Team:DTU-Denmark/Design

• Aspergillus niger
• Engineering Tools
• Morphology
• Protein Secretion
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• Aspergillus niger
• Engineering Tools
• Morphology
• Protein Secretion

Selecting a Chassis Fungal Organism: Aspergillus niger

In order to design a cell factory, we had to consider the choice of organism carefully. Through lectures at the university, investigation of literature, and talks with stakeholders in our Human Practices work, it became obvious that filamentous fungi have a high potential for industrial production. To make our project as industrially relevant as possible, we chose the filamentous fungi Aspergillus niger as our host, as it is commonly used in enzyme and secondary metabolite producing companies, such as Novozymes, with whom we had close contact. In order to make the work with A. niger easily accessible, we decided to provide a structured workflowof the work with A. niger with attached protocols, which can also be found on the contribution page.

The reference strain that we have used is ATCC 1015, in which we have performed all our genetic engineering studies. This strain has been altered so it is Non-Homologous End Joining (NHEJ) pathway deficient, by being ΔkusA. This forces the strain to use Homologous Direct Repair (HDR) to repair breaks in the DNA, enabling insertions/deletions (indels) to occur at targeted locations. The strain also has a deletion in the pyrG gene, which encodes for the Orotidine 5'-phosphate decarboxylase protein. Therefore, the de novo biosynthesis pathway of UMP is truncated, and the strain requires uracil or uridine to be added as supplements in the media to survive, which is used as a selection marker (Nødvig et al., 2015).

Engineering Principles

To modify the genome of A. niger, we decided to use USER cloning for plasmid construction, and CRISPR-Cas9 for genome engineering.

USER Cloning and Fusion

USER stands for Uracil-Specific Excision Reagent. It is a method to directionally clone inserts into a USER compatible vector by the use of a USER nicking enzyme. The sequence for insert is created through primers, with one uracil and a tail that is compatible with a uracil cloning site in the vector, amplified through PCR. This product is treated with Uracil-Specific Excision Reagent, removing the U and the attached strand from the double-stranded DNA, creating an overhang ready for cloning.

A USER cloning cassette within the USER compatible vector can be digested with the restriction enzymes PacI and Nt.BbvCI to create overhangs that match the insert’s USER overhangs. PacI creates a small overhang whereafter Nt.BbvCI makes a single-strand nick, which creates an eight bp long 5’ overhang. When mixed, the insert and vector will assemble and create the designed vector.

To assemble multiple fragments, one can use USER fusion where several products for insertion can be created with compatible user overhangs and assembled in a USER compatible vector (Nour-Eldin et al., 2010), as seen in the figure below. The USER tails in grey will match with the USER compatible vector and the USER tails that will assemble the fragments to each other are designed so when combined they will create the crispr RNA (crRNA), explained in the section below. The region is delimited by a promoter and a terminator to ensure transcription of the insert into messenger RNA (mRNA), and the sgRNA (which is the combination of the crRNA and the backbone of the gRNA) is flanked by two tRNA. These are located here due to their ability to auto-cleave from the mRNA after transcription, which frees the sgRNA making it able to bind to Cas9 and edit the genome.

CRISPR-Cas9

The CRISPR-Cas9 system is a viral defense mechanism originating from Streptococcus pyogenes. During the last decade, the system has been discovered and investigated, where it has shown high efficiency in genomic engineering. Since its discovery, it has been adapted for genomic engineering for several species. Recently, it has been adapted to fungal systems, specifically Aspergillus niger (Nødvig et al., 2015). Cas9 (CRISPR associated protein 9) is an RNA-guided endonuclease. The crispr RNA (crRNA) is what makes the system so specific, as it guides the Cas9 along with the tracrRNA (gRNA backbone) to its target in the genome, where it cuts 3 nucleotides (nt) upstream of the PAM sequence (NGG). Since the working strain is NHEJ deficient, it has to use HDR to repair the break, allowing the creation of indels.

When the deletion is the desired result, the fungus must be transformed with the plasmid containing both the Cas9 and the two sgRNAs to make the two cuts, one upstream and another downstream of the gene. It has to be transformed with a repair oligo as well, of around 90 nucleotides long. Its sequence must consist of regions located close to where the gap has been created. In this way, by HDR using this oligomer as a template, the break is mended with the previous sequence removed.

