Part:BBa_K5314002

AmAGS2 (AGS2)

AGS2-1 is a specific isoform of α-glucan synthase encoded by the AmAGS2 (AGS2) gene in Aureobasidium melanogenum. The protein exhibits a complex architecture, featuring the amylase domain (Amy_D), which is crucial for catalyzing the hydrolysis of starch and related polysaccharides, and the glycogen synthetase domain (Gys_D), responsible for transferring glucose units to growing α-glucan chains. Additionally, the protein has transmembrane regions that house the exopolysaccharide transporter domain (EPST_D), which facilitates the transport of synthesized polysaccharides out of the cell.[1]

This enzyme plays a crucial role in the biosynthetic pathway for pullulan, a polysaccharide known for its film-forming and stabilizing properties, widely used in food and pharmaceutical applications. AGS2-1 catalyzes the elongation of carbon chains through the addition of UDP-glucose, resulting in the polymerization of α-glucan, which is then secreted extracellularly.[2]

In our project, we employed a gene knockdown strategy to silence the AGS2-1 gene, leading to the cessation of pullulan production. This genetic modification resulted in a significant increase in β-glucan yield, a polysaccharide with numerous health benefits, including immunomodulatory and cholesterol-lowering effects. Furthermore, the reduction in pullulan synthesis minimized the accumulation of unwanted byproducts, thereby improving the overall efficiency of the metabolic pathway.

Looking ahead, future research teams could explore targeted modulation of AGS2-1 expression levels to fine-tune the balance between pullulan and β-glucan production. By optimizing this pathway, we can enhance the yield of valuable polysaccharides.

By Cre-Lox recombination, we replaced the Pullulan key synthesis gene (AGS2-1) in the genome of Aureobasidium melanogenum BZW (knock out PKS controlling melanin synthesis) with an antibiotic resistance gene (NAT) with promoter and polyA tail. The NAT gene will be removed by the action of Cre enzyme.

Fig 1. PKS (A) and AGS2-1(B) knock-out agarose gel electrophoresis verification diagram. The right-hand side of BZ-11 is adjacent to 1 kb DNA ladder：From top to bottom 10 kb、8 kb、7 kb、6 kb、5 kb、4 kb、3 kb、2 kb、1 kb

Fig 2. Infrared spectroscopy results (BZ-11 is wild-type, BZW Δags2-1/2 knockdown of PKS, controlling the synthesis of melanin AGS2-1 and AGS2-2 genes).

Fig 3. HPLC analysis (BZ-11 is wild-type, BZW Δags1/2 is knockdown of PKS, AGS2-1 and AGS2-2 genes).

Source

Organism: Aureobasidium melanogenum BZ-11
GenBank: WNV28174.1

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1461
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1461
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 580
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1461
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1461
  Illegal NgoMIV site found at 2155
  Illegal AgeI site found at 357
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 641
  Illegal BsaI site found at 2315
  Illegal BsaI site found at 2434
  Illegal BsaI site found at 3073
  Illegal BsaI.rc site found at 1741
  Illegal SapI site found at 2758
  Illegal SapI.rc site found at 2248
  

References:
[1] Chen, T.-J., Liu, G.-L., Wei, X., Wang, K., Hu, Z., Chi, Z., & Chi, Z.-M. (2020b). A multidomain α-glucan synthetase 2 (AmAgs2) is the key enzyme for pullulan biosynthesis in Aureobasidium melanogenum P16. International Journal of Biological Macromolecules, 150, 1037–1045. https://doi.org/10.1016/j.ijbiomac.2019.10.108

[2] Chen, S., Zheng, H., Gao, J., Song, H., & Bai, W. (2023). High-level production of pullulan and its biosynthesis regulation in Aureobasidium pullulans BL06. Frontiers in bioengineering and biotechnology, 11, 1131875. https://doi.org/10.3389/fbioe.2023.1131875