Modified xylanase exhibiting improved expression
A modified Family 11 xylanase enzyme comprising a sequence that introduces a functional consensus glycosylation site is provided. Non-limiting examples of introduced glycosylation sites include mutation of the amino acid at position 34, 131, 180, 182, or a combination thereof, to an asparagine. The indicated amino acid position in the Family 11 xylanase is determined from sequence alignment of the xylanase of interest with that of a Trichoderma reesei xylanase II amino acid sequence. The introduced consensus glycosylation site facilitates increased expression efficiency of the modified xylanase when compared to the expression efficiency of a corresponding xylanase from which the modified xylanase was derived, using similar host strains and growth conditions.
1. A method for increasing the expression efficiency of a Family 11 xylanase from a Trichoderma host comprising:
(a) introducing a functional consensus N-glycosylation site into a Family 11 xylanase coding region that is not found in a parental Family 11 xylanase coding region by substitution of the native amino acid at one or more of positions 34, 131, 180 and 182 with an asparagine, the position being determined by alignment of the Family 11 xylanase with the amino acid sequence of SEQ ID NO: 1, to produce a modified Family 11 xylanase coding region;
(b) incorporating a DNA encoding the modified Family 11 xylanase coding region produced in step (a) into a genetic construct comprising a promoter and a secretion signal sequence operatively linked to said modified Family 11 xylanase coding region;
(c) transforming a Trichoderma host microbe with the genetic construct of step (b) and selecting for genetically modified Trichoderma expressing the modified Family 11 xylanase; and
(d) culturing the genetically modified Trichoderma of step (c) in suitable medium to induce expression and secretion of the modified Family 11 xylanase
wherein the expression efficiency of the modified Family 11 xylanase is at least 40% higher than that of a parental Family 11 xylanase when expressed using the same method.
2. The method of claim 1 , wherein the Trichoderma host microbe is a member of the genus Trichoderma or Hypocrea.
3. The method of claim 1 , wherein the Trichoderma host microbe is Trichoderma reesei.
4. The method of claim 1 , wherein the secretion signal is a Trichoderma secretion signal.
5. The method of claim 4 , wherein the Trichoderma secretion signal is a xylanase secretion signal.
6. The method of claim 5 , wherein the xylanase secretion signal is a Trichoderma xylanase I secretion signal or a Trichoderma xylanase II secretion signal.
7. The method of claim 1 , wherein the promoter is selected from the group consisting of a Trichoderma cbh1 promoter, a cbh2 promoter, an eg1 promoter, an eg2 promoter, an eg3 promoter, an eg5 promoter, a xln1 promoter, a xln2 promoter, and a combination of at least two of these promoters.
8. The method of claim 1 , wherein the modified Family 11 xylanase coding region has the amino acid sequence of SEQ ID NO: 1 with the following amino acid substitutions:
N10H, Y27M, N29L, S75A, L105H, S125A, I129E, T131N, H144R, N157D, Q161R, Q162H and T165R;
N10H, Y27M, N29L, S75A, L105H, S125A, I129E, T131N, Y135R, H144R, N157D, Q161R, Q162H and T165R;
N10H, Y27M, N29L, S75A, L105H, S125A, I129E, H144R, N157D, Q161R, Q162H, T165R and F180N;
N10H, Y27M, N29L, S75A, L105H, S125A, I129E, H144R, N157D, Q161R, Q162H, T165R, F180N and S182T;
N10H, Y27M, N29L, S75A, L105H, S125A, I129E, H144R, N157D, Q161R, Q162H, T165R and S182N;
N10H, Y27M, N29L, S75A, L105H, S125A, I129E, H144R, N157D, Q161R, Q162H, T165R, S182N and S184T;
N10H, Y27M, N29L, Q34N, S75A, L105H, S125A, I129E, H144R, N157D, Q161R, Q162H and T165R; and
N10H, Y27M, N29L, Q34N, S36T, S75A, L105H, S125A, I129E, H144R, N157D, Q161R, Q162H and T165R.
9. The method of claim 1 , wherein the modified Family 11 coding region is Trichoderma reesei xylanase I with a substitution of threonine at position 118 with asparagine.
10. The method of claim 1 , wherein the modified Family 11 coding region is Streptomyces lividans xylanase C with a substitution of threonine at position 128 with asparagine.
11. The method of claim 1 , wherein the modified Family 11 coding region comprises X34N.
12. The method of claim 1 , wherein the modified Family 11 coding region comprises X131N.
13. The method of claim 1 , wherein the modified Family 11 coding region comprises X180N.
14. The method of claim 1 , wherein the modified Family 11 coding region comprises X182N.