Genetically modified bacterial cells and methods useful for producing indigoidine
The present invention provides for a genetically modified bacterial host cell capable of producing indigoidine, wherein the host cell comprises a non-ribosomal peptide synthetase (NRPS) that converts glutamine to indigoidine, and the bacterial host cell is reduced in its expression of one or more of the sixteen indicated enzymes.
1. A genetically modified Pseudomonas host cell capable of producing indigoidine, wherein the host cell comprises a Bacillus subtilis 4′-phosphopantetheinyl transferase (Sfp) and a non-ribosomal peptide synthetase (NRPS) comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:1, and the amino acid sequence SRRLEREV (SEQ ID NO:3), or SRRLEREVAQESSRLVRLHAE (SEQ ID NO:4), at the position corresponding to 1005 to 1012, or 1005 to 1025, of SEQ ID NO:1, respectively, wherein the NRPS converts glutamine to indigoidine, and the Pseudomonas host cell is reduced in its expression of the following enzymes: Glucose dehydrogenase (ubiquinone 8 as acceptor, periplasm); Phosphoenolpyruvate synthase; Malate dehydrogenase; Malate dehydrogenase (ubiquinone 8 as acceptor); Malic enzyme NADP (malate dehydrogenase (oxaloacetate-decarboxylating)(NADP+)); and Transaldolase.
2. The genetically modified Pseudomonas host cell of claim 1 wherein the Pseudomonas cell is a Pseudomonas putida.
3. The genetically modified Pseudomonas host cell of claim 1 , wherein the NRPS is a bacterial NRPS.
4. The genetically modified Pseudomonas host cell of claim 3 , wherein the NRPS is a Streptomyces lavendulae NRPS (BpsA).
5. The genetically modified Pseudomonas host cell of claim 1 , wherein the NRPS comprises a conserved domain, such as the amino acid sequence APRTETEKEI AEVWAKSLRR ESVSVQDDFF ESGGNSLIAV GLIRELNSRL GVSLPLQSVL ESPTVEKLSR RLEREV (SEQ ID NO:2), at the position corresponding to 937 to 1012 of SEQ ID NO:1.
6. A method for a genetically modified Pseudomonas host cell producing indigoidine, comprising (a) providing the genetically modified Pseudomonas host cell of claim 1 , (b) culturing or growing the host cell in a suitable culture or medium such that indigoidine is produced, and (c) optionally extracting or separating the indigoidine from the rest of the culture or medium, and/or host cell.
7. The method of claim 6 , wherein the providing step (a) comprises introducing a nucleic acid encoding the NRPS operatively linked to a promoter capable of expressing the NRPS in the host cell into the host cell.
8. The method of claim 6 , wherein the culturing or growing step (b) comprises the host cell growing by respiratory cell growth.
9. The method of claim 6 , wherein the culturing or growing step (b) takes place in a batch process or a fed-batch process, such as a high-gravity fed-batch process.
10. The method of claim 6 , wherein the culture or medium comprises hydrolysates derived or obtained from a biomass, such as a lignocellulosic biomass.
11. The method of claim 6 , wherein the culture or medium comprises one or more carbon sources, such as a sugar, such as glucose or galactose, or glycerol, or a mixture thereof.
12. The method of claim 11 , wherein the carbon source is fermentable; in some embodiments, the carbon source is non-fermentable.
13. The method of claim 6 , wherein the culture or medium comprises urea as a nitrogen course.
14. The method of claim 6 , comprising introducing a nucleic acid encoding the NRPS operatively linked to a promoter capable of expressing the NRPS in the host cell into the host cell.
15. The genetically modified Pseudomonas host cell of claim 1 , wherein the NRPS comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:1.
16. The genetically modified Pseudomonas host cell of claim 15 , wherein the NRPS comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:1.
17. The genetically modified Pseudomonas host cell of claim 16 , wherein the NRPS comprises the amino acid sequence of SEQ ID NO:1.
18. The genetically modified Pseudomonas host cell of claim 1 , wherein the Pseudomonas host cell is further reduced in its expression of one or more of the following enzymes or enzymes catalyzing the indicated reactions: N acetylornithine deacetylase; Ornithine Decarboxylase; Proline dehydrogenase; Poly 3 hydroxyalkanoate polymerase 3 Hydroxybutanoyl CoA (poly(3-hydroxyalkanoate) polymerase 1); 1,6 anhydrous N Acetylmuramate kinase; D lactate transport via proton symport periplasm; Carboxylic acid dissociation; and HCO3 equilibration reaction.