Quantitative analysis of Penicillium chrysogenum Wis54-1255 transformants overexpressing the penicillin biosynthetic genes

The low penicillin-producing, single gene copy strain Wis54-1255 was used to study the effect of overexpressing the penicillin biosynthetic genes in Penicillium chrysogenum. Transformants of Wis54-1255 were obtained with the amdS expression-cassette using the four combinations: pcbAB, pcbC, pcbC-penDE, and pcbAB-pcbC-penDE of the three penicillin biosynthetic genes. Transformants showing an increased penicillin production were investigated during steady-state continuous cultivations with glucose as the growth-limiting substrate. The transformants were characterized with respect to specific penicillin productivity, the activity of the two pathway enzymes delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and isopenicillin N synthetase (IPNS) and the intracellular concentration of the metabolites: delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (bisACV), isopenicillin N (IPN), glutathione (GSH), and glutathione disulphide (GSSG). Transformants with the whole gene cluster amplified showed the largest increase in specific penicillin productivity (r(p))-124% and 176%, respectively, whereas transformation with the pcbC-penDE gene fragment resulted in a decrease in r(p) of 9% relative to Wis54-1255. A marked increase in r(p) is clearly correlated with a balanced amplification of both the ACVS and IPNS activity or a large amplification of either enzyme activity. The increased capacity of a single enzyme occurs surprisingly only in the transformants where all the three biosynthetic genes are overexpressed but is not found within the group of pcbAB or pcbC transformants. The indication of the pcbAB and pcbC genes being closely regulated in fungi might explain why high-yielding strains of P. chrysogenum have been found to contain amplifications of a large region including the whole penicillin gene cluster and not single gene amplifications. Measurements of the total ACV concentration showed a large span of variability, which reflected the individual status of enzyme overexpression and activity found in each strain. The ratio ACV:bisACV remained constant, also at high ACV concentrations, indicating no limitation in the capacity of the thioredoxin-thioredoxin reductase (TR) system, which is assumed to keep the pathway intermediate LLD-ACV in its reduced state. The total GSH pool was at a constant level of approx. 5.7 mM in all cultivations.     

Transformation in Penicillium chrysogenum

An auxotrophic mutant of Penicillium chrysogenum with a DNA rearrangement that affects the trpC region has been transformed to the Trp+ phenotype by using a plasmid that contains the trifunctional wild-type gene. A frequency of 40-80 transformants per microgram of input DNA was usually achieved. A low frequency of plasmid integration at the recipient mutated trpC gene was detected; however, most of the transformants integrated the plasmid DNA elsewhere into the genome. Some of the transformants contain multiple rearranged copies of the vector integrated in a tandem fashion.     

Isolation of deacetoxycephalosporin C from fermentation broths of Penicillium chrysogenum transformants: construction of a new fungal biosynthetic pathway.

Deacetoxycephalosporin C (DAOC), a precursor of cephalosporins excreted by Cephalosporium and Streptomyces species, has been produced in Penicillium chrysogenum transformed with DNA containing a hybrid penicillin N expandase gene (cefEh) and a hybrid isopenicillin N epimerase gene (cefDh). DAOC from a P. chrysogenum transformant was identified by ultraviolet light (UV), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) and mass spectrum analyses. P. chrysogenum transformed with DNA containing cefEh without cefDh did not produce DAOC. Untransformed P. chrysogenum produced penicillin V (phenoxymethylpenicillin) but not DAOC. Transformants also produced penicillin V but, in general, less than untransformed P. chrysogenum. The cefEh and cefDh genes were constructed by replacing the open reading frame (ORF) of cloned P. chrysogenum pcbC and penDE genes with the ORF of the Streptomyces clavuligerus expandase gene, cefE, and the ORF of the Streptomyces lipmanii epimerase gene, cefD, respectively. Analyses of representative transformants suggested that production of DAOC occurred via cefEh and cefDh genes stably integrated in the P. chrysogenum genome. DNA from untransformed P. chrysogenum did not hybridize to cefE or cefD gene probes.     