On the contrary, if the objective of the transformation is to produce an insertion, the fungus has to be transformed with a linearized sequence containing the construct to be inserted, flanked by two homology regions, of between 1 and 2 kb long. These regions can be designed to target specifically one of the studied insertion sites in A. niger. As seen in the figure below, the homology arms in the linearized plasmid can therefore recombine with the genome, introducing the desired construct within.

In order to improve efficiency, a double-stranded break can be introduced in between both homology regions to force the fungus to repair the break via HDR with the linearized plasmid. This is done similarly to the knockout strategy, with a plasmid containing Cas9 and in this case only one sgRNA that targets the sequence in between the homology regions.

Morphology

In order to optimize fungal morphology, the team conducted research within published studies relevant to this topic. This led to the discovery of seven interesting genes with diverging morphological traits when engineered; interesting for industrial setups. Some of these gene-knockouts had already been tested in A. niger, and some had only been tested within other strains. To make novel research, we decided to not only create single gene mutated strains, but also make double knockout strains. Additionally, the novelty of the project would lie within the data created through the bioreactors, BioLector and the simulation and parameter determination from the microscopic pictures.

Genes for Morphology Engineering

The genes chosen to be engineered were aplD, arfA, chsC, gul-1, pkaR, racA and spaA. Information about the individual genes and previous engineering performed can be found by clicking on the images of the genes below.

ΔaplD

aplD is a predicted gamma-adaptin encoding gene in A. niger, which might be related to endosome cargo transport and it plays a crucial role in the filamentous growth and pellet formation of A. niger. Through a study they controlled the gene by the tet-on system. During reduced expression the growth was hyper branching and the pellet formation was defected with a putative protein secretion increase which might be related to an increase of dispersed mycelia (Cairns et al., 2019).

ΔarfA

arfA is an essential gene for A. niger as it encodes a small GTPase gene and has a critical role in growth rates and hyphal morphology. Through tet-on experiments, a study found that loss- and gain-of-function mutations had an impact on hyphal growth rates, morphology of hyphal tip, protein secretion and demonstrated multiple functions in the secretory pathway of A. niger. The overexpression reduced the pellet diameter and increased the protein secretion. During loss-of-function, the strains showed abnormal hyphal branching, swelling and curved growth as well as punctate localisation of intracellular glucoamylase (Fiedler et al., 2018).

ΔchsC

chsC encodes a chitin synthase and plays an important role in the integrity of the hyphal wall as well as conidiophore development. A study showed that silencing of the gene resulted in shorter hypha, less dispersed mycelia and more dense pellets and a higher accumulation of citric acid. This trait might be beneficial in industrial setups, as the long hyphal elements will be less likely to intertwine and affect the medium (Sun et al., 2018).

Δgul-1

gul-1 in Neurospora crassa encodes an mRNA binding protein that is related to cell wall remodelling and it was suggested to be important for the mycelial morphology in submerged cultivation. It affects transcription of cell wall-related genes, and therefore influences the structure of the mycelial morphology. When disrupting the gene, it caused pellet formation during fermentation and hyper branching, thereby decreasing the viscosity of the broth. Additionally, increased protein secretion was determined which might be due to defects in the cell wall. However, the strain also exhibited slow growth rates. Overexpression of the same gene caused increase in viscosity instead which would not be beneficial. We found an uncharacterised homolog to gul-1 in A. niger that was interesting to investigate the mutation effect of during fermentation (Lin et al., 2018).

ΔpkaR

The gene mcb in Neurospora crassa encodes a regulatory subunit of protein kinase A (PKA). Through a temperature-sensitive mcb mutant, it was found that at higher temperatures the growth polarity was lowered and the amount of extracellular protein was increased. Additionally, the hyphal tips were swollen. They state that secretion of protein can be increased by altering the polarity of growth and hypothesise that the subunit will release from the catalytic subunit when defected, resulting in increased PKA activity. As this gene was found in N. crassa a homolog gene pkaR in A. niger was found and selected for further work (Sun & Su, 2019; Lee et al., 1998).

ΔracA

racA is the Rho GTPase in A. niger, which controls actin dynamics and thereby regulates polarity. Through deletion and overexpression, the localization of actin was defected. In the knockout strain, the branching was more often dichotomous and the branching frequency increased resulting in less secretory vesicles per hyphea. It affected the endocytotic processes and the endocytotic ring was mislocated (Kwon et al., 2013).

