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Tajalli Keshavarz, Applied Biotechnology Research Group, Department of Molecular and Applied Biosciences, University of Westminster, London, UK. E-mail: T.Keshavarz@wmin.ac.uk
Aim: To investigate the effects of single and multiple additions of the oligosaccharide elicitors, obtained from alginate and locust bean gum, on penicillin G production and the transcript level of penicillin G biosynthetic genes.
Methods and Results: The transcript copy numbers and penicillin G concentration in liquid cultures of Penicillium chrysogenum grown under control and elicited conditions were compared using quantitative PCR and HPLC assay respectively. An increase in the penicillin G production rate and transcript copy numbers of the three major penicillin G biosynthetic genes pcbAB, pcbC and penDE was observed in the elicited cultures compared to control cultures. The effects were observed to be higher in multiple elicitor added cultures compared to single elicitor supplemented and control cultures.
Conclusions: The results show, for the first time in bioreactor cultures, the enhancement of penicillin G transcript copy number of the penicillin biosynthetic genes using qPCR with a corresponding increase in the penicillin G production upon multiple elicitor addition of two different types of elicitors.
Significance and Impact of the Study: Establishment of the effect of multiple elicitor addition on penicillin G production and investigating the role of oligosaccharide elicitors as transcriptional activators has wide spread impact for antibiotic industry.
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Penicillin biosynthesis in P. chrysogenum has been investigated at the molecular level over the past few decades (Gomez-Pardo and Peñalva 1990; Alvarez et al. 1993; Brakhage et al. 2004). The three major enzymatic steps in the penicillin biosynthetic pathway are catalysed by the enzymes δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase (ACVS), isopenicillin N synthase (IPNS) and acetyl CoA: isopenicillin N acyltransferase (IAT), which together catalyses the formation of penicillin from its amino acid precursors (Brakhage et al. 2004). These three major enzymes are encoded by the structural genes denoted pcbAB, pcbC and penDE (Petruccioli et al. 1999). pcbAB encodes a large multifunctional nonribosomal peptide synthetase (ACVS) which is involved in the formation of the first intermediate δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (LLD-ACV) from the three amino acid precursors l-α-aminoadipic acid, l-cysteine, and l-valine. pcbC encodes IPNS which catalyses the oxidative ring closure of the linear tripeptide leading to the first β-lactam, isopenicillin N (IPN). penDE encodes IAT which catalyses the final step of exchanging the l-α-aminoadipic acid side chain for a hydrophobic acyl group (Alvarez et al. 1993).
The biosynthetic genes in a pathway can be either simultaneously expressed or sequentially induced. The expression of penicillin biosynthetic genes is most probably simultaneous as supported by the simultaneous appearance of the three penicillin biosynthetic gene transcripts in P. chrysogenum, at the same time during a fermentation run to ensure the synthesis of penicillin (Renno et al. 1992). Previous reports have shown promoters of different strengths found upstream of penicillin biosynthesis genes, in P. chrysogenum. It was shown that penDE has a lower level of expression than pcbC and a threefold-higher level of expression than pcbAB due to differing promoter strengths (Feng et al. 1994). Penicillin production is also subject to pH regulation mediated by the transcriptional factor, PacC, which acts as a wide-domain regulator. PacC binds to the intergenic region between the pcbAB and pcbC genes and enhances transcription of the pcbC gene (Hillenga et al. 1995). There are various other factors such as dissolved oxygen (Rollins et al. 1991), carbon concentration (Kozma et al. 1993) and nitrogen concentration (Feng et al. 1994) that regulate penicillin biosynthesis at several stages, for instance activation of side chain precursor synthesis, transcription of penicillin biosynthesis genes and post-transcriptional regulation of penicillin biosynthesis genes (Brakhage 1998).
Gang et al. (2001) showed through Northern analysis and reporter gene studies that the transcript levels of the three penicillin biosynthetic genes correlate well with increases in production of penicillin in cultures supplemented with alginate and OM as single elicitors at a concentration of 1 g l−1. However, both these techniques have their limitations. Northern blotting is a semi-quantitative method with results based on band intensity (Aerts et al. 2001; Dean et al. 2002; Vandesompele et al. 2002), while reporter gene analysis is dependent on successful expression of the target enzyme using the designed construct and is an indirect measure of transcriptional activation (Heuvel 1997). In comparison, qPCR is a more precise and reproducible method for measuring levels of specific RNAs (Murphy et al. 2007b). It also allows absolute quantification of the transcript levels.
