC ENGINEERING AND APPLIED SYSTEMS BIOLOGY
2. Comparative metabolomics in lactic acid bacteria
1. X-omics data for in depth understanding of polyketide production in Aspergillus nidulans
Marieke Pastink a,∗ , Bas Teusink b , Willem de Vos c , Jeroen Hugenholtz d
Gianni
Panagiotou ∗ ,
Lisbeth Olsson, Jens Nielsen
Center for Microbial Biotechnology-DTU, Building 223, kgs2800 Lyngby, Denmark The genus Aspergillus includes a member of great relevance in fundamental research, namely A. nidulans, which represents an important model organism for studies of cell biology and gene regulation. The fungus Penicillium patulum encode a gene for the protein 6-methylsalicylic acid polyketide synthase (MSAS) which produces the secondary metabolite 6-MSA. MSAS is an excellent model system for studying the mechanisms of chain length control, regiospecificity of reduction, and building block specificity by PKSs. Clearly, heterologous expression of functional PKSs in organisms such as A. nidulans would be advantageous since it would enable use of the advanced knowledge and technology available with this microorganism. Our group has very recently constructed three recombinant A. nidulans strains aiming to develop a robust platform for 6MSA production. In the first stage, MSAS from P. patulum has been functionally expressed in A. nidulans. Furthermore, the gene encoding phosphoketolase has been overexpressed in order to increase precursor supply. A double mutant was constructed as well to test our hypothesis that with these defined changes will reach our goals of improved yield and productivity in our process. Considering the high degree of flexibility in the metabolism of aspergilli, physiological studies on different carbon sources accompanied by the analysis of transcriptome, metabolome and fluxome data of the three recombinant A. nidulans strains were performed for the evaluation of the function of the metabolic network after genetic perturbations and the optimization of 6MSA production. doi:10.1016/j.jbiotec.2007.07.348
0168-1656/$ – see front matter doi:10.1016/j.jbiotec.2007.07.347
a
NIZO/WCFS/Kluyver Centre, Kernhemseweg 2, 6718 ZB, Netherlands b NIZO food research, Ede, Netherlands c Laboratory of microbiology, Wageningen University, Wageningen, Netherlands d Kluyver Centre for Genomics of Industrial fermentations, Wageningen, Netherlands Many industrially relevant activities in lactic acid bacteria are directly linked to amino acid metabolism. Best known examples are the production of flavour and the production of vitamins (folate, riboflavin, vitamin B12). In this research project metabolomics will be used to compare amino acid metabolism in different lactic acid bacteria and under different conditions. The metabolome analysis will be performed for three dairy microorganisms – Lactococcus lactis, Lactobacillus plantarum and Streptococcus thermophilus – under different growth conditions. Complete genome sequences of these three bacteria are available and the metabolome data will be projected on the predicted metabolic network based on the DNA sequences. Different software tools like KEGG, ERGO and Simpheny are used to predict and compare metabolic networks based on DNA sequences. Complete Simpheny models for Lb. plantarum and L. lactis are available. The model for S. thermophilus is under construction and is based on the annotated genome of strain LMG18311 (Bolotin et al., 2004; Hols et al., 2005). All genes essential for growth have been identified and selected. Subsequently, continuous fermentations were performed to produce cells for accurate biomass analysis and to get detailed information about amino acid stoichiometry and about fermentation products. During steady state of these fermentations, samples were taken for biomass- and HPLC analysis. The formation of volatile metabolites in growing cultures under controlled conditions has been measured. These volatiles were measured using Purge and Trap coupled with GC–MS.
Industrial Biotechnology
METABOLIC ENGINEERING AND APPLIED SYSTEMS BIOLOGY
2. Comparative metabolomics in lactic acid bacteria
1. X-omics data for in depth understanding of polyketide production in Aspergillus nidulans
Marieke Pastink a,∗ , Bas Teusink b , Willem de Vos c , Jeroen Hugenholtz d
Gianni
Panagiotou ∗ ,
Lisbeth Olsson, Jens Nielsen
Center for Microbial Biotechnology-DTU, Building 223, kgs2800 Lyngby, Denmark The genus Aspergillus includes a member of great relevance in fundamental research, namely A. nidulans, which represents an important model organism for studies of cell biology and gene regulation. The fungus Penicillium patulum encode a gene for the protein 6-methylsalicylic acid polyketide synthase (MSAS) which produces the secondary metabolite 6-MSA. MSAS is an excellent model system for studying the mechanisms of chain length control, regiospecificity of reduction, and building block specificity by PKSs. Clearly, heterologous expression of functional PKSs in organisms such as A. nidulans would be advantageous since it would enable use of the advanced knowledge and technology available with this microorganism. Our group has very recently constructed three recombinant A. nidulans strains aiming to develop a robust platform for 6MSA production. In the first stage, MSAS from P. patulum has been functionally expressed in A. nidulans. Furthermore, the gene encoding phosphoketolase has been overexpressed in order to increase precursor supply. A double mutant was constructed as well to test our hypothesis that with these defined changes will reach our goals of improved yield and productivity in our process. Considering the high degree of flexibility in the metabolism of aspergilli, physiological studies on different carbon sources accompanied by the analysis of transcriptome, metabolome and fluxome data of the three recombinant A. nidulans strains were performed for the evaluation of the function of the metabolic network after genetic perturbations and the optimization of 6MSA production. doi:10.1016/j.jbiotec.2007.07.348
0168-1656/$ – see front matter doi:10.1016/j.jbiotec.2007.07.347
a
NIZO/WCFS/Kluyver Centre, Kernhemseweg 2, 6718 ZB, Netherlands b NIZO food research, Ede, Netherlands c Laboratory of microbiology, Wageningen University, Wageningen, Netherlands d Kluyver Centre for Genomics of Industrial fermentations, Wageningen, Netherlands Many industrially relevant activities in lactic acid bacteria are directly linked to amino acid metabolism. Best known examples are the production of flavour and the production of vitamins (folate, riboflavin, vitamin B12). In this research project metabolomics will be used to compare amino acid metabolism in different lactic acid bacteria and under different conditions. The metabolome analysis will be performed for three dairy microorganisms – Lactococcus lactis, Lactobacillus plantarum and Streptococcus thermophilus – under different growth conditions. Complete genome sequences of these three bacteria are available and the metabolome data will be projected on the predicted metabolic network based on the DNA sequences. Different software tools like KEGG, ERGO and Simpheny are used to predict and compare metabolic networks based on DNA sequences. Complete Simpheny models for Lb. plantarum and L. lactis are available. The model for S. thermophilus is under construction and is based on the annotated genome of strain LMG18311 (Bolotin et al., 2004; Hols et al., 2005). All genes essential for growth have been identified and selected. Subsequently, continuous fermentations were performed to produce cells for accurate biomass analysis and to get detailed information about amino acid stoichiometry and about fermentation products. During steady state of these fermentations, samples were taken for biomass- and HPLC analysis. The formation of volatile metabolites in growing cultures under controlled conditions has been measured. These volatiles were measured using Purge and Trap coupled with GC–MS.