Mycotoxin-free Aspergillus oryzae strain lineage for alternative and novel protein production at industrial scale

Abstract

Advanced industrial strains of Aspergillus oryzae have been used for decades for the production of recombinant proteins including food and feed enzymes at large scale. The A. oryzae strain lineage evaluated in this review derives from the proprietary Novozymes (now Novonesis) strain collection. A. oryzae wild-type strains have the potential to produce three different mycotoxins (aflatoxins (AFL), cyclopiazonic acid (CPA), and 3-nitropropionic acid (3-NPA)). Here, we review the work originally performed at Novozymes to identify a strain (BECh1) that contained a large chromosomal deletion comprising both AFL and CPA gene clusters, significantly improving the safety of the lineage. The description of the deleted region is presented here. As the genetic basis for 3-NPA biosynthesis was recently revealed, we describe here that this A. oryzae lineage contains an additional large deletion that encompasses the 3-NPA biosynthetic genes npaA and npaB, thereby rendering the strains unable to produce any mycotoxin. Further strain development has resulted in strains devoid of penicillin production by inactivation of the penicillin G gene cluster (penG). This strain lineage represents the first example of mycotoxin-free A. oryzae for production of recombinant (alternative) novel food proteins. Recently, bovine beta-lactoglobulin made using a strain of this lineage has received GRAS status and can be commercialized for use in food in the USA. With its history of safe use in food and feed, the lack of toxigenic potential and the ability to differentiate strains with modern technologies, this A. oryzae strain can be considered safe as other organisms with a Quality Presumption of Safety (QPS) status in Europe. QPS is not applicable to filamentous fungi and only granted at the species level to bacteria and a few yeast species. We suggest modernizing the QPS concept to become strain rather than species specific and present arguments to qualify this strain lineage as QPS or QPS-like. KEY POINTS: • The biosynthetic genes for 3-nitropropionic acid have been recently characterized. • An industrial strain lineage for food protein production lacks all known mycotoxin genes. • Proteins produced in this mycotoxin-free lineage should entail lower regulatory requirements.

Keywords: Aspergillus oryzae; Mycotoxin; Novel food protein; Precision fermentation; Regulatory approval.

Fig. 1

The potential for mycotoxin production in A. oryzae includes cyclopiazonic acid (CPA), aflatoxin (e.g., aflatoxin B₁), and 3-nitropropionic acid (3-NPA)

Fig. 2

A. oryzae strain lineage derived from the original koji strain A1560/IFO4177, depicting the relevant mutagenesis steps resulting in the loss of AFL and CPA gene clusters (in strain BECh1) and 3-NPA (in strain JaL815). GRAS notification numbers for approved products in the USA are depicted above the strain names (e.g., GRN 90). Only relevant steps of the strain lineage are shown

Fig. 3

The A. oryzae genomic region of chromosome 6, depicting the region deleted in strain JaL815 and derived strains, spanning from AO090138000088 to AO090138000157, and the position of the npaA and npaB genes. The complete 390 kb region (Deletion in JaL815) is shown (top). The central part of the deleted region is also shown to display the location and organization of npaA and npaB (below)

Fig. 4

Growth differences of wildtype strain (A1560 (A)) and an optimized host strain (derived from AT604 (B)) on minimal solid medium. The plates were prepared by spot inoculation of approx. 100 spores and incubated 6 days at 30 °C

Fig. 5

Genome analysis of the A. oryzae genomic region of chromosome 3, depicting the region deleted in strain BECh1 spanning from AO090026000062 to the telomeric region, together with the position of the AFL (green box) and CPA gene clusters (yellow box). The picture does not include gene names for all the genes of each cluster, for clarity. The position of chromosomal breakage for BECh1 and for A. oryzae Group 2 strains is also depicted

Fig. 6

The A. oryzae genomic region of chromosome 6, depicting the penG gene clusters, the deleted region that contains part of the aatA and acvA and the full ipnA coding region