ΔspaA

spaA might ensure polarity maintenance and thereby polar growth, and is required for maximal polar growth rate. Studies have shown that deletion of spaA caused lower radial colony growth rate and growth in more dense colony formations. The mutant also produced more apical and subapical branching and caused hyperbranching. Though important, the formation of hyphae is still occurring. When overexpressing the gene, the growth rate of hyphae decreases. Additionally, the hyphal polarity axes are destabilized affecting the growth direction. This happens when the amount of spaA is increased to the optimum or when it fails to localize only at the polar growth site. Interestingly, both mutants showed increased hyphal diameter (Meyer et al., 2008).

To study the effect of changing the expression level of the genes of interest, we designed to knock the genes out, as well as re-insert them using 2 different promoters from the DTU iGEM 2019 team LEAP’s promoters, to test down- and up-regulation. However, due to time limitations, only the knockouts were feasible, whereas the plasmids for inserts were designed but not finished. Below a table can be found which include previous engineering strategies and the novel engineering work performed during this project.

	Information	Previous Genetic Engineering	Novel Engineering Preformed
aplD (Cairns et al., 2019)	Gamma-adaptin	Knockout, Down- and Upregulation
arfA (Fiedler et al., 2018)	Small GTPase	Knockout, Down- and Upregulation
chsC (Sun et al., 2018)	Chitin synthase	Knockout	Double Knockout
gul-1 (Lin et al., 2018)	mRNA binding protein involved in cell wall remodelling	Knockout, Down- and Upregulation in N. crassa	Knockout and Double Knockout in A. niger
pkaR (Sun & Su, 2019; Lee et al., 1998)	Regulatory subunit of PKA	Downregulation in N. crassa	Knockout in A. niger
racA (Kwon et al., 2013)	Rho GTPase	Knockout, Down- and Upregulation
spaA (Meyer et al., 2008)	Might ensure polarity maintenance	Knockout, Down- and Upregulation	Double Knockout

Design of Plasmids for Knockout and Insertion

Each gene had properties that should be handled in different ways. To design the genetic modifications, we chose to use CRISPR/Cas-9 and USER cloning for both knockouts and insertions.

Knockouts

To create the knockouts, the crRNA sequences were designed to match each of the seven genes. For this, two protospacer sequences were found in the beginning and end of each gene and used as overhangs on the primers to create the components of gRNA and tRNA’s. Through PCR with these primers and the template pFC902 (BBa_K3385001) provided by DTU Bioengineering, we created biobricks to assemble the CRISPR knockout vector. The backbone of this vector was pFC330_pCas9_pyrG (BBa_K3385000), which encoded pyrG as a selection marker and contained the AMA1 sequence for plasmid replication in fungi (Nødvig et al., 2018). The mutants created through knockout engineering were tested. More information can be found under the section Measurements.

Insertions for Down- and Upregulation

For the creation of different expression levels, promoters from the DTU iGEM 2019 team, LEAP, were used . We chose a strong and a weak promoter in order to create up- and down-regulations. The strong promoter was PLEAPmstA_1 (BBa_K3046005) and the weak promoter was PLEAPunk_1 (BBa_K3046006).

The backbone of the plasmid was an inhouse plasmid. From this plasmid, we used the homologous recombination regions IS-NIG1 up and down for integration site 1 in A. niger. Additionally, the plasmid included the terminator (TtrpC), the ori sequence for E.coli replication and the ampR gene.

Biobricks were designed for the two promoters and inserted into the plasmid, creating a USER cloning site between promoter and terminator creating the plasmids pIGEM1_IS-NIG1_PLEAPmstA_USER_ttrpC and pIGEM1_IS-NIG1_PLEAPunk_1_USER_ttrpC. The final plasmids were then ready for insertion of the genes. The genes were amplified with USER overhangs and the final plasmids could be assembled together (pIGEM1_IS-NIG1_PLEAP(mstA/unk)_genename_ttrpC).

Another CRISPR-Cas9 vector was designed, which contained a crRNA targeting the integration site IS1. Hereby facilitating homologous recombination when adding the linear DNA of these HR regions and the construct in between. This would insert the up- or down-regulated genes, to investigate morphology changes. Due to time limitations, the plasmids were assembled but not inserted into A. niger.