The major differences in antibiotic yield brought about by the elicitor additions have major economical relevance to the industrial sector. Hence, an in depth understanding of the process is of significant interest. In this study we have carried out, for the first time, accurate quantitative estimation of the effect of two different oligosaccharide elicitors on the transcriptional levels of the penicillin biosynthetic genes, using qPCR. In addition, the effect of multiple and single addition of elicitors was also studied with the view to investigate the potential for further enhancement of penicillin G production.
Materials and methods
Micro-organism, media and culture conditions
Penicillium chrysogenum ATCC 48271 from American Type Culture Collection, Rockville, Maryland, USA was used in this study. Two media compositions were used in this study: Penicillium Growth Medium and Penicillin Production Medium (Nair et al. 2005). Both media were adjusted to pH 6·5 with 2 mol l−1 KOH before sterilization. Calcium carbonate was added as a buffering agent to a final concentration of 10 g l−1 to the shaken flask cultures only. The growth medium for both shaken flask and bioreactor studies were inoculated with spores at a final concentration of 106 spores ml−1. The flasks were maintained at 26°C in a rotary shaking incubator at 200 rpm with a 2 cm throw for 48 h. For penicillin G production, 10% of the seed culture was added to the production medium and maintained at the same conditions as for growth. Phenylacetic acid was added to 24 h old cultures as a precursor for penicillin G synthesis and maintained at 1–1·5 g l−1. Three parallel stirred tank bioreactors (5 l STR, FerMac310/60; Electrolab Ltd, Tewkesbury, UK) with identical parameters were used for bioreactor studies. Sterilization of bioreactors was carried out at 121°C for 40 min while containing only the salt solution of the production medium. Carbon and nitrogen sources were sterilized separately at 115°C for 15 min and FeSO4·7H2O solution was filter sterilized. These were added to the bioreactors aseptically before inoculation with 48 h old inoculum cultures. Control (no oligosaccharide supplemented) and test (oligosaccharide supplemented) fermentations were carried out in parallel using the same inoculum and the same batch of medium. The pH of the fermentations were automatically maintained between 6·45–6·55 using sterile 2 mol l−1 KOH and 2 mol l−1 H2SO4. Dissolved oxygen tension was controlled automatically above 30% air saturation with stirrer speeds ranging 250–600 rev min−1. The changes in the agitation rates for the control and the elicited fermentations were similar. The temperature and airflow-rate were kept at 26°C and l·0 vvm, respectively. Biomass production was measured as cell dry weight per litre of culture broth. The concentrations of penicillin G in culture broth samples were determined by HPLC (Adlard et al. 1991).
Preparation and addition of oligosaccharide elicitors
Two oligosaccharide elicitors were prepared. Mannan oligosaccharides (MO) were prepared from locust bean gum by enzymatic hydrolysis (Ariyo et al. 1997). Oligomannuronate (OM) was prepared from sodium alginate by partial acid hydrolysis (Asilonu et al. 2000). Oligosaccharide elicitors were dissolved in distilled water at the required concentrations and sterilised at 115°C for 15 min. No elicitor was added to the control culture. For single elicitor addition studies mannan oligosaccharide at 150 mg l−1 were added 48 h after inoculation to the production media. For multiple elicitor addition studies, 150 mg l−1 of mannan oligosaccharides at 48 h followed by 75 mg l−1 of oligomannuronate at 96 h were added after inoculation of the production media.
Total RNA was isolated from 0·5 ml of fungal samples using RNeasy Plant Mini Kit (Qiagen, UK). The manufacturer’s protocol was followed for total RNA isolation from fungal cultures. A further on-column DNase digestion with RNase-free DNase (Qiagen) set was used to remove any residual trace of DNA which would be detrimental for further studies including qPCR. The total RNA isolated from samples was stored at −80°C. All samples demonstrated high purity as measured by the A260/A280 ratio, generally yielding ratios around 1·8–2·0. The integrity of the RNA samples was determined by agarose gel electrophoresis run along with the standard RNA markers Riboladder long (Bioline, London, UK).
Complementary DNA synthesis
Total RNA was quantified using A260 measurements using the Eppendorf BioPhotometer and later combined with random hexamer primers. Reverse transcription reactions of up to 1 μg of total RNA was performed in 20 μl reactions comprising components of the ImProm-IITM Reverse Transcription System (Promega, UK). The manufacturer’s protocol was followed for the cDNA synthesis.
