Regulation, transport aspects and degeneration of penicillin biosynthesis in Penicillium chrysogenum

Penicillin has been produced on an industrial scale for several decades. The improvements in its production process, in terms of product yields and production rates, present an unprecedented success in fermentation technology. However, the obtained product yields still remain far from their theoretical maximum. More insight in the regulation of the penicillin biosynthesis pathway and connected central metabolic pathways, as well as in the mechanisms of penicillin and side chain precursor transport, which are still incompletely understood, could provide leads for further improvement. As has been observed for other high producing organisms, industrial Penicillium chrysogenum strains appear to loose their high productivity in extended fermentations (degeneration), making the implementation of a continuous fermentation process impossible. The recent sequencing of its genome and advancements in analytical techniques enable researchers to get more insights in these issues using a systems biology approach, which is the topic of this thesis. In general there is a relation between the rate of product formation of a micro organism and its growth rate under substrate limiting conditions. In case of catabolic products, that is, compounds which are an end product of a catabolic pathway, e.g. alcohol as an end product of the fermentation of sugars, the rate of product formation is proportional to the growth rate. If product formation is not coupled to catabolism, any relation between growth and product formation may exist. Such a non-linear relation has also been determined for penicillin production in P. chrysogenum. However, if such a relation is determined under certain specific cultivation conditions, e.g. in steady state chemostat cultures, it does not automatically hold under dynamical (non steady state) conditions, e.g. in a fed-batch cultivation were the growth rate changes in time. To be able to describe the relation between growth and penicillin production under different (steady state as well as dynamic) conditions a mathematical model is required in which the genetic regulation of enzymes levels of the penicillin biosynthesis pathway is taken into account. In Chapter 2 an analysis is performed of enzyme activities in the penicillin production pathway at different growth rates which indicated that IPNS has a rate-limiting role for penicillin production. A model based on the regulation of the gene encoding for such a rate-limiting enzyme in the penicillin pathway, describing the dynamics of penicillin production from gene to flux, was developed and showed a significantly improved description of the specific production rate during steady state and dynamic phases of penicillin fermentations. In general, not only enzyme levels but also intracellular penicillin pathway metabolites and transport steps can control productivity. To study these aspects an accurate method is needed to measure intracellular metabolite levels. The traditional cold methanol quenching method with subsequent washing by centrifugation was found to be inappropriate to measure intracellular penicillin and phenylacetic acid levels, because their extracellular amount was too high. In chapter 3 the development of a new sample quenching and filtration based washing method for quantification of intracellular metabolites was described. This method was found to have a superior washing efficiency compared with the standard centrifugation based washing method, making it possible to measure intracellular levels of metabolites which are extracellularly abundant. Such measurements are especially useful in transport studies. The method was validated by successfully measuring the intracellular levels of metabolites related to penicillin biosynthesis, including the transported PAA and PenG metabolites. Chapter 4 describes the successful application of the method developed in chapter 3, in a study on transport mechanisms and transport kinetics of phenylacetic acids (PAA) and penicillin-G (PenG) in P. chrysogenum. PAA was found to be taken up rapidly by passive diffusion and simultaneously exported by an energy consuming ABC transporter. The PenG anion was found to be reversibly transported over the cell membrane by a facilitated transporter, driven by the negative electrochemical potential difference. The estimate capacity of the PenG transporter was found to be larger than the penicillin flux, but not much larger. It has been observed that upon prolonged cultivation, P. chrysogenum gradually looses its capacity to produce penicillin. This phenomenon, called degeneration, was studied in ethanol limited chemostats at a systems level (from gene to flux), of which the results are presented in chapter 5. Degeneration was found to be a reproducible phenomenon leading to a 10-fold reduction in the biomass specific penicillin production rate after about 30 generations of growth in chemostat culture. No indications were found that the observed massive decrease in penicillin production was caused by a decrease in the number of copies of the penicillin gene cluster, a decrease in the number of peroxisomes (in which part of the penicillin pathway is located) or changes in metabolite levels in central metabolism. The expression levels of genes related to sulfur and nitrogen metabolism decreased significantly during degeneration, which corresponds with the decreased demand for the precursor amino acids cysteine and valine. Also energy charge and changed concentrations of penicillin pathway precursors (valine, cysteine, ?-aminoadipic acid and PAA) could be ruled out as causes for degeneration. In contrast, the enzyme amounts of IPNS and ACVS and the transport PenG capacity decreased significantly and the IPNS amount and PenG export capacity correlated well with the decrease in the specific penicillin production rate, which is in agreement with the results from chapter 2. This indicates that degeneration is due to decreased amount of penicillin pathway related protein levels (enzymes, transporters). The reason of this decrease is most probably a changed regulation (translation efficiency, post-translational modification efficiency) and/or a higher protein degradation rate of these penicillin pathway enzymes.BiotechnologyApplied Science

