Improvement of penicillin yields in solid-state and submerged fermentation of Penicillium chrysogenum by amplification of the penicillin biosynthetic gene cluster

Abstract

Penicillium chrysogenum low and high penicillin producing strains were transformed with a cosmid containing the whole penicillin biosynthetic gene cluster. The cosmid library was constructed in a newly developed cosmid vector, IztapaCos, which allows cloning and direct introduction of large DNA fragments in fungal recipients using phleomycin resistance as selection marker. The effect of increased gene dosage on penicillin production was evaluated both in submerged (SmF) and solid-state fermentation (SSF). Transformants from the low-producing strain Wis 54-1255, showed a 67.3 and 28.3% increased penicillin titer in SSF and SmF, respectively. In transformants from the high-producing strain P2-32 the increase was 92.9 and 158.4% respectively. Strain P2-32 already contains originally about 14 copies of the penicillin biosynthetic cluster, which shows that the strategy of increasing the gene dosage is still valid for high copynumber strains. The different behavior of the two strains in each type of culture is discussed, along with the practical implications for industrial penicillin production.

Strategies for strain improvement in Fusarium fujikuroi: overexpression and localization of key enzymes of the isoprenoid pathway and their impact on gibberellin biosynthesis

“…In A. nidulans, overexpression of the penicillin biosynthesis gene acvA resulted in elevated penicillin production up to 30-fold (Kennedy and Turner 1996). Similarly, an increased gene dosage of the three penicillin biosynthesis structural genes pcbAB, pcbC, and penDE in Penicillium chrysogenum resulted in improved production yields in liquid as well as in solid stage fermentation (Campos et al 2008). Interestingly, molecular analysis of the best P. chrysogenum production strains generated by classical mutagenesis and selection methods revealed increased copy numbers of the whole gene cluster, often obtained in tandem repeats (Barredo et al 1989;Fierro et al 1995).…”

Surface‐enhanced Raman spectroscopy and ultrastructural analysis of penicillin‐producing Penicillium rubens strains

“…A high penicillin-producing strain, P. rubens P2-32-T, 25 was used (average penicillin production of 380 μg m −1 at 24 h of culture) obtained by amplifying the number of copies of the genes that code for penicillin. 1 As a control, P. rubens Wisconsin 54-1255 npe10 was used. This strain was obtained by mutagenesis and did not produce penicillin due to the complete deletion of the penicillin biosynthetic gene cluster.…”

“…In some improved strains, a significant but delicate rebalancing of intracellular metabolism has occurred, leading to impressive high titres of penicillin production. 1 Due to their importance as producers of secondary metabolites and, sometimes, for their pathogenic capacity, their morphology and structure have been studied using UV-Vis spectroscopy and Raman spectroscopy. 2 Raman spectroscopy is an analytical technique applied to the identification of macromolecules, capable of providing information at the molecular level 3 and for the in vitro and in vivo characterisation of individual cells.…”

Efecto de las sobreexpresión del gen lovE sobre la fisiología de Aspergillus terreus, en fermentación sólida y líquida

“…La clonación del clúster completo de penicilina en un cosmido (IztapaCos) para ser transformado en Penicillium chrysogenum para FL fue evaluada tanto en FS como en FL, obteniéndose producciones del 158.4% y del 93%, respectivamente (Campos et al, 2008).…”

“…Esto es algo que no habíamos visto en trabajos anteriores en los que se habían estudiado métodos de mejoramiento genético molecular para penicilina y para lovastatina. En estos estudios se han encontrado transformantes buenas para FS o para FL, pero no para los dos (Campos et al, 2008;Baños, 2010).…”