The primers for the genes of interest were designed using the Primer3 software (Rozen and Skaletsky 2000) as shown in Table 1. The genes of interest are primarily the three major penicillin biosynthetic genes namely, pcbAB, pcbC and penDE. 18S rRNA was used as an internal control for normalisation in qPCR conditions as it is less likely to vary under conditions that affect the expression of mRNA and always present in cells at reasonably constant levels (Suzuki et al. 2000). Gene quantification was carried out with the ABI Prism 7000 Sequence Detection system (Applied Biosytems) using SYBR Green Jumpstart Taq ReadyMix (2X) (Sigma, UK). All reactions were performed in 25 μl reaction volumes that contained 1X SYBR Green Jumpstart Taq ReadyMix, 1X internal reference dye, specific primers and template cDNA made up to final reaction volume using nuclease free water. ROX was used as a passive reference dye for normalization of reporter signal. The final concentrations of primers and Mg2+ were 0·4 μmol l−1 and 1·5 mmol l−1 respectively. Thermo cycling conditions were as follows: 2 min at 50°C, 10 min at 95°C and 40 cycles of 15 s at 95°C and 1 min at 60°C. In all experiments, appropriate negative controls containing no template DNA (negative control), or total-RNA (RT-negative control) were subjected to the same procedure as the test to exclude or detect any possible contamination or carryover of genomic DNA respectively (Semighini et al. 2002). For each of the RNA extractions, the samples were diluted, measurements of gene expression were obtained in triplicate, and the mean of these values was used for further analysis. All reaction mixtures were analysed using 3·5% agarose gel electrophoresis using Hyper ladder V (Bioline) as the DNA marker to confirm that only one PCR product was synthesised per sample. The threshold cycle, Ct, is defined as the first amplification cycle at which fluorescence indicating PCR products becomes detectable. In real-time PCR analysis, quantification is based on the threshold cycle, which is inversely proportional to the logarithm of the initial copy number. The number of target copies contained in an unknown sample was determined by extrapolation from the linear regression of the standard curve obtained for each gene. A calibration curve (transcript copy number versus Ct) was plotted using serial dilution of each gene of interest extracted from the gel after PCR using the QIAquick gel extraction kit (Qiagen). The standard curve produces a linear relationship between Ct and initial amounts of total RNA or cDNA allowing the determination of the concentration of unknowns based on their Ct values (Bustin 2000). The copy numbers of the mRNA corresponding to the gene of interest were calculated using the following equation assuming that each genome had only one gene of interest and that molecular weight of 1 bp was 660 g mol−1: number of copies per microlitre = (6 × 1023)(DNA concentration)/molecular weight of one genome), where 6 × 1023 is the number of copies per mole, the DNA concentration is given in grams per microlitre, and the molecular weight of one genome is given in grams per mole (Ritalahti et al. 2006). The technique was optimized by using triplicate reactions and running independently diluted standard curves repeatedly under identical conditions. To enumerate gene copy numbers of unknown samples, one calibration curve was routinely run with each sample set and compared with previous curves to check for consistency between the runs. Standard calibration curves for the genes pcbAB, pcbC and penDE are shown in Fig. 1a–c, respectively. The correlation coefficients, slopes and efficiencies of the standard curves are presented in Table 2. An ABI-Prism 7000 melting curve analysis was performed for all the reactions and each reaction resulted in single product-specific melting curve.
Table 1. Primers used in qPCR study of penicillin biosynthetic and 18S rRNA genes
Table 2. Correlation coefficient (R2), slope and efficiency of standard curves obtained from 10-fold serial dilutions of the genes of interest from P. chrysogenum ATCC 48271
*Efficiency was estimated by the formula E = (10−1/slope) −1.
All statistical analyses were performed using Graph Pad InStat 3 software (Graph Pad Software, San Diego, CA, USA). Pair-wise multiple comparisons between the studied conditions were made using one-way analysis of variance (anova) followed by Tukey–Kramer multiple comparison test. P values <0·05 were considered significant. All the Ct values are the averages of at least three repetitions.
Earlier studies on fungal elicitation have reported that single elicitor addition resulted in an increase in penicillin G production. Higher levels of penicillin G production were observed in P. chrysogenum cultures supplemented with 150 mg l−1 of mannan oligosaccharides at 48 h (Ariyo et al. 1998). Optimisation studies on multiple elicitor addition demonstrated that highest elicitation in penicillin G production was obtained by adding 150 mg l−1 of MO at 48 h followed by 75 mg l−1 of OM at 96 h (Nair et al. 2008). Based on the initial studies, systematic sets of experiments were carried out simultaneously in shaken flasks and bioreactors to investigate the effect of single and multiple elicitors at the transcriptional level using real-time PCR.
