Metabolic engineering of the a-aminoadipic acid pool, a precursor of secondary metabolites, in fungi
Martín, J.F.1,2 ; Bañuelos, O.1; Gutiérrez, S.1,2; Martín-Valmaseda, E.M.1; Naranjo, L.1; Teves, F.1 and Casqueiro, J.1,2
1Area
de Microbiología, Facultad de Biología, Universidad de León,
24071 León
2Instituto de biotecnología (INBIOTEC), Avda. del Real
Nº1, 24006 León.
The b-lactam producing fungi need a large supply of a-aminoadipic acid through the lysine pathway for penicillin biosynthesis. Overexpression of a-aminoadipic acid biosynthetic genes and directed disruption of genes of the branching pathways leads to penicillin overproduction. The previously cloned lys1 gene encoding the homocitrate synthase (HS-) (the first enzyme of the lysine pathway) was disrupted in Wisconsin 54-1255 pyrG strain. To target inactivate lys1 we have used a double-crossover strategy by transformation with a plasmid containing a fragment of 9 kb, homologous to the lys1-containing target region of P. chrysogenum Wis 54-1255. Three lysine auxotrophs were obtained from a total of 2004 transformants studied. Mutants HS- lacked homocitrate synthase activity.
The lys1 ORF was fused in frame to the green fluorescent protein gene (GFP). HS- mutants transformed with a plasmid containing the lys1-GFP hybrid gene restored prototrophy and contained similar levels of homocitrate synthase than the Wisconsin 54-1255 control strain. Inmunoblot studies with antibodies against GFP demonstrated that the hybrid protein is kept intact. Analysis at the microscope showed that the homocitrate synthase was mainly located at the cytosol.
A P. chrysogenum genomic library was constructed using the high copy number plasmid pAMPF9L. A 4.7 kb fragment complemented the mutation in P. chrysogenum L2, a lysine auxotroph blocked in the first half of the lysine biosynthetic pathway. The 4.7 kb fragment has been partially sequenced and the deduced amino acid sequence showed homology with homoaconitases of Aspergillus nidulans (45.3% identity) and yeast. The P. chrysogenum L2 mutant has been complemented with the lysF gene of A. nidulans encoding homoaconitase.
a-Aminoadipic acid can be supplied for penicillin production throught a-aminoadipate biosynthesis or through lysine catabolism. There are more than eight different lysine catabolism pathways described in plant and microorganisms. One of these is the pipecolic pathway described for some fungi such as Metarhizium anisopliae and Rhizoctonia leguminicola. Other microorganisms including some yeasts catabolize lysine through saccharopine. In order to characterize the lysine catabolic pathway in P. chrysogenum several experimental approaches have been used. Most of the enzymatic activities belonging to different lysine catabolic pathways have been assayed and the only one that it is induced by the presence of lysine is the saccharopine dehydrogenase. HPLC analysis revealed that when P. chrysogenum Wisconsin 54-1255 is grown with lysine as sole nitrogen source the intracellular concentration of sacharopine is increased 5 to 6-fold as compared to control strain grown with ammonium. HPLC analysis, showed that HS- mutants (lacking homocitrate synthase activity, the first enzyme of the lysine biosynthetic pathway) were able to synthesize a-aminoadipic acid from lysine probably through saccharopine. The contribution of the a-aminoadipic acid biosynthetic pathway from a-ketoglutarate and the lysine-catabolic pathway will be discussed.
