1 Department of Systems Biology, Technical University of Denmark2 Fermentation Platform, Department of Systems Biology, Technical University of Denmark3 Fungal Physiology and Biotechnology, Department of Systems Biology, Technical University of Denmark4 Eucaryotic Molecular Cell Biology, Department of Systems Biology, Technical University of Denmark5 Metabolic Signaling and Regulation, Department of Systems Biology, Technical University of Denmark6 Eukaryotic Molecular Cell Biology, Department of Biotechnology and Biomedicine, Technical University of Denmark7 Department of Chemical and Biochemical Engineering, Technical University of Denmark
Natural products constitute one of the largest sources of therapeutics known to mankind. Among the natural products polyketides such as erythromycin (antibiotic) and lovastatin (cholesterol lowering) have long proven their immense value to patients around the world. Polyketides are naturally produced by plants, fungi and bacteria. However, the natural producers often do not achieve commercial titers of the polyketide therapeutic. Thus the natural production must be improved. This can be done by random mutagenesis or heterologous expression of the polyketide gene cluster resulting in production sufficient titers. To improve the production of polyketides biological engineering principles have been applied for the development and engineering of microbial polyketide cell factories. The two biological hosts used for heterologous polyketide production were Aspergillus nidulans and Saccharomyces cerevisiae. Both organisms have well-known genetic tools available for gene targeting and heterologous expression. It has been the aim to create a stable expression platform with all genes integrated in the genome. This has been achieved through the use of two advanced genetic engineering systems for A. nidulans and S. cerevisiae. Both systems have been aided by USER™ cloning vectors that were developed for efficiently generating large amounts of gene targeting substrate. Upon integration the targets should lead to high expression of the polyketide synthase (PKS) as well as the activating phosphopantetheinylase (PPTase). This versatile vector system can easily be used for expression of other polyketides of interest as well as extended to express whole gene clusters. After achieving proof of principle in terms of expression, the polyketide cell factory must be optimized. The optimization can be achieved through the use of adaptive evolution, random mutagenesis and screening as well as metabolic engineering. Firstly, in silico guided metabolic engineering was used as a tool to direct metabolism towards higher levels of 6-MSA production in A. nidulans. 6-MSA was stably expressed in the A. nidulans genome and bioreactor cultivations resulted in high titers of 6-MSA. The genome scale model of A. nidulans and the optimization algorithm OptGene was used to predict a knockout strategy designed to increase the production of 6-MSA in A. nidulans. Among the predicted targets deletion of the NADPH dependent glutamate dehydrogenase (gdhA) was selected as it should result in greater availability of NADPH for polyketide production. The deletion resulted in decreased growth rate of A. nidulans, which was partially rescued by the insertion of an extra copy of the NADH dependent glutamate dehydrogenase (gdhB). Physiological characterization in bioreactors revealed that the yields of 6-MSA on biomass increased albeit not significantly. As a result of this it may be argued that there is still more work to be done in terms of model building in A. nidulans. Utilizing another well-established cell factory S. cerevisiae the capabilities of a novel gene amplification system was demonstrated. The system was aimed at creating up to ten copies of a gene integrated in specific targeting sites of the S. cerevisiae genome. First, large amounts of gene targeting substrates were generated through the construction of a USER® vector. Through the use of one, two and four copy amplification strains the stable production of 6-MSA was established. The 10.5 kb fragment of genes was successfully amplified. The constructed strains were evaluated in Erlenmeyer flasks. The results showed that the copy number of the genes and the 6-MSA titer correlated well. This indicates that even more copies of the genes for 6-MSA production could yield even higher titers. Thus the acyl-CoA substrates do not appear to be limiting the production of 6-MSA. Construction of a cell factory and engineering it to increase production is one approach to obtaining an efficient cell factory. To aid the strain development further, it was sought to demonstrate the usefulness of a microtiter plate based cultivation system that uses CCDflatbed scanners and image analysis as a tool to follow microbial growth and product formation. This CCD-flatbed scanning platform can be used for both process optimization as well as screening libraries of mutants generated through random mutagenesis. The experiments validated the CDD-flatbed scanning platform as a tool for quantifying microbial biomass from both bacteria and yeasts. Furthermore, the platform can be used to detect onset of production as well as volumetric productivities of the colored polyketide actinorhodin in Streptomyces coelicolor. It is a system that can be used in industrial settings for optimizing cell factory conditions. The use of microtiter plates makes it high-throughput and inexpensive method. Thus in conclusion significant steps have been taken towards engineering an effective polyketide cell factory.