Penicillin acyltransferase in Penicillium chrysogenum

Isotopic exchange of (35)S between penicillins and 6-amino-penicillanic acid (6-APA) was observed in cell-free extracts of Penicillium chrysogenum. Sulfhydryl-containing compounds were required for activity. Dithiothreitol, dithioerythritol, mercaptoethanol, and glutathione served as activators. The acyltransferase was purified threefold by adsorption on calcium phosphate gel at pH 6 and elution at pH 8. The partially purified enzyme showed maximal activity at pH 8. The enzyme was stable at 25 C for at least 30 min at pH 8. Dissociable inhibitors or activators, other than the sulfhydryl-containing compounds, could not be demonstrated in the enzyme preparation. An apparent Michaelis constant of 1.5 +/- 0.5 mm was determined for penicillin G at a 6-APA concentration of 5 mm. The enzyme did not appear to possess penicillin amidase activity. The exchange mechanism probably involves an acyl-enzyme intermediate. Penicillins V, G, K, X, and dihydro F showed isotopic exchange with (35)S-6-APA. Penicillin N, methylpenicillin, and phenyl-penicillin did not show exchange. The level of acyltransferase in P. chrysogenum 51-20F3 was measured at times during the fermentation. The level of activity increased threefold between 40 and 55 hr, remaining high until about 90 hr.     

Characterization of a phenylacetate-CoA ligase from Penicillium chrysogenum

Enzymatic activation of PAA (phenylacetic acid) to phenylacetyl-CoA is an important step in the biosynthesis of the beta-lactam antibiotic penicillin G by the fungus Penicillium chrysogenum. CoA esters of PAA and POA (phenoxyacetic acid) act as acyl donors in the exchange of the aminoadipyl side chain of isopenicillin N to produce penicillin G or penicillin V. The phl gene, encoding a PCL (phenylacetate-CoA ligase), was cloned in Escherichia coli as a maltose-binding protein fusion and the biochemical properties of the enzyme were characterized. The recombinant fusion protein converted PAA into phenylacetyl-CoA in an ATP- and magnesium-dependent reaction. PCL could also activate POA, but the catalytic efficiency of the enzyme was rather low with k(cat)/K(m) values of 0.23+/-0.06 and 7.8+/-1.2 mM(-1).s(-1) for PAA and POA respectively. Surprisingly, PCL was very efficient in catalysing the conversion of trans-cinnamic acids to the corresponding CoA thioesters [k(cat)/K(m)=(3.1+/-0.4)x10(2) mM(-1).s(-1) for trans-cinnamic acid]. Of all the substrates screened, medium-chain fatty acids, which also occur as the side chains of the natural penicillins F, DF, H and K, were the best substrates for PCL. The high preference for fatty acids could be explained by a homology model of PCL that was constructed on the basis of sequence similarity with the Japanese firefly luciferase. The results suggest that PCL has evolved from a fatty-acid-activating ancestral enzyme that may have been involved in the beta-oxidation of fatty acids.     

Scale-down of penicillin production in Penicillium chrysogenum

In large-scale production reactors the combination of high broth viscosity and large broth volume leads to insufficient liquid-phase mixing, resulting in gradients in, for example, the concentrations of substrate and oxygen. This often leads to differences in productivity of the full-scale process compared with laboratory scale. In this scale-down study of penicillin production, the influence of substrate gradients on process performance and cell physiology was investigated by imposing an intermittent feeding regime on a laboratory-scale culture of a high yielding strain of Penicillium chrysogenum. It was found that penicillin production was reduced by a factor of two in the intermittently fed cultures relative to constant feed cultivations fed with the same amount of glucose per hour, while the biomass yield was the same. Measurement of the levels of the intermediates of the penicillin biosynthesis pathway, along with the enzyme levels, suggested that the reduction of the flux through the penicillin pathway is mainly the result of a lower influx into the pathway, possibly due to inhibitory levels of adenosine monophosphate and pyrophosphate and lower activating levels of adenosine triphosphate during the zero-substrate phase of each cycle of intermittent feeding.