Optimization of cold methanol quenching for quantitative metabolomics of Penicillium chrysogenum

A sampling procedure for quantitative metabolomics in Penicillium chrysogenum based on cold aqueous methanol quenching was re-evaluated and optimized to reduce metabolite leakage during sample treatment. The optimization study included amino acids and intermediates of the glycolysis and the TCA-cycle. Metabolite leakage was found to be minimal for a methanol content of the quenching solution (QS) of 40% (v/v) while keeping the temperature of the quenched sample near -20º C. The average metabolite recovery under these conditions was 95.7% (±1.1%). Several observations support the hypothesis that metabolite leakage from quenched mycelia of P. chrysogenum occurs by diffusion over the cell membrane. First, a prolonged contact time between mycelia and the QS lead to a somewhat higher extent of leakage. Second, when suboptimal quenching liquids were used, increased metabolite leakage was found to be correlated with lower molecular weight and with lower absolute net charge. The finding that lowering the methanol content of the quenching liquid reduces metabolite leakage in P. chrysogenum contrasts with recently published quenching studies for two other eukaryotic micro-organisms. This demonstrates that it is necessary to validate and, if needed, optimize the quenching conditions for each particular micro-organism.BT/BiotechnologyApplied Science

Ultrasonic Imaging of the Onset and Growth of Fractures Within Partially Saturated Whitby Mudstone Using Coda Wave Decorrelation Inversion

Using active ultrasonic source survey data, coda wave decorrelation (CWD) time-lapse imaging during the triaxial compression of Whitby Mudstone cores provides a 3-D description of the evolution and redistribution of inelastic strain concentrations. Acoustic emissions (AEs) monitoring is also performed between any two consecutive surveys. From these data, we investigate the impact of initial water saturation Sw on the onset, growth, and reactivation of inelastic deformation, compared to the postdeformation fracture network extracted from X-ray tomography scans. Our results indicate for the applied strain rate and degree of initial water saturation, and within the frequency range of our ultrasonic transducers (0.1 to 1 MHz), that inelastic strain localization and propagation in the Whitby Mudstone does not radiate AEs of sufficient magnitude to be detected above the average noise level. This is true for both the initial onset of inelasticity (strain localization) and during macroscopic failure. In contrast, the CWD results indicate the onset of what is interpreted as localized regions of inelastic strain at less than 50% of the peak differential stress the Whitby Mudstone can sustain. The seemingly aseismic nature of these clay-rich rocks suggests the gradual development of inelastic strain, from the microscopic diffuse damage, up until the macroscopic shear failure.Applied Geophysics and Petrophysic

An Engineered Yeast Efficiently Secreting Penicillin

BiotechnologyApplied SciencesThis study aimed at developing an alternative host for the production of penicillin (PEN). As yet, the industrial production of this β-lactam antibiotic is confined to the filamentous fungus Penicillium chrysogenum. As such, the yeast Hansenula polymorpha, a recognized producer of pharmaceuticals, represents an attractive alternative. Introduction of the P. chrysogenum gene encoding the non-ribosomal peptide synthetase (NRPS) δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) in H. polymorpha, resulted in the production of active ACVS enzyme, when co-expressed with the Bacillus subtilis sfp gene encoding a phosphopantetheinyl transferase that activated ACVS. This represents the first example of the functional expression of a non-ribosomal peptide synthetase in yeast. Co-expression with the P. chrysogenum genes encoding the cytosolic enzyme isopenicillin N synthase as well as the two peroxisomal enzymes isopenicillin N acyl transferase (IAT) and phenylacetyl CoA ligase (PCL) resulted in production of biologically active PEN, which was efficiently secreted. The amount of secreted PEN was similar to that produced by the original P. chrysogenum NRRL1951 strain (approx. 1 mg/L). PEN production was decreased over two-fold in a yeast strain lacking peroxisomes, indicating that the peroxisomal localization of IAT and PCL is important for efficient PEN production. The breakthroughs of this work enable exploration of new yeast-based cell factories for the production of (novel) β-lactam antibiotics as well as other natural and semi-synthetic peptides (e.g. immunosuppressive and cytostatic agents), whose production involves NRPS's

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

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-?-(?-aminoadipyl)-L-?-cystenyl-D-?-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.BT/BiotechnologyApplied Science