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.