Catalog
In order to enhance Aureobasidium melanogenum BZ-11 as a more efficient β-glucan producer, we have engineered it accordingly. Our workflow is outlined in Figure 1. It primarily involves the identification of the strain, knockout of genes related to melanin and pullulan synthesis, optimization of the culture medium, scaled-up cultivation, and the design of VHb.
 |
|
Figure 1: Our Design Approach |
We employ Aureobasidium melanogenum BZ-11 as our chassis strain, which is a safe and established microbial platform. Strain BZ-11 is known for its high yield of extracellular polysaccharides, including a significant content of β-glucans, making it a promising candidate for β-glucan production. However, BZ-11 also produces melanin and pullulan, which, when incorporated into the extracellular polysaccharides, can adversely affect the purity of the glucan product[1][2].
 |
|
Figure 2: Melanin and pullulan limited BZ-11 to become a better β-glucan producer |
After reviewing the literature and experimental validation, we discovered that BZ-11 secretes melanin extracellularly. The incorporation of melanin into the extracellular polysaccharides makes it difficult to separate, affecting the purity and final color of the glucan product. Therefore, we designed a strategy to inhibit its melanin synthesis pathway while ensuring the normal survival of the strain. Through the modeling and analysis of the dry laboratory group, we ultimately decided to knock out the PKS gene in its genome. The PKS gene encodes polyketide synthase, which primarily catalyzes the reaction Acetyl-CoA + Malonyl-CoA → 1,3,6,8-THN. In the absence of polyketide synthase enzyme, the synthesis of melanin is inhibited. The strain lacking the PKS gene can no longer secrete melanin and does not significantly affect the normal life activities of the strain.
 |
|
Figure 3: Schematic representation of the synthesis pathways for pullulan, β-glucan, and melanin in Aureobasidium melanogenum BZ-11. |
In addition to this, the synthesis of pullulan is also one of the limiting factors for strain BZ-11. The synthetic pathways for β-glucan and pullulan include a common precursor substance—UDPG. After dry laboratory group modeling and analysis, we designed the knockout of two copies of the AGS2 gene. The AGS2 gene encodes alpha-glucan synthase, which primarily catalyzes the reaction Short Alpha-1,3-glucan → Precursor-pullulan. With the knockout of AGS2, UDPG, the common precursor for pullulan and β-glucan synthesis, will be utilized more for the synthesis of β-glucan. This not only increases the purity of β-glucan but also enhances its production. The strain lacking the AGS2 gene can no longer secrete pullulan and does not significantly affect the normal life activities of the strain.
We have designed a homologous recombination approach to replace the PKS and AGS2 genes in the BZ-11 genome with resistance genes from the knockout vector, thereby achieving the goal of knocking out the target genes. The initial intention of the BEST AB-Free method is related to reducing antibiotic resistance; therefore, we have incorporated the Cre-loxp system into the knockout vector design. By introducing this system, we will significantly reduce the use of resistance genes during the experimental process and ensure that the final BZWΔags2-1/2 strain does not carry any introduced resistance genes. This has a positive impact on both laboratory safety and microbial safety.
Based on the Cre-loxp system, we have designed a "homologous recombination-antibiotic elimination-homologous recombination" cycle for the consecutive knockout of PKS, AGS2-1, and AGS2-2.
 |
|
Figure 4: homologous recombination-antibiotic elimination-homologous recombination cycle |
We plan to introduce the NAT gene between two homologous arms for the selection of successfully recombined strains, and on both sides of the NAT gene, we have also designed two loxp sites facing the same direction. Once homologous recombination is successful, the PKS gene will be disrupted and unable to function properly, replaced by the NAT gene located between the two directly oriented loxp sites. After strain selection, the Cre expression vector PAMCRG-1 plasmid will be introduced. At this point, the plasmid entering the cell will express the Cre gene, and the Cre enzyme can mediate the cyclization of the NAT gene between the two loxp sites. The cyclized NAT gene, like the Cre expression vector, will undergo plasmid loss. Ultimately, a knockout strain without the NAT gene and without expressing the Cre enzyme will be obtained. At this time, the knockout vector for the AGS2-1/2 gene can be introduced again, and the NAT gene can continue to be used for strain selection, reducing the introduction of resistance genes.