Protein Secretion

Signal Peptides

Signal peptides (SPs) are relatively short amino acid sequences, 16-30 amino acids, located at the N-terminal of protein sequences. They are used by cells as a recognition signal for proteins, to localize the proteins in their functioning location.

SPs are important for exporting products out of the cell, through a process called exocytosis, easing the downstream processing as the cells do not need to be lysed to extract the product. Product extraction is often a costly process accounting for up to 70% of production costs in biopharmaceuticals. Therefore, finding a better SP - one that exports proteins in higher titers, and is able to export both homologous and heterologous proteins - is industrially relevant for cost optimization purposes. It should also be possible to introduce the SPs before any protein. Eukaryotes, such as A. niger, naturally produce and secrete proteins using signal peptides for degradation of substrates in their surrounding environment. Among the secreted proteins are carbohydrate degrading enzymes, such as cellobiohydrolases, β-xylosidases, β-glucosidases and endoglucanases (Borin et al., 2015).

Library Design

We wanted to improve protein secretion in A. niger. To select native signal peptides, we looked into the literature to find highly expressed proteins in A. niger (Borin et al., 2015). The protein sequence was found on aspgd.org (Cerqueira et al., 2014) and analyzed in SignalP-5.0 (Almagro Armenteros et al., 2019) to find the SP sequence. Our drylab team made a model to predict optimal SP sequences for secreting any protein, SignalPrepper, from which we selected 5 signal peptides.

Protein	Signal amino acid sequence
Native signal peptides in A. niger (Borin et al., 2015)
Feruloylesterase A (faeA)	MKQFSAKYALILLATAGQALA
Pectinesterase (pmeA)	MVKSILASVFFAATALA
Endopolygalacturonase I (pgaI)	MHSYQLLGLAAVGSLVSA
Exo-1,4-beta-xylosidase (xlnD)	MAHSMSRPVAATAAALLALALPQALA
Xyloglucan-specific endo-beta-1,4-glucanase A (eglA)	MKLPVTLAMLAATAMG
Beta-galactosidase (lacA)	MKLSSACAIALLAAQAAG
Endo-beta-1,4-glucanase B (eglB)	MKFQSTLLLAAAAGSALA
1,4-beta-D-glucan cellobiohydrolase A (cbhA)	MHQRALLFSALLTAVRA
1,4-beta-D-glucan cellobiohydrolase B (cbhB)	MSSFQVYRAALLLSILATANA
Glucoamylase (glaA)	MSFRSLLALSGLVCTGLA
Synthetic signal peptides - predicted by SignalPrepper
SigPower	MYQRFSILFLVLLFLLFDEALA
SigPilot	MRLHMLFLLAVALCLTLDTALA
SigPineapple	MVKMFSLAALIFLLITLDQALA
SigPeanut	MFRCGSLFLVLCLLLVVDRALA
SigPuppy	MSRWLLLCAILCICILMEPALA

To characterize these we had to design an expression platform. We chose to work with the native glucoamylase enzyme (encoded by glaA) to measure the secretion levels for the different signal peptides, as its activity can easily be measured through an assay, see our measurement page.For the promoter, we chose one from the DTU-Denmark 2019 teams big promoter library, PLEAPmstA_1 part BBa_K3046005. We used Ttef as the terminator, part BBa_K3046018. We wanted the expression platform to be integrated into the A. niger genome. An integration platform for A. niger was already designed by the DTU-Denmark 2019 team. Part BBa_K3046030 pGIA2P1 contains homologous regions for integration in the albA conidial pigment gene. Inserting into this gene therefore also serves as a selection marker as the colonies will have light conidiophores instead of dark ones.

As glucoamylase is a native enzyme in A. niger, we had to knock out the glaA gene to ensure that the activity we were seeing was due to our insert and not the natural production. For this we again used the CRISPR-Cas9 system as described in morphology section.

The first construct of our expression platform contained the native signal peptide for glaA. The platform is designed so that you can easily replace the signal peptide by primer extending PCR amplification and USER cloning. Primers, which were designed with overhangs containing the replacing signal peptide, were used to amplify the expression platform. It was then cloned together to form the expression platform containing the new signal peptides.

As it can be seen in the figure below, all the different signal peptide vectors were then linearized through PCR and inserted into A. niger in order to obtain all the mutants.

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