Effective lead selection for improved protein production in Aspergillus niger based on integrated genomics.

The filamentous fungus Aspergillus niger is widely exploited for industrial production of enzymes and organic acids. An integrated genomics approach was developed to determine cellular responses of A. niger to protein production in well-controlled fermentations. Different protein extraction methods in combination with automated sample processing and protein identification allowed quantitative analysis of 898 proteins. Three different enzyme overproducing strains were compared to their isogenic fungal host strains. Clear differences in response to the amount and nature of the overproduced enzymes were observed. The corresponding genes of the differentially expressed proteins were studied using transcriptomics. Genes that were up-regulated both at the proteome and transcriptome level were selected as leads for generic strain improvement. Up-regulated proteins included proteins involved in carbon and nitrogen metabolism as well as (oxidative) stress response, and proteins involved in protein folding and endoplasmic reticulum-associated degradation (ERAD). Reduction of protein degradation through the removal of the ERAD factor doaA combined with overexpression of the oligosaccharyl transferase sttC in A. niger overproducing beta-glucuronidase (GUS) strains indeed resulted in a small increase in GUS expression.

Heterologous protein production in the yeast Kluyveromyces lactis.

Kluyveromyces lactis is both scientifically and biotechnologically one of the most important non-Saccharomyces yeasts. Its biotechnological significance builds on its history of safe use in the food industry and its well-known ability to produce enzymes like lactase and bovine chymosin on an industrial scale. In this article, we review the various strains, genetic techniques and molecular tools currently available for the use of K. lactis as a host for protein expression. Additionally, we present data illustrating the recent use of proteomics studies to identify cellular bottlenecks that impede heterologous protein expression.

Proteome analysis of the medicinal plant Catharanthus roseus.

A proteomic approach is undertaken aiming at the identification of novel proteins involved in the alkaloid biosynthesis of Catharanthus roseus. The C. roseus cell suspension culture A11 accumulates the terpenoid indole alkaloids strictosidine, ajmalicine and vindolinine. Cells were grown for 21 days, and alkaloid accumulation was monitored during this period. After a rapid increase between day 3 and day 6, the alkaloid content reached a maximum on day 16. Systematic analysis of the proteome was performed by two-dimensional polyacrylamide gel electrophoresis. After day 3, the proteome started to change with an increasing number of protein spots. On day 13, the proteome changed back to roughly the same as at the start of the growth cycle. 88 protein spots were selected for identification by mass spectrometry (MALDI-MS/MS). Of these, 58 were identified, including two isoforms of strictosidine synthase (EC 4.3.3.2), which catalyzes the formation of strictosidine in the alkaloid biosynthesis; tryptophan synthase (EC 4.1.1.28), which is needed for the supply of the alkaloid precursor tryptamine; 12-oxophytodienoate reductase, which is indirectly involved in the alkaloid biosynthesis as it catalyzes the last step in the biosynthesis of the regulator jasmonic acid. Unique sequences were found, which may also relate to unidentified biosynthetic proteins.

The Catharanthus alkaloids: pharmacognosy and biotechnology.

The Catharanthus (or Vinca) alkaloids comprise a group of about 130 terpenoid indole alkaloids. Vinblastine is now marketed for more than 40 years as an anticancer drug and became a true lead compound for drug development. Due to the pharmaceutical importance and the low content in the plant of vinblastine and the related alkaloid vincristine, Catharanthus roseus became one of the best-studied medicinal plants. Consequently it developed as a model system for biotechnological studies on plant secondary metabolism. The aim of this review is to acquaint a broader audience with the recent progress in this research and with its exciting perspectives. The pharmacognostical aspects of the Catharanthus alkaloids cover botanical (including some historical), phytochemical and analytical data. An up-to-date view on the biosynthesis of the alkaloids is given. The pharmacological aspects of these alkaloids and their semi-synthetic derivatives are only discussed briefly. The biotechnological part focuses on alternative production systems for these alkaloids, for example by in vitro culture of C. roseus cells. Subsequently it will be discussed to what extent the alkaloid biosynthetic pathway can be manipulated genetically ("metabolic engineering"), aiming at higher production levels of the alkaloids. Another approach is to produce the alkaloids (or their precursors) in other organisms such as yeast. Despite the availability of only a limited number of biosynthetic genes, the research on C. roseus has already led to a broad scientific spin-off. It is clear that many interesting results can be expected when more genes become available.