It is worth mentioning that in our "homologous recombination-antibiotic elimination-homologous recombination" cycle, the expression of Cre enzyme needs to be phased rather than constantly working [3]. Otherwise, during the next homologous recombination, the knockout vector that has just entered the cell has not had time to undergo homologous recombination and has already been circularized by Cre enzyme, failing to achieve the purpose of gene knockout. In addition, Cre enzyme itself has certain toxicity, and its long-term presence can be harmful to the cell[4].
 |
|
Figure 5: Figure: Potential outcomes if the Cre recombinase continues to function. |
Previous studies often used episomal plasmids and/or inducible promoters to control the expression of Cre gene, but this method is difficult to implement in Aureobasidium melanogenum. Therefore, we chose to control the expression of Cre by taking advantage of the characteristic of plasmid loss. We designed the ColE1 Ori in the expression vector to help E. coli amplify the plasmid, while we did not design a replication origin recognized by Aureobasidium melanogenum. Therefore, during the amplification process of the strain, the Cre expression vector will not be amplified and will eventually be lost, terminating the expression of Cre gene.
Through literature review, we have discovered that the introduction of hemoglobin proteins such as Vitreoscilla Hemoglobin (VHb) can significantly increase the production of pullulan in Aureobasidium melanogenum[5]. Given the significant overlap in the synthesis pathways of β-glucan and pullulan in BZ-11 (from glucose-6-phosphate to UDP-G), we hypothesize that after blocking the pullulan synthesis pathway, the introduction of an hemoglobin protein could increase the production of β-glucan in the BZWΔags2-1/2 strain. Moreover, in actual fermentation processes, the viscous extracellular polysaccharides may affect the efficiency of oxygen uptake by the strain. Ultimately, combining the modeling and analysis of the dry laboratory group, we designed to introduce the VHb protein into BZWΔags2-1/2. Considering the viscous extracellular polysaccharide environment inside the fermentation tank, we innovatively designed the targeted introduction of VHb, locating some of the VHb on the cell wall and some in the cytoplasm to achieve continuous oxygen transport and maximize the strain's oxygen uptake capacity。
 |
|
Figure 6: Cell wall and intracellular VHb were introduced to increase oxygen uptake. |
The Glycosylphosphatidylinositol (GPI) anchor constitutes a complex structure that comprises a phosphoethanolamine linkage, a carbohydrate core, and a lipid moiety, playing a pivotal role in the subcellular positioning and modulation of certain proteins[6]. This anchor is a common feature in eukaryotic organisms, predominantly found among numerous membrane-bound proteins, where it secures proteins to the extracellular aspect of the plasma membrane.
Proteins that are affixed with a GPI anchor are designated as GPI-anchored proteins. In the process of synthesizing these proteins, an N-terminal leader sequence guides the synthesis towards the endoplasmic reticulum[7].
The synthesis and attachment of GPI to proteins occur within the endoplasmic reticulum. The linkage of GPI to proteins is facilitated by the GPI-transamidase (GPI-TA) enzyme complex, which identifies and removes the C-terminal GPI attachment signal from the precursor proteins[8]. Subsequently, GPI is appended to the newly revealed C-terminus of the proteins[8].
 |
|
Figure 7: The formation process of mature GPI-anchored proteins. (adapted from previous work[8]) |
A significant number of glycosylphosphatidylinositol (GPI)-anchored proteins in fungi ultimately localize to the outermost compartment, the cell wall [9]. Enzymes belonging to the Dfg5 subfamily are responsible for the critical process of transferring GPI-anchored substrates from the plasma membrane to the cell wall. These enzymes also distinguish between GPI-anchored proteins that remain within the plasma membrane and those that are relocated to the cell wall (GPI-CWPs)[9]. Using this whole set of mechanisms, we designed the introduction vector of GPI-anchored cell wall VHb.
Through sequence comparisons, we have identified several types of GPI-anchored cell wall proteins (GPI-CWPs) in Aureobasidium melanogenum[10][11]. Subsequently, we designed a fusion protein in which the C-terminus of the VHb is linked to the GPI attachment signal peptide via a linker, and the N-terminus is connected to the amino-terminal signal peptide.
The fusion protein we constructed theoretically can assist VHb in binding to the cell wall of BZWΔags2-1/2, providing the cell with a more oxygen-rich microenvironment, thereby enhancing the production of glucan.
