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

Abstract

Background: In 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.

Results: During 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.

Conclusions: Our 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.

Figure 1

Penicillin pathway in P. chrysogenum. L-cysteine (Cys), L-valine (Val) and α-amino adipic acid (AAA) are produced from ethanol in central metabolism. These three precursor amino acids are converted into L-α-(δ-aminoadipyl)-L-α-cystenyl-D-α-valine (ACV) by the enzyme L-α-(δ-aminoadipyl)-L-α-cystenyl-D-α-valine synthetase (ACVS, which is coded for by the gene pcbAB). ACV is subsequently converted to isopenicillin-N (IPN) by the enzyme isopenicillin-N synthase (IPNS, which is coded for by the gene pcbC). IPN is then transported into the peroxisome where it is converted into PenG with the precursor PAA, which is added to the medium and imported in the cell and then activated by phenylacetyl CoA ligase to PAA-CoA which is used by the enzyme acyl coenzyme A: Isopenicillin N acyltransferase (AT, which is coded for by the gene penDE). The product PenG is then transported out of the peroxisome and out of the cell into the cultivation medium.

Figure 2

Time patterns of the concentrations of biomass, PenG, PAA and the biomass specific PenG production rate q_p during ethanol limited chemostat cultivation. (A) PenG, (B) biomass and (C) PAA concentrations in chemostat 1 (filled circles), 2 (open squares), 3 (filled triangles) and 4 (diamonds) and (D) biomass specific PenG production in chemostat 1 (solid line), 2 (small stripes), 3 (dots) and 4 (large stripes).

Figure 3

Calculated flux distribution in the central metabolism of P. chrysogenum, assuming pseudo steady-state. The numbers next to the arrows represent the average flux of chemostat 3 and 4 in mmol/Cmol/h at the penicillin production peak (t = 75 h, upper number) and the degenerated culture (t = 500 h, lower number). The direction of the arrow indicates the direction of the flux. Not all fluxes in the network are shown. The standard errors of the fluxes shown are smaller than 5% unless indicated otherwise.

Figure 4

Results of the penicillin gene cluster and microbody quantifications. (A) Copy number of the penicillin gene cluster in the reference strain (Wisconsin 54-1255, 1 penicillin gene cluster, white bar) and in two prolonged chemostat runs of P. chrysogenum DS17690 at the penicillin production peak (t = 75 h, black bars) and the degenerated state (t = 500 h, grey bars). (B) Average number of microbodies per sub apical cell during chemostat cultivation: (filled circles) chemostat 5, (open squares) chemostat 6.

Figure 5

Mean normalized expression of genes throughout ethanol limited chemostat cultivation. The numbers are averages for chemostats 1 and 2. (A) The penicillin pathway genes pcbAB (ACVS, filled circles), pcbC (IPNS, open squares) and penDE (AT, filled triangles). (B) The gene Pc22g14600 (filled circles).

Figure 6

The 6 different transcriptome clusters in which the differentially expressed genes were divided and subjected to a functional category analysis.

Figure 7

Genes of which the expression is positively correlated with PenG production. (A) Venn diagram of the genes comprising cluster 5 and of the genes of which the transcript level was reported to be specifically higher under PenG producing conditions (group 5 in Harris et al. [6]). (B) the 53 genes that are overlapping the two sets. The genes underlined contained at least one cis-regulatory motif, as indicated in (C), in their promoter sequences (promoter sequences includes the region -900, -1). (C) overrepresented cis-regulatory motif in the set of the 53 genes. The motif has been identified using the MEME software and the logo has been edited using the Weblogo software.

Figure 8

Levels of proteins and metabolites associated with PenG production, plotted against the specific penicillin production q_P. Left panel: Relative protein amounts of the enzymes of the penicillin biosynthesis pathway (see Figure 1) compared to the reference condition at the penicillin production peak (first time point) in chemostat 1 (filled circles) and chemostat 4 (open diamonds). Middle panel: Intracellular levels of penicillin precursors and intermediates in chemostat 2 (open squares), chemostat 3 (filled triangles) and chemostat 4 (open diamonds). Right panel: Energy charge, calculated ratio of NADH/NAD from the Mannitol6P/F6P sensor reaction, intracellular PAA and the concentration ratio of PAA in chemostat 2 (open squares), chemostat 3 (filled triangles) and chemostat 4 (open diamonds).

Figure 9

Heat map of the transcript level of the genes comprising cluster 5. The heat map displays the mean normalized data at the penicillin production peak (t = 75 h), of the degenerated culture (t = 500 h) and during the steady state of the continued sub cultivation (t = 75 h) obtained by inoculation of a new chemostat with biomass of the degenerated culture.