RECOMBINANT NONPATHOGENIC BACTERIUM

PSEUDOMONAS CHLORORAPHIS

BACKGROUND OF THE INVENTION

Field of the Invention

[0001] This invention relates to the genes encoding enzymes and regulatory proteins involved in dirhanmose-lipi biosynthesis. This invention also relates to a recombinant, nonpathogenic Pse domonas chlororaph which is capable of producing RiL and R ₂ L.

Description of Related Art

[0002] Rhamnolipids are a family of rhamnose-eontamrog glycolipids produced mainly by bacteria in the Pseudomonadaceae family, especially those belonging to the Pseudomonas genus. The lipid portion of most rhamnolipids contain 3-hydroxyaikaiioyl-3-iiydiOxyalkanoate (C _x -C _y , where x and y are the carbon chain lengths of the alkaiioate) moiety, though some rhamnolipids may contain only a monomelic 3-hydroxyalkanoate. Furthermore, rhanmolipid could also be synthesized with either one (RiL) or two (R ₂ L) rliamnose molecules. Abdel- Mawgoud et o/., 2010, Applied Microbiology and Biotechnology, 86:1323-1336, contains a smrmiary of ihaiiinolipid varieties synthesized by various organisms. The structure of an R ₂ L, i.e., a-L-ihanniopyranosyl-(l→2)-a-L-riiaiiinopyranosyl-3-hydiOx y

hydroxydecanoate (R2-C10-C10), as an illustration, follows:

[0003] Rhamnolipids have many potential uses, most of which are associated with rhanmolipid's excellent surface-active properties. See, e.g., Faivre and Rosilio 2010, Expert. Opinion on Drug Delivery, 7: 1031-1048; Lourith and Kaniayavattanakiil 2009; Nguyen and Sabatini 2011,

Internationa! Journal of Cosmetic Science, 31:255-261 ; and Pinzon el al. 2009 In Hayes et al. (ed.). Biobased Surfactants and Detergents: Synthesis. Properties, and Applications, Chapter 4, pp. 77-105. AOCS Press, Urbana, IL. Rhanmolipids may also possess valuable biological activities useful in wound healing (see Stipeevie, et t ^' . 2006. Burns 32:24-34; see also U.S. Patent 7.262,171), antibacterial (Sotkova, et al. 2008. Curr. Microbiol. 56:639-644; Vatsa, et ah 2010. Int. J. Mol. Set 11:5095-5108), and fungicidal (Takemoto. et al 2010. Am. J. Enol Vitic. 61 : 120 - 124; Yoo _. , et al 2005. J. Microbiol Biotechnol. 15:1164-1169) applications.

[0004] A putative metabolic pathway of rhaumolipid biosynthesis in P. aeruginosa is shown in Figure 1. The precursor pool for rhanmoiipid synthesis is proposed to be the fatty acid de novo biosynthesis pathway that could provide 3-ketoacyl-acyl carrier-protein (-ACP) metabolites with varying chain length of the acyl group. A β-fcetoacyl reductase enzyme (RlilG) coded by the rhlG gene reduces the keto functional group into a hydroxyl group (Campos-Garcfa, et al. 1998. J. of Bacteriology, 180:4442-4451 ; Miller, et al. 2006. J. of Biological Chemistry 281:18025- 18032). Two molecules of 3-hydroxyacyl-ACP are then condensed to yield 3-hydroxyalkanoyl- 3-hydiOxyaikanoate (3-HHA) by the rhaii osjd transferase A enzyme (RhlA) (Deziel, et al. 2003. Microbiology 149:2005-2013; Zhu and Rock 2008. J. of Bacteriology 190:3147-3154]. A riianmose moiety from an activated sugar precursor,. dTDP-rhanmose, is attached to 3-HHA to form RiL via the enzymatic action of rhan osy .transferase B (RhlB) (Cabrera- Valladares, et al. 2006. App . Microbiol. Biotechnol. 73:187-194). Finally, biosynthesis of R?L is accomplished by the transfer of another rhanmose moiety from dTDP-rhanmose to RiL by the action of rhanniosyltransferase C (RhlC) (Cabrera- Valladares, et al 2006). Biochemical details of certain reaction steps in the pathway are still unclear. For example, it is not clear whether the active form of 3-FfHA is attached to an ACP or not. Furthermore, the chemical-energy requirement in terms of high bond-energy molecules (e.g., ATP or dTTP) is not understood, leaving unanswered the question of how the dTDP-rhamnose molecule used as a substrate by RhlB and RhlC is regenerated. [0005] Nevertheless, the overall picture of the metabolic pathway has allowed the genetic manipulation of bacteria to affect rhaiimolipid synthesis. An early study demonstrated that several r anmolipid-nonproducing bacteria (i.e. P. aeruginosa PG201 , P.flnorescens ATC