Based on the work of the dry laboratory group, we selected three cell wall anchor proteins and constructed a test vector for these anchor proteins. Since there is limited research on cell wall localization in Aureobasidium melanogenum, we initially designed to use EGFP for the functional validation of the anchor function.
 |
|
Figure 8: Construction of anchor protein test vectors |
After fluorescence observation, we can select the best performing anchor protein for the construction of VHb expression vector. Eventually, we can achieve the anchoring of VHb on the cell wall.
[1] Zhou, R., Ma, L., Qin, X., Zhu, H., Chen, G., Liang, Z., & Zeng, W. (2023). Efficient Production of Melanin by Aureobasidium Melanogenum Using a Simplified Medium and pH-Controlled Fermentation Strategy with the Cell Morphology Analysis. Applied Biochemistry and Biotechnology, 196(2), 1122–1141. https://doi.org/10.1007/s12010-023-04594-8
[2] Jiang, H., Chen, T., Chi, Z., Hu, Z., Liu, G., Sun, Y., Zhang, S., & Chi, Z. (2019). Macromolecular pullulan produced by Aureobasidium melanogenum 13-2 isolated from the Taklimakan desert and its crucial roles in resistance to the stress treatments. International Journal of Biological Macromolecules, 135, 429–436. https://doi.org/10.1016/j.ijbiomac.2019.05.190
[3] Zhang, Z., Lu, Y., Chi, Z., Liu, G., Jiang, H., Hu, Z., & Chi, Z. (2019b). Genome editing of different strains of Aureobasidium melanogenum using an efficient Cre/loxp site-specific recombination system. Fungal Biology, 123(10), 723–731. https://doi.org/10.1016/j.funbio.2019.06.001
[4] Rashbrook, V. S., Brash, J. T., & Ruhrberg, C. (2022). Cre toxicity in mouse models of cardiovascular physiology and disease. Nature Cardiovascular Research, 1(9), 806–816. https://doi.org/10.1038/s44161-022-00125-6
[5] Xue, S., Jiang, H., Chen, L., Ge, N., Liu, G., Hu, Z., Chi, Z., & Chi, Z. (2019b). Over-expression of Vitreoscilla hemoglobin (VHb) and flavohemoglobin (FHb) genes greatly enhances pullulan production. International Journal of Biological Macromolecules, 132, 701–709. https://doi.org/10.1016/j.ijbiomac.2019.04.007
[6] Ikezawa, H. (2002). Glycosylphosphatidylinositol (GPI)-Anchored proteins. Biological and Pharmaceutical Bulletin, 25(4), 409–417. https://doi.org/10.1248/bpb.25.409
[7] Howell, S., Lanctôt, C., Boileau, G., & Crine, P. (1994b). A cleavable N-terminal signal peptide is not a prerequisite for the biosynthesis of glycosylphosphatidylinositol-anchored proteins. Journal of Biological Chemistry, 269(25), 16993–16996. https://doi.org/10.1016/s0021-9258(17)32508-5
[8] Liu, S., Jin, F., Liu, Y., Murakami, Y., Sugita, Y., Kato, T., Gao, X., Kinoshita, T., Hattori, M., & Fujita, M. (2021). Functional analysis of the GPI transamidase complex by screening for amino acid mutations in each subunit. Molecules, 26(18), 5462. https://doi.org/10.3390/molecules26185462
[9] Vogt, M. S., Schmitz, G. F., Silva, D. V., Mösch, H., & Essen, L. (2020). Structural base for the transfer of GPI-anchored glycoproteins into fungal cell walls. Proceedings of the National Academy of Sciences, 117(36), 22061–22067. https://doi.org/10.1073/pnas.2010661117
[10] Fujii, T., Shimoi, H., & Iimura, Y. (1999). Structure of the glucan-binding sugar chain of Tip1p, a cell wall protein of Saccharomyces cerevisiae. Biochimica Et Biophysica Acta (BBA) - General Subjects, 1427(2), 133–144. https://doi.org/10.1016/s0304-4165(99)00012-4
[11] Jaafar, L., & Zueco, J. (2003). Characterization of a glycosylphosphatidylinositol-bound cell-wall protein (GPI-CWP) in Yarrowia lipolytica. Microbiology, 150(1), 53–60. https://doi.org/10.1099/mic.0.26430-0