Parallel runs (shaken flasks and bioreactors) were carried out and similar observations were made. In the shaken flask runs the rate of penicillin G production was 133% higher in single elicitor added cultures compared to control cultures and an increase of 71% was attained in the multiple elicitor added culture compared to the single addition cultures. The variation in transcriptional level in the shaken flask runs was similar to that observed in bioreactor studies with an increase of 216%, 61% and 200% in the pcbAB, pcbC and penDE transcript levels for single elicitor added cultures over the control cultures. Addition of the second elicitor resulted in an increase of 116%, 39% and 33% in pcbAB, pcbC and penDE transcript levels compared to the single elicitor cultures (data not shown).
5 l Stirred Tank Reactor (STR) studies
Penicillin G analysis
Addition of single and multiple elicitors resulted in an increase in the penicillin G concentration over the control fermentation to which no elicitor was added (Fig. 2). The highest penicillin G concentrations for the control, single and multiple elicitor addition were 0·85, 1·57 and 1·92 g l−1 respectively (P < 0·001). The rate of penicillin G production was 100% (20 mg l−1 h−1) higher in the single elicitor added culture compared to the control cultures (10 mg l−1 h−1). In multiple elicitors added cultures the rate of penicillin g production (25 mg l−1 h−1) was 150% and 25% higher compared to the control and single elicitor added cultures respectively. All analyses for these results were carried out in triplicate and the overall difference between the control and the elicited cultures was statistically significant (P < 0·05).
The variation in transcriptional levels in the 5 l STR is shown in Fig. 3. The highest levels of pcbAB and pcbC transcripts were detected at 48 h in control cultures, while accumulation of penDE was maximal at 24 h (Fig. 3a–c). A gradual decline in transcript levels was observed from 48 h to 96 h. The treatment of P. chrysogenum cultures with MO caused differential penicillin biosynthetic gene transcript levels. In the elicited cultures the highest transcriptional level of pcbAB and pcbC genes were observed at 96 h and 72 h respectively. For the pcbAB gene, the transcript copy number at 96 h in cultures supplemented with MO was 25 × 106 compared to 6 × 106 in the control cultures, an increase of 316% (P < 0·001). An increase of 183% and 43% in the pcbC and penDE transcript levels was detected at 72 h (P < 0·001). In contrast to the rapid increases in the transcriptional levels after the addition of MO, the supplementation of the second elicitor (OM) caused a slower, but more prolonged stimulation of the gene transcript levels. Percentage increases in pcbAB, pcbC and penDE transcript levels of 44%, 83% and 50% respectively were observed after the second elicitor (OM) treatment.
Although significant changes were observed in the transcript copy number of the penicillin biosynthetic genes, the transcription of the 18S rRNA was maintained at the same level for all the tested cultures (data not shown). This confirms the specific effect of the elicitors on the transcriptional levels of the penicillin biosynthetic genes.
The single addition of alginate and locust bean gum-derived oligosaccharides has been found to increase the production of penicillin G and its synthetic intermediates (Ariyo et al. 1997; Tamerler et al. 2001). The most potent of these elicitors has been MO derived from locust bean gum which has shown considerable increase (66%) in the penicillin G concentration on single addition (Radman et al. 2004).
However repeated addition of the same elicitor at different concentrations and addition times in P. chrysogenum has failed to show any significant increase in the penicillin G production (Iturbe 2005; Nair et al. 2008). This correlates with the hypothesis observed in plant cells where various specific perception systems for signalling molecules have been described to show a refractory effect when treated with the same elicitor for a second time (Felix et al. 1993). Investigation of this phenomenon in Lycopersicon esculentum established that repeated addition of chitin fragments causes desensitization of cells in time and concentration-dependent manner, although they still reacted to further addition of xylanase (Felix et al. 1998) or ergosterol (Granado et al. 1995). Reciprocally, when cells were treated with ergosterol, they were refractory to further stimulation with ergosterol but still responded to chitin fragments and xylanase (Felix et al. 1998). Desensitization was not associated with increased inactivation of the stimulus or with a disappearance of receptor binding sites and thus appears to be caused by an intermediate step in signal transduction. Hence, in this work, for the first time, multiple additions of two different elicitors were studied in the context of penicillin G production. This resulted in a further significant increase of 25% in penicillin G production in the bioreactor culture. A similar observation has been made in bacterial (Bacillus licheniformis) culture for production of bacitracin where an increase of 23·3% on single addition of oligoguluronate and an increase of 13·2% and 36·5% on multiple elicitor addition of oligoguluronate and MO compared to single elicitor addition and control respectively was reported (Murphy et al. 2007a). This observation is in agreement with the desensitisation theory. It is plausible that the addition of a second different elicitor, with a completely different structure, has been perceived by a different set of receptors. This interaction would then stimulate a different signal pathway and result in further stimulation of the penicillin G production.
