An Engineered Yeast Efficiently Secreting Penicillin

Abstract

This 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.

Biosynthesis of the Antibiotic Nonribosomal Peptide Penicillin in Baker’s Yeast

“…We observed that this always required strong expression of the peroxisomally-located enzyme pclA. As engineered heterologous biosynthesis of penicillin has only previously been observed with Hansenula polymorpha, a methylotrophic yeast with large peroxisomes 20 , this suggests that boosting peroxisomal expression further, for example by increasing peroxisome formation, by may be a future approach to improve yields from S. cerevisiae. Further yield improvements could also be achieved using other metabolic engineering strategies 21,22 .…”

Peroxisomes Are Required for Efficient Penicillin Biosynthesis in Penicillium chrysogenum

“…Peaks were separated on a ShimPack XR-ODS 3.0-mm-internal-diameter by 75-mm column (Shimadzu) at a flow rate of 0.5 ml/min and detected at a wavelength of 254 nm. Intracellular ␤-lactam levels were determined by using mass spectrometry (5).…”

Exploring antibiotic biosynthesis: Leo Vining’s insights lead to new strategies in the quest for ‘The 10 × '20 Initiative’

“…Although this approach sounds very attractive on the surface, a significant investment in developing a suite of strains that have been modified to generate common precursors is essential. Heterologous hosts such as S. coelicolor, 25,26 S. lividans, 27 Streptomyces avermitilis, 28 Escherichia coli 29 and Hansenula polymorpha 30 have been used as heterologous hosts for the production of antibiotics. Expression of antibiotic biosynthesis pathways in E. coli 29 and yeasts 30 have resulted in very low yields, indicating that heterologous hosts need further manipulation in order to enhance their capability to supply precursors.…”

“…Heterologous hosts such as S. coelicolor, 25,26 S. lividans, 27 Streptomyces avermitilis, 28 Escherichia coli 29 and Hansenula polymorpha 30 have been used as heterologous hosts for the production of antibiotics. Expression of antibiotic biosynthesis pathways in E. coli 29 and yeasts 30 have resulted in very low yields, indicating that heterologous hosts need further manipulation in order to enhance their capability to supply precursors. Lee et al 26 observed that aloesaponarin II production was detected only when the regulatory gene actII-ORF4 was present in the S. coelicolor host and the production level was higher in a strain, in which the actinorhodin gene cluster was deleted.…”