Modified gram positive bacteria and uses thereof
The present invention relates to gram positive bacteria with increased stress resistance and/or improved storage characteristics. In particular, the invention relates to gram positive bacterium which accumulate intracellular trehalose. The gram positive bacterium according to the invention lack cellobiose-specific PTS system IIC component (PtcC)activity. The gram positive bacterium may further lack trehalose 6-phosphate phosphorylase (TrePP)activity. The gram positive bacterium may further overexpress trehalose transporters. The invention further relates to compositions comprising such gram positive bacterium as well as methods and uses thereof.
1. A method for improving a stress resistance or storage characteristic of a lactic acid bacterium (LAB) or Bifidobacterium , comprising a step of partially or completely deleting, disrupting, or inactivating an endogenous ptcC in the LAB or Bifidobacterium such that the LAB or Bifidobacterium is incapable of producing a functional PtcC;
wherein the LAB or Bifidobacterium further comprises at least one feature selected from the group consisting of:
(a) the LAB or Bifidobacterium constitutively overexpresses a gene encoding a trehalose transporter;
(b) an endogenous trePP has been partially or completely deleted, disrupted, or inactivated such that the LAB or Bifidobacterium is incapable of producing a functional trehalose 6 -phosphate phosphorylase (TrePP); and
(c) the LAB or Bifidobacterium contains a functional heterologous trehalose 6-phosphatase gene.
2. The method of claim 1 , wherein the endogenous ptcC encodes a PtcC having an amino acid sequence that is at least 90% identical to SEQ ID NO: 8.
3. The method of claim 1 , wherein the endogenous ptcC is inactivated by:
i) completely or partially removing the coding region;
ii) completely or partially removing or mutagenizing the promoter region;
iii) disrupting the coding sequence through insertional inactivation;
iv) inserting a premature stop codon;
v) introducing a frame shift mutation; or
vi) introducing one or more missense or nonsense mutations.
4. The method of claim 3 , wherein the endogenous ptcC is inactivated by inserting a premature stop codon.
5. The method of claim 1 , wherein the LAB or Bifidobacterium constitutively overexpresses a gene encoding a trehalose transporter.
6. The method of claim 5 , wherein the trehalose transporter is an endogenous trePTC transporter.
7. The method of claim 5 , wherein the trehalose transporter has an amino acid sequence that is at least 90% identical to SEQ ID NO: 10 or 12.
8. The method of claim 5 , wherein the gene encoding the trehalose transporter is operatively linked to a heterologous promoter.
9. The method of claim 8 , wherein the heterologous promoter has a nucleic acid sequence that is at least 75% identical to SEQ ID NO: 13.
10. The method of claim 5 , wherein the gene encoding the trehalose transporter is operatively linked to a hllA promoter (PhllA).
11. The method of claim 5 , wherein the gene encoding the trehalose transporter is chromosomally located.
12. The method of claim 1 , wherein the LAB or Bifidobacterium lacks trehalose-6-phosphate phosphorylase (TrePP) activity.
13. The method of claim 12 , wherein the endogenous trePP encodes a TrePP having an amino acid sequence that is at least 90% identical to SEQ ID NO: 2.
14. The method of claim 1 , wherein the LAB or Bifidobacterium comprises a functional heterologous trehalose-6-phosphate phosphatase gene.
15. The method of claim 14 , wherein the heterologous trehalose-6-phosphate phosphatase gene is otsB.
16. The method of claim 15 , wherein the otsB is E. coli otsB.
17. The method of claim 15 , wherein the otsB is inserted as a second cistron downstream of an endogenous usp45.
18. The method of claim 1 , wherein the LAB or Bifidobacterium comprises a nucleic acid encoding a heterologous prophylactic and/or therapeutic gene product.
19. The method of claim 1 , wherein the LAB is a Lactococcus or Lactobacillus.
20. The method of claim 19 , wherein the LAB is a Lactococcus lactis.
21. The method of claim 1 , wherein the stress resistance or storage characteristic is selected from the group consisting of: resistance to acid conditions, resistance to bile salts, resistance to drying, freezing, or freeze-drying, and osmotic resistance.
22. The method of claim 2 , wherein the LAB is a Lactococcus lactis.
23. The method of claim 3 , wherein the LAB is a Lactococcus lactis.
24. The method of claim 5 , wherein the LAB is a Lactococcus lactis.
25. The method of claim 6 , wherein the LAB is a Lactococcus lactis.
26. The method of claim 7 , wherein the LAB is a Lactococcus lactis.
27. The method of claim 8 , wherein the LAB is a Lactococcus lactis.
28. The method of claim 9 , wherein the LAB is a Lactococcus lactis.
29. The method of claim 10 , wherein the LAB is a Lactococcus lactis.
30. The method of claim 11 , wherein the LAB is a Lactococcus lactis.
31. The method of claim 12 , wherein the LAB is a Lactococcus lactis.
32. The method of claim 13 , wherein the LAB is a Lactococcus lactis.
33. The method of claim 14 , wherein the LAB is a Lactococcus lactis.
34. The method of claim 15 , wherein the LAB is a Lactococcus lactis.
35. The method of claim 16 , wherein the LAB is a Lactococcus lactis.
36. The method of claim 17 , wherein the LAB is a Lactococcus lactis.
37. The method of claim 18 , wherein the LAB is a Lactococcus lactis.
38. The method of claim 21 , wherein the LAB is a Lactococcus lactis.