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

Abstract

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 β-glucuronidase (GUS) strains indeed resulted in a small increase in GUS expression.

Introduction

The filamentous fungus Aspergillus niger is able to secrete large amounts of a wide variety of enzymes and metabolites. In nature, the enzymes are needed to release nutrients from complex biopolymers while metabolite excretion gives the fungus a competitive advantage. These natural characteristics are exploited by industry in both solid state and submerged fermentations for the production of enzymes and metabolites.

Protein production and secretion in A. niger is a complex and widely studied process and significant differences in titres between homologous and heterologous proteins are frequently observed. While homologous proteins are produced in a 10–50 g/L range, heterologous proteins often are produced 10- to 100-fold less (van den Hondel et al., 1991), indicating that A. niger encounters severe problems in expression and/or secretion of foreign proteins. Homologous protein production strains show high protein fluxes and have a high demand for amino acids, while heterologous protein production results in secretion stress and a typical unfolded-protein response (UPR) response. Activation of the UPR pathway in filamentous fungi due to heterologous protein production has been frequently observed and is manifested by induction of chaperone and foldase genes (e.g., Wiebe et al., 2001, Saloheimo et al., 2003, Collén et al., 2005, Arvas et al., 2006).

Recently, genome sequences of industrial A. niger strains have been published covering both enzyme and metabolite producing strains (Pel et al., 2007, Baker, 2006, Sun et al., 2007, http://genome.jgi-psf.org/Aspni1/Aspni1.home.html). In addition, genomic tools such as transcriptomics and various proteomics technologies have been developed for fungi, which enable a new way of working in order to improve strains and processes for industrial fermentations.

In our studies, strains were grown under strictly controlled fermentation conditions and samples for transcriptomics and proteomics were obtained using a standardised rapid procedure. Based on these reproducible fermentations, differential transcriptomics and proteomics studies were performed on protein production strains and their isogenic control strains. Genes which showed co-regulation at the transcriptome and proteome level were selected as potential targets for strain improvement. To date, only a few papers have been published investigating the responses due to protein production in filamentous fungi (e.g., MacKenzie et al., 2005, Sims et al., 2004, Sims et al., 2005, Levin et al., 2007, Arvas, 2007, Guillemette et al., 2007, Yuan et al., 2008).

Despite their importance, the number of published proteomics studies on filamentous fungi is also rather limited, as was reviewed by Kim et al., 2007, Kim et al., 2008, Carberry and Doyle, 2007. However, fungal proteomics is gathering pace due to the increasing availability of genome sequence information and advances in proteomics technologies. Preparation of protein extracts is a critical step in reliably and reproducibly determining the presence and the abundance of proteins. Since the fungal cell wall is exceptionally robust (Bowman and Free, 2006) and proteases are extremely resistant, cell lysis and protein extraction is challenging. Several protocols have already been published for fungal protein extraction (e.g., Nandakumar and Marten, 2002, Grinyer et al., 2004, Shimizu and Wariishi, 2005, Kniemeyer et al., 2006, Kim et al., 2008). To date, 139 intracellular proteins have been identified for various Aspergillus species (Kim et al., 2008). Using different extraction methods in parallel, a proteome which covers 898 proteins was obtained in this study.

As a next step, integration of transcriptomics and proteomics data is challenging since, e.g., turnover of mRNA, initiation of translation and stability of proteins are independent processes controlled at different levels. This might result in dissimilar or even invert relationships for mRNA and protein levels, a phenomenon which is studied among others in Saccharomyces cerevisiae (Daran-Lapujade et al., 2007, De Groot et al., 2007). Nevertheless, pathways and functionally related factors that responded similar on both levels are potential targets for strain improvement. Moreover, discrepancies between proteomics and transcriptomics levels can give indications for more complex regulatory (posttranslational) mechanisms which do not justify simple gene overexpression as de-bottlenecking strategy.

Here, we use a proteomics-based integrated genomics approach of controlled A. niger fermentations to determine the cellular responses of strains producing different homologous and heterologous enzymes. Our in house developed protein extraction protocols were used and almost 900 intracellular proteins were identified from 2D gels using automated spot excision, tryptic digestion and MALDI-TOF-MS peptide mass fingerprinting. Genes up-regulated both in the proteome and the corresponding transcriptome were selected as leads for generic strain improvement. Modulation of two of the identified lead factors resulted in an increased expression of the model protein β-glucuronidase (GUS). To our knowledge, this is one of the first applications of an integrated genomic approach based on controlled fermentations of A. niger to improve protein production in a generic way.