15453, P. oleavorans GPol, P. putida KT2442, Escherichia coli DH5ot, Mid E. coli W21 0) could be genetically engineered to produce rhaiimolipids through the expressicai of heterologous rhlA and rh!B genes from . aeruginosa (Ochsner, et al. 1995. AppL Environ. Microbiol.

61 :3503-3506: Cabrera- Valiadares, et al. 2006). Wang, et l. integrated . aeruginosa rhlA and rhlB genes into the chromosome of E. coli BL21(DE3) and P. aeruginosa PAQl-rhlA ^~ (Wang, ei ah 2007. Biotechnology and Bioengineering 98:842-853). While the rhlA-rhlB -complemented P. aeruginosa transformant synthesized the same rhaiimolipid mixture as that found in the wild- type P. aeruginosa, the E. coli irensformant produced predominantly rhamnolipids having CKT Cio (ca. 60%) as the lipid component yields. Cha, et at expressed rhlABRI gene cluster from P. aeruginosa EMSI in P. putida 1067 to show production of rhamnolipids without detailing the compositions of the products (Cha, et al 2008. Bioresource Technology' 99:2192-2199).

[0006] P. aeruginosa is the most commonly studied organism for rhaiimolipid biosynthesis. Various high-yield strains of P. aeruginosa have been adopted for large-scale produc tion (Mtiller. et ah 2011. Applied Microbiology and Biotechnology 89:585-592). In view of

rhamno lipid's potential applications in food (see U.S. Patent 5,658,793) and medical (Stipe evic, et al. 2006; see U.S. Patent 7,262,171) areas, a method for producing RiL using Pseudomonas chlororaphis, a nonpathogenic bacterium was previously developed (Gunther, et al. 2005. AppL Environ. Microbiol. 71: 2288-2293; U.S. Patent 7,202,063]. Despite this progress, there is still a need to develop a method for R ₂ L in nonpathogenic bacterium. This invention identifies several genes involved in rhamiiolipid biosynthesis and covers recombinant nonpathogenic bacteria capable of producing RjL in a 10- fo d improved yield (in comparison to the original P.

chlororaphis strain described in U.S. Patent 7,202,063) or producing ?L, as a result of introduction of the appropriate DNA into the bac terium, thereby significantly lowering the production cost of RiL and broadening the application sphere of rhamnolipids.

BRIEF SUMMARY OF THE INVENTION [0007] it is an object of this invention to have a polynucleotide encoding riiamnosyltransferase A. It is a further object of this invention that the polynucleotide has the sequence set forth in SEQ ID NO: 12, the full length complement of SEQ ID NO: 12, and the reverse complement of SEQ ID NO: 12. It is also an object of this invention that the polynucleotide has a sequence that is at least 95 % identical to the sequence in SEQ ID NO: 12, at least 90% identical to the sequence in SEQ ID NO: 12 and at least 85% identical to the sequence in SEQ ID NO: 12.

[0008] It is an object of this invention to have a polynucleotide encoding rhamnosyltransferase A. It is a further object of this invention that the polynucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO: 1 1, a sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 11 , a sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 11 ; and a sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: I 1.

[0009] It is another object of t is invention to have an expression vector containing a

polynucleotide that encodes for rhamnosyltransferase A, the nucleotide sequence of SEQ ID NO: 12, the full-length complement of SEQ ID NO: 12, the reverse complement of SEQ ID NO: 12, a sequence that is at least 95% identical to SEQ ID NO: 12, a sequence thai is at least 90%

identical to SEQ ID NO: 12, a sequence that is at least 85% identical to SEQ ID NO: 12, that encodes the amino acid sequence set forth in SEQ ID NO: 11, a sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 11, a sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO; 1 1; and a sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 11.

