Synthetic Biology Tools for Engineering Aspergillus oryzae

Abstract

For more than a thousand years, Aspergillus oryzae has been used in traditional culinary industries, including for food fermentation, brewing, and flavoring. In recent years, A. oryzae has been extensively used in deciphering the pathways of natural product synthesis and value-added compound bioproduction. Moreover, it is increasingly being used in modern biotechnology industries, such as for the production of enzymes and recombinant proteins. The investigation of A. oryzae has been significantly accelerated through the successive application of a diverse array of synthetic biology techniques and methodologies. In this review, the advancements in biological tools for the synthesis of A. oryzae, including DNA assembly technologies, gene expression regulatory elements, and genome editing systems, are discussed. Additionally, the challenges associated with the heterologous expression of A. oryzae are addressed.

Keywords: Aspergillus oryzae; DNA assembly; genome editing; synthetic biology.

Figure 3

Pathway of heterologous protein secretion by A. oryzae. The secretion of foreign proteins begins with protein translation in ribosomes and then complex assembly in the endoplasmic reticulum (ER). The polypeptide entering the ER cavity needs to be folded with the assistance of the binding protein BiP1 and the protein disulfide isomerase PDI1. The correct protein is transported to the Golgi apparatus by vesicles for further modification and packaging. Finally, the protein is transported to the extracellular space through vesicles (red arrows); incompletely folded or misfolded proteins promote protein degradation through the endoplasmic reticulum stress response (gray arrows). In the process of protein degradation, the UPR can trigger the downstream UPR cascade through membrane-bound IRE1 and subsequently couple and autophosphorylate to form activated IRE1. Activated IRE1 matures the mRNA of the transcription factor HAC1 by splicing, leading to the expression of downstream protein-folding genes or degradation by ERAD [94]. The cell structure is represented by purple. The heterologous protein secretion pathway is shown in red font. The yellow dotted line represents an enlarged endoplasmic reticulum lumen. It is worth noting that this figure only shows what we mentioned in this article. The specific process needs to be further explored.

Figure 1

DNA assembly techniques used for A. oryzae. A. oryzae is indicated by a green module (a) Conventional DNA manipulation through the restriction/ligation method. The linear vector and gene fragments are digested with EcoRI and HindIII to obtain fragments and vectors with sticky ends. The gene is successfully assembled into a vector with ligase.The purple module is indicated. (b) In the Gateway method, targeted recombination through the att locus enables the transfer of target genes. The yellow module is indicated. (c) In Gibson assembly, the 5′ exonuclease activity of the T5 exonuclease is used to cut fragment A and fragment B, and then Phusion DNA polymerase is used to fill the vacancy, and Taq ligase is used to repair the incision. The red module is indicated. (d) In TAR, efficient homologous recombination in yeast cells is achieved using the free ends of DNA fragments containing homologous sequences. The blue module is indicated.

Figure 2

Two repair pathways mediated by the engineered nucleases TALENs and CRISPR/Cas9 in cells. (a) In A. oryzae, TALENs and CRISPR/Cas9 can recognize and bind to specific DNA sequences, resulting in double-strand breaks (DSBs) that induce NHEJ or HR. NHEJ introduces random insertions and deletions into the genome. HR is the use of the homologous recombination principle to achieve precise integration of exogenous sequences. Notably, Cas9 introduces blunt breaks, while FokI, the TALEN endonuclease, introduces a staggered cut (for simplicity, this difference is not shown in the figure). (b) The TALE monomer of TALENs specifically recognizes the DNA sequence and binds to double strands. The fused Fok dimer, composed of two monomers, cleaves the target DNA sequence to produce DSBs. TALE is composed of an N-terminal transport signal, a transcriptional activation domain, a DNA-specific recognition binding domain and a C-terminal nuclear localization signal peptide. FokI: a restriction endonuclease. Shear activity can be exerted only as dimers. (c) The CRISPR/Cas9 system containing the Cas9 protein and sgRNA (single-guide RNA). The sgRNA recognizes a 20 bp sequence followed by PAM, after which the Cas9 protein is used to cut the DNA double strand to produce DSBs. PAM sequence: Usually, the PAM sequence is composed of three bases, NGG.