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 composition comprising a lactic acid bacterium (LAB) or a Bifidobacterium , wherein the LAB or the Bifidobacterium lacks PtcC activity, wherein the LAB or the Bifidobacterium contains a functional heterologous trehalose-6-phosphate phosphatase gene, wherein the LAB or the Bifidobacterium further (a) constitutively overexpresses a gene encoding a trehalose transporter, or (b) lacks trehalose 6-phosphate phosphorylase (TrePP) activity, and wherein the composition is a food additive, a probiotic composition, or a starter culture.
2. The composition of claim 1 , wherein an endogenous ptcC gene encodes a PtcC having an amino acid sequence that is at least 90% identical to SEQ ID NO: 8.
3. The composition of claim 1 , wherein the endogenous ptcC gene 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 within the coding region;
v) introducing a frame shift mutation within the coding region; or
vi) introducing one or more missense or nonsense mutations within the coding region.
4. The composition claim 3 , wherein the endogenous ptcC gene is inactivated by inserting a premature stop codon within the coding region.
5. The composition of claim 1 , wherein the heterologous trehalose-6-phosphate phosphatase gene is an otsB gene.
6. The composition of claim 5 , wherein the otsB gene is an E. coli otsB gene.
7. The composition of claim 5 , wherein the otsB gene is inserted as a second cistron downstream of an endogenous usp45.
8. The composition of claim 1 , wherein the LAB or the Bifidobacterium constitutively overexpresses a gene encoding a trehalose transporter.
9. The composition of claim 8 , wherein the trehalose transporter is an endogenous trePTC transporter.
10. The composition of claim 8 , wherein the trehalose transporter has an amino acid sequence that is at least 90% identical to SEQ ID NO: 10 or 12.
11. The composition of claim 8 , wherein the gene encoding the trehalose transporter is operatively linked to a heterologous promoter.
12. The composition of claim 11 , wherein the heterologous promoter has a nucleic acid sequence that is at least 75% identical to SEQ ID NO: 13.
13. The composition of claim 8 , wherein the gene encoding the trehalose transporter is operatively linked to a hllA promoter (PhllA).
14. The composition of claim 8 , wherein the gene encoding the trehalose transporter is chromosomally located.
15. The composition of claim 1 , wherein the LAB or the Bifidobacterium lacks trehalose-6-phosphate phosphorylase (TrePP) activity.
16. The composition of claim 15 , wherein an endogenous trePP encodes a TrePP having an amino acid sequence that is at least 90% identical to SEQ ID NO: 2.
17. The composition of claim 1 , wherein the LAB or the Bifidobacterium comprises a nucleic acid encoding a heterologous prophylactic and/or therapeutic gene product.
18. The composition of claim 1 , wherein the LAB or the Bifidobacterium is dried, spray-dried, frozen, or freeze-dried.
19. The composition of claim 1 , wherein the LAB is a Lactococcus or a Lactobacillus.
20. The composition of claim 19 , wherein the LAB is a Lactococcus lactis.