Gene Targeting inPenicillium chrysogenum: Disruption of thelys2Gene Leads to Penicillin Overproduction

Abstract

Two strategies have been used for targeted integration at thelys2 locus of Penicillium chrysogenum. In the first strategy the disruption of lys2 was obtained by a single crossing over between the endogenous lys2 and a fragment of the same gene located in an integrative plasmid.lys2-disrupted mutants were obtained with 1.6% efficiency when the lys2 homologous region was 4.9 kb, but no homologous integration was observed with constructions containing a shorter homologous region. Similarly,lys2-disrupted mutants were obtained by a double crossing over (gene replacement) with an efficiency of 0.14% by using two lys2 homologous regions of 4.3 and 3.0 kb flanking thepyrG marker. No homologous recombination was observed when the selectable marker was flanked by short lys2 homologous DNA fragments. The disruption of lys2 was confirmed by Southern blot analysis of three different lysine auxotrophs obtained by a single crossing over or gene replacement. Thelys2-disrupted mutants lacked α-aminoadipate reductase activity (encoded by lys2) and showed specific penicillin yields double those of the parental nondisrupted strain, Wis 54-1255. The α-aminoadipic acid precursor is channelled to penicillin biosynthesis by blocking the lysine biosynthesis branch at the α-aminoadipate reductase level.

Amplification and disruption of the phenylacetyl-CoA ligase gene of Penicillium chrysogenum encoding an aryl-capping enzyme that supplies phenylacetic acid to the isopenicillin N-acyltransferase

“…It was grown in LB (Luria-Bertani) medium with ampicillin (100 µg/ml), chloramphenicol (30 µg/ml) or kanamycin (50 µg/ml). P. chrysogenum spores were obtained from plates of Power medium [23] (n = 18) grown for 5 days at 28 • C. Seed cultures were initiated by inoculating fresh spores from one sporulated Petri dish into CI (complex inoculation) medium (20 g/l corn steep solids, 10 g/l yeast extract, 58 mM sucrose, 50 mM calcium carbonate, pH 5.7) or DI (defined inoculation) medium [18] after incubation for 24 h. Penicillin production was studied in cultures in CP (complex production) medium [4 g/l potassium phenylacetate, 20 g/l Pharmamedia (Traders Protein, Memphis, TN, U.S.A.), 50 g/l lactose, 0.03 M ammonium sulphate and 0.05 M calcium carbonate, pH 6.6] or DP (defined production) medium [18]. Cultures (100 ml in 500 ml flasks) were inoculated with 5 % seed cultures and incubated in an orbital shaker (at 250 rev./min for up to 96 h at 25 • C).…”

“…Gene disruption in P. chrysogenum is difficult to perform because of frequent ectopic integration of the exogenous DNA during disruption experiments, but gene inactivation is required to prove unequivocally the involvement of a specific gene in a biosynthetic reaction. We have previously optimized gene disruption in P. chrysogenum [18] using the two-marker strategy [19].…”

The fungal α‐aminoadipate pathway for lysine biosynthesis requires two enzymes of the aconitase family for the isomerization of homocitrate to homoisocitrate

“…Especially for penicillin production the detailed characterization of the second aconitase in production strains appears important. As α‐aminoadipate formation is a rate‐limiting step in penicillin production (Jaklitsch et al ., ; Casqueiro et al ., ), the introduction of a highly expressed synthetic codon‐adapted version of S. cerevisiae Aco2p could possibly enhance production rates.…”

“…Nevertheless, filamentous fungi not only use the α‐aminoadipate pathway for the de novo synthesis of lysine, but also require α‐aminoadipate for penicillin production (Brakhage, ). Here, the internal α‐aminoadipate concentration directly correlates with the rate of penicillin production (Jaklitsch et al ., ; Casqueiro et al ., ). Interestingly, despite the importance of the α‐aminoadipate pathway for penicillin production, not all enzymatic reactions leading to the formation of α‐aminoadipate have been studied in detail, but, at least, all intermediates leading to the formation of α‐aminoadipate are known.…”

A novel convenient procedure for extractive work‐up of whole‐cell biotransformations using de‐emulsifying hydrolases

“…This problem is typical for high-cell-density cultures as they are often used in the whole-cell biocatalytic pro-duction of fine chemicals. Here, cell densities exceed that of conventional fermentation protocols (e.g., for the production of penicillin for which extractive work-up procedures are established) by a factor of 10 to 100 (Casqueiro et al, 1999). The undesired phenomenon of gel and slime formation, which is caused by emulsification, is typically solved by centrifugation of the organic solvent -water mixtures (Davoli et al, 1999).…”