Production of recombinant proteins by filamentous fungi

Several Aspergillus species, in particular Aspergillus niger and Aspergillus oryzae, are widely used as protein production hosts in various biotechnological applications. In order to improve the expression and secretion of recombinant proteins in these filamentous fungi, several novel genetic engineering strategies have been developed in recent years. This review describes state-of-the-art genetic manipulation technologies used for strain improvement, as well as recent advances in designing the most appropriate engineering strategy for a particular protein production process. Furthermore, current developments in identifying bottlenecks in the protein production and secretion pathways are described and novel approaches to overcome these limitations are introduced. An appropriate combination of expression vectors and optimized host strains will provide cell factories customized for each production process and expand the great potential of Aspergilli as biotechnology workhorses to more complex multi-step industrial applications.

Expression and export: recombinant protein production systems for Aspergillus

Aspergilli have a long history in biotechnology as expression platforms for the production of food ingredients, pharmaceuticals and enzymes. The achievements made during the last years, however, have the potential to revolutionize Aspergillus biotechnology and to assure Aspergillus a dominant place among microbial cell factories. This mini-review will highlight most recent breakthroughs in fundamental and applied Aspergillus research with a focus on new molecular tools, techniques and products. New trends and concepts related to Aspergillus genomics and systems biology will be discussed as well as the challenges that have to be met to integrate omics data with metabolic engineering attempts.

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Aspergillus as a multi-purpose cell factory: current status and perspectives.

The filamentous fungus Aspergillus niger is well-known as a producer of primary metabolites and extracellular proteins. For example, glucoamylase is the most efficiently secreted protein of Aspergillus niger, thus the homologous glucoamylase (glaA) promoter as well as the glaA signal sequence are widely used for heterologous protein production. Xylose is known to strongly repress glaA expression while maltose is a potent inducer of glaA promoter controlled genes. For a more profound understanding of A. niger physiology, a comprehensive analysis of the intra- and extracellular proteome of Aspergillus niger AB1.13 growing on defined medium with xylose or maltose as carbon substrate was carried out using 2-D gel electrophoresis/Maldi-ToF and nano-HPLC MS/MS. The intracellular proteome of A. niger growing either on xylose or maltose in well-aerated controlled bioreactor cultures revealed striking similarities. In both cultures the most abundant intracellular protein was the TCA cycle enzyme malate-dehydrogenase. Moreover, the glycolytic enzymes fructose-bis-phosphate aldolase and glyceraldehyde-3-phosphate-dehydrogenase and the flavohemoglobin FhbA were identified as major proteins in both cultures. On the other hand, enzymes involved in the removal of reactive oxygen species, such as superoxide dismutase and peroxiredoxin, were present at elevated levels in the culture growing on maltose but only in minor amounts in the xylose culture. The composition of the extracellular proteome differed considerably depending on the carbon substrate. In the secretome of the xylose-grown culture, a variety of plant cell wall degrading enzymes were identified, mostly under the control of the xylanolytic transcriptional activator XlnR, with xylanase B and ferulic acid esterase as the most abundant ones. The secretome of the maltose-grown culture did not contain xylanolytic enzymes, instead high levels of catalases were found and glucoamylase (multiple spots) was identified as the most abundant extracellular protein. Surprisingly, the intracellular proteome of A. niger growing on xylose in bioreactor cultures differed more from a culture growing in shake flasks using the same medium than from the bioreactor culture growing on maltose. For example, in shake flask cultures with xylose as carbon source the most abundant intracellular proteins were not the glycolytic and the TCA cycle enzymes and the flavohemoglobin, but CipC, a protein of yet unknown function, superoxide dismutase and an NADPH dependent aldehyde reductase. Moreover, vacuolar proteases accumulated to higher and ER-resident chaperones and foldases to lower levels in shake flask compared to the bioreactor cultures. The utilization of xylose or maltose was strongly affecting the composition of the secretome but of minor influence on the composition of the intracellular proteome. On the other hand, differences in culture conditions (pH control versus no pH control, aeration versus no aeration and stirring versus shaking) have a profound effect on the intracellular proteome. For example, lower levels of ER-resident chaperones and foldases and higher levels of vacuolar proteases render shake flask conditions less favorable for protein production compared to controlled bioreactor cultures.

https://link.springer.com/content/pdf/10.1186%2F1475-2859-9-23.pdf

The intra- and extracellular proteome of Aspergillus niger growing on defined medium with xylose or maltose as carbon substrate

Germination of conidia of aspergillus niger is accompanied by major changes in RNA profiles

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

The invention relates to a nucleotide sequence comprising: a synonymous nucleotide coding sequence with optimized codon frequency such that a native codon has been exchanged with a synonymous codon, said synonymous codon encoding the same amino acid as the native codon and having a higher frequency in codon usage as defined in Table 1 than the native codon; and optionally said nucleotide sequence comprises control sequences such as: one translational termination sequence orientated in 5' towards 3' direction selected from the following list of sequences: TAAG, TAGA and TAAA, preferably TAAA, and/or one translational initiator coding sequence orientated in 5' towards 3' direction selected from the following list of sequences: gctnccyyc, using ambiguity codes for nucleotides: v (A/C/G); n (A/C/G/T), preferably 5'-GCT TCC TTC-3'. The invention further relates to a consensus translational initiator sequence: 5'-mwChkyCAmv-3', preferably the translational initiator sequence is selected from the list consisting of: 5'-mwChkyCAAA-3', 5'-mwChkyCACA-3', and 5'-mwChkyCAAG-3'.

Method for Producing a Compound of Interest in a Filamentous Fungal Cell

L'invention concerne des polypeptides qui possedent une activite correspondant a au moins une activite du polypeptide SEC61, des polynucleotides codant ces polypeptides ainsi que leur utilisation dans la preparation de cellules hotes convenant a la production du polypeptide recherche. Ces cellules hotes peuvent presenter une capacite secretoire accrue du polypeptide recherche.

Production de polypeptides par stimulation de la capacite secretoire

Verfahren zur Herstellung einer Verbindung in einer filamentösen Pilzzelle

L'invention porte sur une sequence nucleotidique comprenant une sequence de codage nucleotidique synonyme presentant une frequence codon synonyme, ce codon synonyme codant le meme acide amine que le codon natif et presentant une frequence plus elevee dans l'usage codon tel que defini dans le tableau 1 que le codon natif. Facultativement, cette sequence nucleotidique comprend des sequences de controle telles qu'une sequence de terminaison translationnelle orientee dans le sens 5' vers 3' choisie dans la liste de sequences suivantes : TAAG, TAGA et TAAA, de preference TAAA, et/ou une sequence de codage initial translationnelle orientee dans le sens 5' vers 3' choisie dans la liste de sequences suivante : gctnccyyc, au moyen des codes d'ambiguite pour des nucleotides : v(A/C/G); n (A/C/G/T), de preference 5'-GCT TCC TTC-3'. Cette invention porte aussi sur une sequence initiale translationnelle consensus : 5'-mwChkyCAmv-3', la sequence d'initiateur translationnelle etant choisie de preference dans la liste: 5'-mwChkyCAAA-3', 5'-mwChkyCACA-3', et 5'-mwChkyCAAG-3'.

Serge Petrus Donkers

Papers

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

Method for Producing a Compound of Interest in a Filamentous Fungal Cell

Production de polypeptides par stimulation de la capacite secretoire

Verfahren zur Herstellung einer Verbindung in einer filamentösen Pilzzelle

Procede de fabrication d'un compose d'interet dans une cellule fongique filamenteuse