| Penicillin is one of most important broad-spectrum(?lactam antibiotics and its intermediate,6APA,can be used as the precursor for the production of ampicillin and amoxicillin.Penicillium chrysogenum is the main industrial penicillin producer.Although extensive research has been made on strain improvement,medium optimization and process modeling,few study has ever gathered information regarding shear stress gradients caused by mechanical agitation and substrate gradients caused by fresh medium feeding in a large-scale fermentor.which retards further increase of the Titer.Rate and Yield(T.R.Y)of the penicillin production.To investigate this,based on the simulation results of a 54 m3 industrial-scale penicillin fermentor,we cost-effectively explored the physiological response to industrial-scale fluid conditions simulated in laboratory-scale scale-down simulators.Firstly,we customized a fast sampling device,which can satisfy rapid sampling within one second,and established a well-defined protocol for acquisition of both intra-and extra-cellular metabolome.U-13C-labeled cell extracts were obtained from a P.chrysogenum fed-batch culture,grown on 100%U-13C-labeled glucose and ?,?-13C2-phenylacetic acid.Isotope dilution mass spectrometry(IDMS)-based analytical procedures were further established.Secondly,dynamic feeding strategies(Pulse,Ramp and Oscillation,PRO)were employed for fast physiological characterization of a high-producing P.chrysogenum strain in response to the perturbed conditions.At the same time,intracellular metabolome was acquired for the comparison with the predictions by a 9-pool metabolic structured model.These metabolomics data can in future be an indispensable input for model extension and/or structure optimization.Thirdly,by performing chemostat experiments at 400 RPM and 600 RPM,two typical power inputs representative of industrial penicillin fermentation(P/V,1.00 kW/m3 in more remote zones and 3.83 kW/m3 in the vicinity of the impellers,respectively)were scaled-down to bench-scale bioreactors.It was found that at 400 RPM applied in prolonged glucose-limited chemostat cultures,the previously reported degeneration of penicillin production using an industrial Penicillium chrysogeum strain was virtually absent.To investigate this,the cellular response was studied at flux(stoichiometry),residual glucose,intracellular metabolite and transcript levels.At 600 RPM,20%more cell lysis was observed and the increased degeneration of penicillin production was accompanied by a 22%larger ATP gap and an unexpected 20-fold decrease in the residual glucose concentration(Cs,out).At the same time,the biomass specific glucose consumption rate(qs)did not change but the intracellular glucose concentration was about 6-fold higher,which indicates a change to a higher affinity glucose transporter at 600 RPM.In addition,power input differences cause differences in the diffusion rates of glucose and the calculated Batchelor diffusion length scale suggests the presence of a glucose diffusion layer at the glucose transporting parts of the hyphae,which was further substantiated by a simple proposed glucose diffusion-uptake model.By analysis of calculated mass action ratios(MARs)and energy consumption,it indicated that at 600 RPM glucose sensing and signal transduction in response to the low Cs,out appear to trigger a gluconeogenic type of metabolic flux rearrangement,a futile cycle through the pentose phosphate pathway(PPP)and a declining redox state of the cytosol.In support of the change in glucose transport and degeneration of penicillin production at 600 RPM.the transcript levels of the putative high-affinity glucose/hexose transporter genes Pc12g()2880 and Pc06g01340 increased 3.5 and 3.3-fold,respectively,and those of the pcbC gene encoding isopenicillin N-synthetase(IPNS)were more than 2-fold lower in the time range of 100 h to 200 h of the chemostat cultures.Summarizing,changes at power input have unexpected effects on degeneration and glucose transport,and result in significant metabolic rearrangements,These findings are highly relevant for further investigation of the industrial production of penicillin.Fourthly,by using two different scale-down simulators,industrial-scale substrate gradients were mimicked at lab-scale continuous cultures through a)imposing an intermittent feeding regime(IFR)in a single reactor and b)applying a two-compartment reactor(TCR)with continuous feeding in one compartment and broth recirculation between the two compartments.The IFR is applied to simulate substrate dynamics experienced by the cells at full-scale at timescales of tens of seconds to minutes(30 s,3 min and 6 min),while the TCR is designed to simulate substrate gradients at an applied mean residence time(?c)of 6min.The performance of these simulators was compared to the conventional chemostat.A biological systems analysis(flux,metabolites and mRNA)of the response of an industrial high-yielding P.chrysodgenum strain has been performed in these continuous cultures.In the IFRs,the highest substrate feast levels were 30.167 and 333 ?M with feeding cycles of 30 s,3 min and 6 min,respectively.About halfway during the 3min and 6min feeding cycles the culture shifted from a feast to a famine regime,showing about 50%feast and 50%famine and hardly any limitation regime.which deviated significantly from the simulation result at the 54 m3 full-scale penicillin fermentor.In the 30 s cycle,however,starvation and feast regimes were absent and 100%limitation regime was observed.In the TCR system with the ?e=6 min,the average residual Cs was nearly 3-fold higher in the feed compartment(57 ?M)than in the non-feed compartment(19 ?M).This showed that both compartments were in the limitation regime,although the full-scale simulated total broth average Cs value(Cs,avg ? 34.4 ?M)was well in between the values in the TCR system.The results showed that compared to an undisturbed continuous feeding regime in a single reactor,the specific penicillin production rate(qPenG)became 10%,2-fold,and 2.6-fold lower in the 30 s,3 min and 6 min IFR chemostat cultures,respectively.In the systems analysis of the IFRs.dynamic metabolomics data showed that in the IFRs.this high-yielding P.chryogenum strain used the surplus of carbon during the feast phase to accumulate high levels of the central metabolites,rather than in carbohydrate storage pools,and consumed these accumulated metabolites during the famine phase to cope with external substrate deprivation.Striking was that qPenG in the TCR system at ?c=6 min showed resemblance to the 3 min IFR,although the metabolic regimes were very different(in IFR 50/50 famine/feast,in the TCR 100%limitation).In contrast,the metabolomics data in the TCR system indicated that besides the metabolite carbon pool,the storage pool(e.g.,mannitol and arabitol)may have a large contribution of carbon supply in the non-feed compartment.Further,transcript analysis revealed that the IFR and the TCR systems gave different expression levels of the glucose transporter genes and the penicillin gene clusters.The results showed that qPenG did not correlate well with exposure to the substrate regimes(excess,limitation and starvation),but there was a clear inverse relation between qPenG and the intracellular glucose level(Cs,in).Finally,analysis of quantitative metabolomics data showed that the penicillin production capacity was significantly impaired in the presence of high power input and substrate gradient conditions,while,the intracellular amount of trehalose was significantly increased.In order to further investigate the actual relation between the presence of the trehalose biosynthesis and the penicillin production,we used Agrobacterium-Mediated Transformation method to construct two knock-out strains,i.e.,P.chrysogenum Atpsl and P.chrysogenum Atps2,and explored the effect of these knock-outs on cell growth,sporulation process and penicillin production.The results,unexpectedly,showed that these knock-outs did not benefit penicillin production and sporulation,and also influenced the kinetics of glucose uptake and the amounts of intracellular metabolites. |
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