Optimization of a multi-stage, multi-subunit malaria vaccine candidate for the production in Pichia pastoris by the identification and removal of protease cleavage sites.

We demonstrated the successful optimization of a recombinant multi-subunit malaria vaccine candidate protein for production in the methylotrophic yeast Pichia pastoris by the identification and subsequent removal of two protease cleavage sites. After observing protein degradation in the culture supernatant of a fed-batch fermentation, the predominant proteolytic fragment of the secreted recombinant protein was analyzed by mass spectrometry. The MS data indicated the cleavage of an amino acid sequence matching the yeast KEX2-protease consensus motif EKRE. The cleavage in this region was completely abolished by the deletion of the EKRE motif in a modified variant. This modified variant was produced, purified, and used for immunization of rabbits, inducing high antigen specific antibody titers (2 × 10(6) ). Total IgG from rabbit immune sera recognized different stages of Plasmodium falciparum parasites in immunofluorescence assays, indicating native folding of the vaccine candidate. However, the modified variant was still degraded, albeit into different fragments. Further analysis by mass spectrometry and N-terminal sequencing revealed a second cleavage site downstream of the motif PEVK. We therefore removed a 17-amino-acid stretch including the PEVK motif, resulting in the subsequent production of the full-length recombinant vaccine candidate protein without significant degradation, with a yield of 53 mg per liter culture volume. We clearly demonstrate that the proteolytic degradation of recombinant proteins by endogenous P. pastoris proteases can be prevented by the identification and removal of such cleavage sites. This strategy is particularly relevant for the production of recombinant subunit vaccines, where product yield and stability play a more important role than for the production of a stringently-defined native sequence which is necessary for most therapeutic molecules.

Development of carbon plasma-coated multiwell plates for high-throughput mass spectrometric analysis of highly lipophilic fermentation products.

Classical approaches to strain improvement and metabolic engineering rely on rapid qualitative and quantitative analyses of the metabolites of interest. As an analytical tool, mass spectrometry (MS) has proven to be efficient and nearly universally applicable for timely screening of metabolites. Furthermore, gas chromatography (GC)/MS- and liquid chromatography (LC)/MS-based metabolite screens can often be adapted to high-throughput formats. We recently engineered a Saccharomyces cerevisiae strain to produce taxa-4(5),11(12)-diene, the first pathway-committing biosynthetic intermediate for the anticancer drug Taxol, through the heterologous and homologous expression of several genes related to isoprenoid biosynthesis. To date, GC/MS- and LC/MS-based high-throughput methods have been inherently difficult to adapt to the screening of isoprenoid-producing microbial strains due to the need for extensive sample preparation of these often highly lipophilic compounds. In the current work, we examined different approaches to the high-throughput analysis of taxa-4(5),11(12)-diene biosynthesizing yeast strains in a 96-deep-well format. Carbon plasma coating of standard 96-deep-well polypropylene plates allowed us to circumvent the inherent solvent instability of commonly used deep-well plates. In addition, efficient adsorption of the target isoprenoid product by the coated plates allowed rapid and simple qualitative and quantitative analyses of the individual cultures.

Metabolic engineering of taxadiene biosynthesis in yeast as a first step towards Taxol (Paclitaxel) production.

Metabolic engineering in microbes could be used to produce large amounts of valuable metabolites that are difficult to extract from their natural sources and too expensive or complex to produce by chemical synthesis. As a step towards the production of Taxol in the yeast Saccharomyces cerevisiae, we introduced heterologous genes encoding biosynthetic enzymes from the early part of the taxoid biosynthetic pathway, isoprenoid pathway, as well as a regulatory factor to inhibit competitive pathways, and studied their impact on taxadiene synthesis. Expression of Taxus chinensis taxadiene synthase alone did not increase taxadiene levels because of insufficient levels of the universal diterpenoid precursor geranylgeranyl diphosphate. Coexpression of T. chinensis taxadiene synthase and geranylgeranyl diphosphate synthase failed to increase levels, probably due to steroid-based negative feedback, so we also expressed a truncated version of 3-hydroxyl-3-methylglutaryl-CoA reductase (HMG-CoA reductase) isoenzyme 1 that is not subject to feedback inhibition and a mutant regulatory protein, UPC2-1, to allow steroid uptake under aerobic conditions, resulting in a 50% increase in taxadiene. Finally, we replaced the T. chinensis geranylgeranyl diphosphate synthase with its counterpart from Sulfolobus acidocaldarius, which does not compete with steroid synthesis, and codon optimized the T. chinensis taxadiene synthase gene to ensure high-level expression, resulting in a 40-fold increase in taxadiene to 8.7+/-0.85mg/l as well as significant amounts of geranylgeraniol (33.1+/-5.6mg/l), suggesting taxadiene levels could be increased even further. This is the first demonstration of such enhanced taxadiene levels in yeast and offers the prospect for Taxol production in recombinant microbes.

Introduction of the early pathway to taxol biosynthesis in yeast by means of biosynthetic gene cluster construction using SOE-PCR and homologous recombination.

Metabolic engineering of plant natural product pathways in heterologous systems requires the highly concerted action of several biosynthetic genes. Besides the functional heterologous expression of the genes encoding the natural product biosynthetic pathway, often additional extensive modifications in the host primary metabolism are also needed, in order to obtain efficient supply of the required biosynthetic building blocks to support the engineered natural product biosynthesis. Selection markers in heterologous expression systems, like baker's yeast (Saccharomyces cerevisiae), are often limited and the chromosomal insertion prevents later modifications of engineered pathway, e.g. exchange of gene promoters, or the introduction of additional genetic regulatory elements in a timely manner. Thus the construction of biosynthetic gene clusters on episomal expression vectors seems a logical solution for this dilemma. Although manipulation of long DNA fragments still represents a challenge, by using PCR and in vitro homologous recombination, we assembled a biosynthetic gene cluster for the concerted heterologous expression of three important genes for the metabolic engineering of taxoid biosynthesis in yeast.