Part:BBa_K3075003

LXYL-P1-2- SpyT-His

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1462
  Illegal PstI site found at 1499
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1462
  Illegal PstI site found at 1499
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1062
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1462
  Illegal PstI site found at 1499
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1462
  Illegal PstI site found at 1499
  Illegal NgoMIV site found at 168
  
• 1000
  COMPATIBLE WITH RFC[1000]

Introduction

LXYL-p1-2-SpyT-His consists of the enzyme Beta-D-xylosidase fused to a C-terminal short polypeptide tag (Spytag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. The additional SpyTag enables the enzyme to be attached to the Assemblase scaffold designed by the 2018 UNSW iGEM team via a Spy-Catcher-Tag conjugation system.

Figure 1: A visualisation of the attachment of LXYL-p1-2-SpyT-His (green) fusion protein to the Assemblase scaffold (red) via the Spy-Catcher-Tag system. Graphic produced by Linda Chen 2019.

The Hexahistidine tag is a common additive due to its high affinity for metal ions used in the purification technique of immobilized metal affinity chromatography (IMAC). Ni2+ ions were used for his-tag purification due to its high yield.

Usage and Biology

In Lentinula edodes, Beta-D-xylosidase (LXYL) catalyses the hydrolysis of O-glycosyl bonds. The native function of LXYL was to catalyse the reaction of 7-beta-xylosyl-10-deacetylbaccatin III (XDB) to 10-deacetylbaccatin III (DB). The LXYL-p1-2 mutant has been optimised to release the β-xylosyl group of 7-β-xylosyltaxanes (3). Specifically, the beta-hydrolase activity has been exploited to catalyse the removal of the xylose group from 7-beta-xylosyl-10-deacetyltaxol (XDT) to produce the intermediate 10-deacetyltaxol (DT) (3) (Figure 2). Recombinant LXYL-P1-2 has a sequence of 803 amino acid residues with a molecular mass of 85,975 Da.

Figure 2: 7-beta-xylosidase catalyses the hydrolysis of 7-beta-xylosyl-10-deacetyltaxol (XDT) to 10-deacetyltaxol (DT). Figure obtained from Ping Zhu (2017).

Characterisation

pET19b vector provided by Dr Dominic Glover was linearised by PCR amplification. Linear gene fragments were purchased from Integrated DNA technologies (IDT). The gene constructs were assembled into the pET19b expression vector at the multiple cloning site via Gibson assembly with a 3-fold excess of insert. Gibson products were transformed into high efficiency T7 Express E. coli by heat shocking at 42°C and plated on ampicillin supplemented agar plates for selection. This resulted in seven (LXYL) transformant colonies, compared to zero colonies on the linear pET19b transformant negative control. Three colonies of LXYL transformants were screened by colony PCR, where LXYL colonies revealed bands of an estimated size 1000 bp, which is below the expected size of 2528 bp, showing the colonies did not contain the desired LXYL gene (Figure omitted).

Assembly of LXYL was re-attempted, by increasing the insert to vector ratio to 5:1 and screening more colonies by colony PCR. Attempts did not succeed.

As LXYL is a larger gene fragment (2.5kb), ligation into pET19b (5kb) would require a 2X excess of LXYL, instead of the 3X excess. However, excessive amounts of insert would unlikely to be the cause for non-assembly. It may be of benefit to increase the Gibson assembly incubation time to ensure there is sufficient time for the complementary overhangs to properly anneal between the insert and vector.

References

1. Lxyl-p1-2 - Beta-D-xylosidase/beta-D-glucosidase - Lentinula edodes (Shiitake mushroom) - Lxyl-p1-2 gene & protein [Internet]. Uniprot.org. 2019 [cited 22 October 2019]. Available from: https://www.uniprot.org/uniprot/G8GLP2
2. Li B, Wang H, Gong T, Chen J, Chen T, Yang J et al. Improving 10-deacetylbaccatin III-10-β-O-acetyltransferase catalytic fitness for Taxol production. 2019.
3. Cheng H, Zhao R, Chen T, Yu W, Wang F, Cheng K et al. Cloning and Characterization of the Glycoside Hydrolases That Remove Xylosyl Groups from 7-β-xylosyl-10-deacetyltaxol and Its Analogues. Molecular & Cellular Proteomics. 2013;12(8):2236-2248.
4. Chen J, Liang X, Wang F, Wen Y, Chen T, Liu W et al. Combinatorial mutation on the β-glycosidase specific to 7-β-xylosyltaxanes and increasing the mutated enzyme production by engineering the recombinant yeast. Acta Pharmaceutica Sinica B. 2019;9(3):626-638.