Article Figures & Data
Figures
- Fig. 1.
Biosynthesis of lysine and penicillin G in P. chrysogenum showing the interconversion of pipecolic acid into lysine. lys2 and lys5 encode two different proteins required for α-aminoadipic acid reductase activity (Lys5 is the cognate phosphopantetheinyl transferase that introduces a phospantetheine group in Lys2). The conversion steps blocked in the 7.2 and 10.25 mutants are indicated by arrows with hatch marks through the stems.
- Fig. 2.
Growth of the P. chrysogenum Wis 54-1255 parental strain and the lysine auxotrophs derived from it on Czapek minimal medium MM, Czapek medium with pipecolic acid, and Czapek medium with l-lysine. Note that growth on pipecolic acid of the three auxotrophs is very slow compared with growth on lysine. Abbreviations: Wis, P. chrysogenum Wis 54-1255; TDX,P. chrysogenum TDX195; HS−, P. chrysogenum HS1−; L2, P. chrysogenum L2. Strain TDX has been disrupted in thelys2 gene, strain L2 is a lysine auxotroph obtained by ethylmethane sulfonate mutation defective in the homoaconitase gene (27a), and strain HS1− has been disrupted in the lys1 gene (5a).
- Fig. 3.
Plasmids present in the T1 and T2 transformants ofP. chrysogenum 10.25 after they were rescued inE. coli DH10B. Plasmids were digested withHindIII. (A) Agarose gel. (B) Hybridization with anAccI probe (650 bp) internal to the lys1gene. Lanes 1 to 13, plasmids isolated from transformant T2; lane 14, λHindIII (size marker); lanes 15 to 20, plasmids isolated from transformant T1. Note that plasmids in lanes 18, 19, and 20 do not hybridize with the lys1 probe. These plasmids contain inserts that complemented the 10.25 mutation.
- Fig. 4.
Plasmids of strain 10.25 complemented by cotransformation with pLARA and p10T1 after rescuing in E. coli DH10B. Plasmids recovered from three 10.25 cotransformants are shown. Lanes 1 to 5, transformant 10.25-1; lanes 6 to 10, transformant 10.25-2; lanes 11 to 15, transformant 10.25-3; lane 16, size markers (HindIII-digested lambda phage); lane 17, pLARA control plasmid; lane 18, p10T1 plasmid. Note that both pLARA and p10T1 (arrows) are present in complemented 10.25 transformants (lysine prototrophs).
- Fig. 5.
Alignment of the amino acid sequence of the protein encoded by lys7 (cloned by complementation of the 10.25 mutation) with proteins in the EBI databases. Note that the protein encoded by lys7 is very similar to the saccharopine reductases of N. crassa, M. grisea,S. cerevisiae, and S. pombe. Identical amino acids are shaded.
- Fig. 6.
Pipecolate oxidase activity in P. chrysogenum Wis 54-1255 in DP medium with pipecolic acid as sole nitrogen source (○), with pipecolic acid and ammonium (▴), and with only ammonium (●). Note that pipecolate oxidase activity is induced in medium with pipecolic acid as the sole nitrogen source and is repressed by ammonium.
Tables
- Table 1.
Growth on pipecolic acid and α-aminoadipic acid of different mutants isolated from HSl− showing their reversion rates
Mutant Genetic defect Growth on: Reversion rate Czapek MMc Czapek medium + lysine Czapek medium + pipecolate Czapek medium + α-aminoadipate P. chrysogenum Wis 54-1255 None Yes Yes Yes Yes NDa P. chrysogenumTDX195 Lys2− No Yes Yes No <4 × 10−9 P. chrysogenumHS1− Lys1− No Yes Yes Yes <3.3 × 10−8 P. chrysogenum8.46 ND No Yes No ND 3.3 × 10−6 P. chrysogenum7.2 p.o.−b No Yes No Yes <6.2 × 10−9 P. chrysogenum10.25 Lys7− No Yes No No <5.8 × 10−9 - Table 2.
Enzymatic activities in P. chrysogenum mutants 10.25 and 7.2
Mutant Activity (mU/mg of protein) Saccharopine dehydrogenase Saccharopine reductase Pipecolate oxidase P. chrysogenum Wis 54-1255 381 44 2.1 P. chrysogenumHS1− 402 46.1 1.9 P. chrysogenum10.25 571 0 2.1 P. chrysogenum7.2 450 35.4 0
