Degeneration of penicillin production in ethanol-limited chemostat cultivations of Penicillium chrysogenum: A systems biology approach

Abstract

BackgroundIn microbial production of non-catabolic products such as antibiotics a loss of production capacity upon long-term cultivation (for example chemostat), a phenomenon called strain degeneration, is often observed. In this study a systems biology approach, monitoring changes from gene to produced flux, was used to study degeneration of penicillin production in a high producing Penicillium chrysogenum strain during prolonged ethanol-limited chemostat cultivations.ResultsDuring these cultivations, the biomass specific penicillin production rate decreased more than 10-fold in less than 22 generations. No evidence was obtained for a decrease of the copy number of the penicillin gene cluster, nor a significant down regulation of the expression of the penicillin biosynthesis genes. However, a strong down regulation of the biosynthesis pathway of cysteine, one of the precursors of penicillin, was observed. Furthermore the protein levels of the penicillin pathway enzymes L-α-(δ-aminoadipyl)-L-α-cystenyl-D-α-valine synthetase (ACVS) and isopenicillin-N synthase (IPNS), decreased significantly. Re-cultivation of fully degenerated cells in unlimited batch culture and subsequent C-limited chemostats did only result in a slight recovery of penicillin production.ConclusionsOur findings indicate that the observed degeneration is attributed to a significant decrease of the levels of the first two enzymes of the penicillin biosynthesis pathway, ACVS and IPNS. This decrease is not caused by genetic instability of the penicillin amplicon, neither by down regulation of the penicillin biosynthesis pathway. Furthermore no indications were obtained for degradation of these enzymes as a result of autophagy. Possible causes for the decreased enzyme levels could be a decrease of the translation efficiency of ACVS and IPNS during degeneration, or the presence of a culture variant impaired in the biosynthesis of functional proteins of these enzymes, which outcompeted the high producing part of the population.

Impact of Velvet Complex on Transcriptome and Penicillin G Production in Glucose-Limited Chemostat Cultures of a β-Lactam High-ProducingPenicillium chrysogenumStrain

“…For each strain, transcriptome analysis was performed from at least two independent replicate chemostat cultures. The average coefficient of variation for transcriptome data from replicated cultures was below 20%, in accordance with previous chemostat-based transcriptome analyses in P. chrysogenum (Douma et al, 2011;Gombert et al, 2011;Harris et al, 2009aHarris et al, , 2009bKoetsier et al, 2010;Snoek et al, 2009;van den Berg et al, 2008).…”

“…Strains DS63171 (hdfAD PcvelAD) and DS67261 (hdfAD PclaeAD) were therefore analyzed by q-PCR, using the strains DS54465 (hdfAD) (six to eight penicillin biosynthetic clusters), Wisconsin54-1255 (one penicillin biosynthetic cluster), and DS50562 (zero penicillin biosynthetic cluster) as references. Furthermore, the comparison of the number of penicillin clusters of the strain DS54465 (hdfAD) (this study) and its ancestor DS17690 (Douma et al, 2011) did not reveal any intermediate loss of cluster between the two strains. Deletion strains DS63171 (hdfAD PcvelAD) and DS67261 (hdfAD PclaeAD) did not showed significant changes in penicillin biosynthesic gene cluster number relative to DS54465 and DS17690 (Fig.…”

Resolving Phenylalanine Metabolism Sheds Light on Natural Synthesis of Penicillin G in Penicillium chrysogenum

“…The average coefficient of variation for biological replicates (triplicates for the carbon-limited cultures with ammonium sul- a Three different network scenario hypotheses were employed to obtain an optimal fit for flux distribution: scenario i, phenylalanine was incorporated into protein, incorporated into penicillin G, metabolized via the homogentisate pathway, or metabolized via tyrosine; scenario ii, an unidentified pathway for phenylalanine metabolism, which did not involve any of the measured metabolites, was introduced into the model together with exchanges of phenylalanine and tyrosine with the protein pool (synthesis and degradation); and scenario iii, hypothetical hydroxylase activities that convert phenylpyruvate to 4-hydroxyphenylpyruvate and phenylacetaldehyde to 4-hydroxyphenylacetaldehyde were included. (14,26,27,36,68,71).…”

Linkage of Oxidative Stress and Mitochondrial Dysfunctions to Spontaneous Culture Degeneration in Aspergillus nidulans

“…10). Phenotypic features of degenerated fungal cultures include non-sporulation and impaired abilities of sexuality, fruiting, and production of secondary metabolites (6,7,15). However, mutation or deletion of different genes involved in controlling sporulation could also result in a fluffy phenotype in A. nidulans and other fungi.…”

“…The mechanism(s) underlying fungal culture degeneration is poorly understood. Genetic mutations were not evident in the degenerated fungal isolates of Penicillium chrysogenum (6). Otherwise, methylation of genomic DNA was reported in a sector of the plant pathogenic fungus Fusarium oxysporum after successive subculturing (16).…”