The absolute quantification of the transcript copy numbers carried out using the qPCR analysis shows a direct correlation between the increase in penicillin G concentration and the levels of mRNA concentration for the penicillin biosynthetic genes. The levels of the pcbAB, pcbC and penDE transcript levels reach maximum levels at time points before the maximum level of penicillin G production is achieved. Also, each elicitor addition has resulted in a simultaneous increase in transcript number of the three biosynthetic genes, although to varying extents, ranging between 33% and 316%. This absolute quantitative estimation of transcript levels has therefore shown precisely that the addition of elicitors affects the transcriptional levels of the penicillin G biosynthetic genes. Hence, the ultimate mode of action of the elicitors is by means of an increase in the transcriptional levels of the genes in the biosynthetic pathway. This result is in agreement with the initial results of single elicitor addition obtained by Gang et al. (2001). The increase in levels of 216%, 61% and 200% observed in the absolute concentration of the transcript copy numbers of the pcbAB, pcbC and penDE in this work were much higher compared to 40%, 61% and 50% than those reported by Gang and co-workers respectively. Since qPCR is a highly accurate method for transcript copy number quantification, the higher numbers are true representations of the actual changes within the cell as compared to the semi-quantitative methods used in the previous analysis. However, this could also be due to different strains of Penicillium chrysogenum, different media compositions and different elicitors being used by the two laboratories.
There could be a number of reasons why the oligosaccharides elicitors instigate the overproduction of penicillin G in P. chrysogenum. The induced synthesis of penicillin G by elicitors suggests that these elicitors may function as activators of a stress signal or internal defence mechanism in this system. van der Holst et al. (2001) presented a compelling argument for a receptor-based oligosaccharide induced mechanism for elicitation. This work is based on a hypothetical model for elicitation in plant cells where oligosaccharides are perceived by membrane-bound receptors. Recognition of elicitation stimulus leads to the activation of specific or early response genes (Radman et al. 2003). Mitogen activated protein kinase (MAPK) cascades are major downstream components of receptor/sensors that transduce external signals into intracellular responses in all eukaryotes. It has been found in suspension cultures of Rubia tinctorum, on the addition of chitosan, signalling is mediated by translocation of MAPK to the nucleus resulting in the stimulation of anthraquinone synthesis. The increase in anthraquinone levels is preceded by an increase in the isochorismate synthase transcript and activity levels (van Tegelen et al. 1999). The MAPK cascade activates the transcription of MAPK-dependent genes and is also involved in induction at transcription, translational or post-translational levels (Kultz and Burg 1998). Another possible mechanism of elicitor function could involve the Toll-like or Nod-like receptors (TLR and NLR) similar to the innate immune system related recognition involved in most multicellular organisms (Jault et al. 2004; Proell et al. 2008). Pathogen binding to these receptors is known to trigger a complex signal transduction pathway leading to the activation of an inflammatory response. Sequences similar to the TLR and NLR were searched for, within the fungal domain. Although, some sequence similarities were obtained, no significant matches were detected. This indicates that elicitors are possibly recognised by receptors different from the Toll and NOD family. Comprehensive accumulation of data on the cell signaling after the addition of elicitors is required to reveal details of the mechanism of elicitation in fungal cultures.
In conclusion, this work, for the first time, has used absolute quantification of transcript copy numbers to establish the transcriptional mode of action of oligosaccharide elicitors, in the context of penicillin G production by Penicillium chrysogenum. This study has also shown that multiple additions of two different elicitors can lead to a further significant increase in penicillin G production, with the mode of action of the second elicitor also being at the transcriptional level. The penicillin production observed in this work compliments many years of laborious strain improvement and media optimization procedures and hence can potentially have significant economical impact. Also, although the effect of oligosaccharide elicitors might not be the same in all fungal cultures, the work carried out so far (Shin et al. 1998; Petruccioli et al. 1999; Nair et al. 2005) suggests serious potential for the use of elicitors for enhanced production of a range of economically important fungal products.
The authors wish to acknowledge Dr Sanjiv Rughooputh for his suggestions on real-time PCR.