Figure 1.
Enzymes: Dxs, 1-deoxy-D-xylulose-5-phosphate synthase; Dxr, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; IspD, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase; IspE, 4-diphosphocytidyl-2-C-methylerythritol kinase; IspF, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; IspG, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase; IspH, 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase; Metabolites: 1, D-glyceraldehyde 3-phosphate; 2, pyruvate; 3, 1-deoxy-D-xylulose 5-phosphate; 4, 2-C-methyl-D-erythritol 4-phosphate; 5, 4-diphosphocytidyl-2-C-methyl-D-erythritol; 6, 2-phospho-4-diphosphocytidyl-2-C-methyl-D-erythritol; 7, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate; 8, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate. The MVA pathway (right). Enzymes: Erg10, acetoacetyl-CoA thiolase; Erg13, 3-hydroxy-3-methylglutaryl-CoA synthase; Hmg1/2, 3-hydroxy-3-methylglutaryl-CoA reductase; Erg12, mevalonate kinase; Erg8, phosphomevalonate kinase; Erg19, mevalonate diphosphate decarboxylase; Idi, isopentenyl diphosphate isomerase; Metabolites: 9, acetyl-CoA; 10, acetoacetyl-CoA; 11, 3-hydroxy-3-methylglutaryl-CoA; 12, mevalonate; 13, phosphomevalonate; 14, diphosphomevalonate; 15, isopentenyl diphosphate; 16, dimethyl allyl diphosphate.
Figure 2.
Schematic representation of genetic engineering strategies for A) genomic integration of the bacterial MEP pathway genes into the yeast genome (chromosome XVI), and B) plasmid-based reconstruction of possible Fe/S trafficking routes involved in maturation of bacterial IspG/IspH, bacterial electron transfer systems and plant-derived ispG/ispH in S. cerevisiae.
For details see text.
Table 1.
List of strains and plasmids used in this study.
Figure 3.
Growth of S. cerevisiae strains CEN.PK 113-13D (black circles) and SCISP06 (gray circles) in SD minimal medium.
Dashed lines represent the growth in 0 g L−1 of lovastatin; solid lines represent the growth in presence of 2 g L−1 of lovastatin. Error bars show the standard deviation from three cultivations.
Figure 4.
Schematic representation of evaluation the functionality of the bacterial MEP pathway in S. cerevisiae in different conditions.
A) Gel electrophoresis of PCR products to confirm deletion of ERG13 (1: SCISP28, 2: SCISP29, 3: SCISP16, 4: CEN.PK 113-13D (wild type), 5: SCISP30, 6: SCISP31, 7: SCISP32, M: 1 kb Plus DNA ladder (Fermentas, Maryland, USA); B) Aerobic cultivation of MEP pathway strains co-expressing erpA, fpr and fldA; C) Aerobic cultivation of MEP pathway strains co-expressing erpA, fpr, fldA, At-IspG, At-IspH with either CpIscA or hISCA1; D) Anaerobic cultivation of MEP pathway strains co-expressing erpA, fpr and fldA; E) Anaerobic cultivation of MEP pathway strains co-expressing erpA, fpr, fldA, At-IspG, At-IspH with either CpIscA or hISCA1. All strains carried an ERG13 deletion and were plated on medium with or without 10 mg L−1 mevalonate (MVA).
Figure 5.
Schematic representation of possible Fe/S trafficking routes involved in maturation of bacterial IspG/IspH in E. coli (left) and reconstruction of possible routes preformed in this study in the yeast cytosol (right).
Dashed arrows represent unknown mechanisms for transferring the Fe-S clusters from mitochondria to cytosol. For more information see text.
Table 2.
List of oligonucleotide primers used in this study.