In a recent publication featured in Nature Communications, scholars have devised a modular synthetic biology toolkit tailored for Aspergillus oryzae, a consumable fungus utilized in fermented foods, protein synthesis, and meat substitutes.

Background The agricultural sector is responsible for approximately one-third of global greenhouse gas emissions, thereby contributing to the decline in biodiversity, environmental degradation, and the emergence of novel diseases.

Transitioning away from industrialized animal husbandry towards sustainable alternatives is imperative to mitigate the environmental impact and provide sustenance for the global populace. Microbial food production offers enhanced safety and efficiency, precise control over production, and alleviation of animal suffering.

Filamentous fungi, encompassing mushrooms and molds, represent a diverse array of microorganisms and confer significant advantages for microbial food production.

Furthermore, their inherent high secretion capacity positions them as ideal hosts for protein synthesis. Additionally, due to their filamentous structure mirroring that of animal muscle, fungal biomass (mycelium) can be crafted into meat alternatives (mycoprotein).

The study and its discoveries In the present investigation, scholars have crafted a modular synthetic biology toolkit tailored for A. oryzae, a safe and palatable fungus with a long history of culinary usage.

They have introduced an alternative using synthetic biology, user-friendly approach utilizing clustered, regularly interspersed short palindromic repeats (CRISPR) – CRISPR-associated protein 9 (Cas9), which is compatible with existing reagents.

This strategy entails the direct transformation of CRISPR-Cas9 ribonucleoprotein complexes, rather than encoding single-guide RNAs (sgRNAs) and Cas9 from a plasmid.

Furthermore, the DNA template employed to repair double-strand breaks incorporates an orotidine-5′-phosphate decarboxylase gene (pyrG) marker for positive and negative selection.

The system has been engineered such that successful pyrG loop out can only occur upon integration of the repair template at the targeted locus, flanked by identical 300 bp sequences.

Ectopic integrations stemming from non-homologous end joining (NHEJ) within this system are incapable of loop-out or survival when subjected to media containing 5-fluoroorotic acid. An essential facet of this blueprint lay in the recyclability of the pyrG marker upon its insertion at the designated locus.

Furthermore, the researchers delved into candidate-neutral loci within A. oryzae to integrate genes for amplification. They scrutinized the intergenic domains within the A. oryzae RIB40 genome, appraising the expression levels of two juxtaposed genes.

A roster of candidate loci foreseen for heightened gene expression was compiled, with ten regions earmarked for further scrutiny.

Subsequently, the team introduced green fluorescent protein (GFP) cassettes under the governance of a robust, constitutive promoter (pTEF1), gauging fluorescence within the conidia of looped-out strains.

Of the ten loci investigated, nine demonstrated remarkably efficient integration, with GFP expression discernible in eight instances. Each locus exhibited superior expression levels compared to the positive control.

Following this, the researchers endeavored to institute a synthetic expression system (SES) within A. oryzae. They evaluated the efficacy of a characterized synthetic transcription factor (sTF) in driving the expression of mCherry from a core promoter (Cp).

The team genetically integrated the sTF, engendering a low basal expression under an A. niger Cp. Simultaneously, an mCherry cassette bearing 6x upstream activating sequences (UAS) was incorporated at a distinct genomic location preceding the Cp.

The researchers observed mCherry expression within conidia and mycelia, with both the sTF and UAS proving requisite for activity.

Subsequent to this, the researchers redirected their focus towards bioengineering edible mycelium, with a particular emphasis on enhancing the presence of the bioactive amino acid, ergothioneine.

They postulated that augmenting the expression of endogenous ergothioneine biosynthetic genes within A. oryzae could lead to heightened production.

Orthologs of Egt1 and Egt2, enzymes from Neurospora crassa implicated in ergothioneine biosynthesis, were identified within A. oryzae.

These orthologs were subsequently inserted into neutral loci; both genes were expressed under the aegis of a bidirectional promoter or individually at distinct sites.

In mycelium derived from the RIB40 strain, ergothioneine levels were modest. However, they experienced an 11- and 21-fold increase in mycelium derived from strains with bidirectional and separate promoter configurations, respectively.

Ergothioneine levels in the bidirectional promoter strain mirrored those found in oyster mushrooms, while in the separate promoter strain, they were 1.5-fold higher.

No discernible differences in protein content were observed between engineered and wild-type strains, although a minor growth impairment was noted with ergothioneine overproduction.

The researchers then harnessed these methodologies to enhance the sensory characteristics of the edible biomass, targeting heme biosynthesis.

Identifying prospective heme biosynthetic genes within A. oryzae, they focused on the expression of five forecasted rate-limiting enzymes.

Additionally, they expressed two copies of soy leghemoglobin as a potential reservoir for heme, as excessive free heme levels could prove cytotoxic.

The biomass yielded by the engineered strain was four times greater than that of the non-engineered counterpart.

Upon harvesting, the biomass displayed a red hue, in stark contrast to the off-white coloration observed in RIB40. This disparity in coloration persisted post-cooking, thereby enhancing the meat-like appearance of the fungal biomass.

The engineered mycoprotein boasted a full complement of essential amino acids, with no compromise in protein content or growth yield when compared to the non-engineered strain.

In conclusion, the researchers developed a synthetic toolkit for the integration and regulation of genes and pathways. Leveraging this toolkit, they engineered A. oryzae mycoprotein to produce ergothioneine at levels surpassing those found in natural dietary sources, such as mushrooms.

Furthermore, the mycelia were engineered to overproduce heme, thereby enhancing both color and flavor. This endeavor, however, represents an initial prototype; further assessments pertaining to sensory attributes, food safety, consumer acceptance, and regulatory frameworks are imperative.

For more information: Edible mycelium bioengineered for enhanced nutritional value and sensory appeal using a modular synthetic biology toolkit, Nature Communications, https://doi.org/10.1038/s41467-024-46314-8