[0010] It is another object of tins invention to have a recombinant cell that contains an expression vector winch contains a polynucleotide that encodes for rhamnosyltransferase A. the nucleotide sequence of SEQ ID NO: 12, the full-length complement of SEQ ID NO: 12, the reverse complement of SEQ ID NO: 12, a sequence that is at least 95% identical to SEQ ID NO 12, a sequence that is at least 90% identical to SEQ ID NO: 12, a sequence that is at least 85% identical to SEQ ID NO: 12, that encodes the amino acid sequence set forth in SEQ ID NO: 11, a sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: l l. a sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 11; and a sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 11.

[0011] it is a further object of this invention to have a polypeptide that is encoded by a

polynucleotide having the sequence of SEQ ID NO: 12, the full-length complement of SEQ ID NO: 12, the reverse complement of SEQ ID NO: 12. a sequence that is at least 95% identical to SEQ ID NO: 12, a sequence that is at least 90% identical to SEQ ID NO: 12, and a sequence that is at least 85% identical to SEQ ID NO: 12.

[0012] It is an object of this invention to have an riiaiimosylhaiisferase A polypeptide having the amino acid sequence set forth in SEQ ID NO: I I, an amino acid sequence that is at least 95% identical to SEQ ID NO: 11 , an amino acid sequence that is at least 90% ideiiticai to SEQ ID NO: 1 1 , and an amino acid sequence that is at least 85% identical to SEQ ID NO: 11.

[0013 j It is another object of this invention to have an expression vector containing a polynucleotide that encodes for the amino acid sequence set forth in SEQ ID NO: 11, an amino acid sequence that is at least 95% identical to SEQ ID NO: 11, an ammo acid sequence thai is at least 90% identical to SEQ ID NO: 11, and an amino acid sequence that is at least 85% identical to SEQ ID NO: 11.

[0014] it is a further object of this invention to have a recombinant cell containing an expression vector containing a polynucleotide that encodes for the amino acid sequence set forth in SEQ ID NO: 11, an amino acid sequence that is at least 95% identical to SEQ ID NO; 11, an amino acid sequence that is at least 90% identical to SEQ ID NO: 11 , and an amino acid sequence that is at least 85% identical to SEQ ID NO: 11.

[0015] it is an object of this invention to have a polynucleotide encoding rhamnosyltransferase B. It is a further object of tins invention that the polynucleotide has the sequence set forth in SEQ ID NO: 14, the full length complement of SEQ ID NO: 14, and the reverse complement of SEQ ID NO: 14. It is also an object of this invention that the polynucleotide has a sequence that is at least 95 % identical to the sequence in SEQ ID NO: 14, at least 90% identical to the sequence in SEQ ID NO: 14, and at least 85% identical to the sequence in SEQ ID NO: 14.

[0016] It is an object of this invention to have a polynucleotide encoding rhamnosyitransferase

B. It is a further object of this invention that the polynucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO: 13, a sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO 13. a sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13, and a sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 13.

[0017] It is another object of t is invention to have an expression vector containing a

polynucleotide that encodes for rharmiosyltransferase B, the nucleotide sequence of SEQ ID NO: 14, the full-length complement of SEQ ID NO: 14. the reverse complement of SEQ ID NO: 1 , a sequence that is at least 95% identical to SEQ ID NO: 14. a sequence thai is at least 90%

identical to SEQ ID NO: 14, a sequence that is at least 85% identical to SEQ ID NO: 14. that encodes the amino acid sequence set forth in SEQ ID NO: 13, a sequence thai is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 13, a sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13, and a sequence thai is at least 85% identical to the amino acid sequence set forth in SEQ ID NO; 13.

[0018] It is another object of this invention to have a recombinant cell that contains an expression vector which contains a polynucleotide that encodes for liia iiosyiiransferase B, the nucleotide sequence of SEQ ID NO: 14, the full-length complement of SEQ ID NO: 14, the reverse complement of SEQ ID NO: 14, a sequence that is at least 95% identical to SEQ ID NO 14, a sequence that is at least 90% identical to SEQ ID NO: 14, a sequence that is at least 85% identical to SEQ ID NO: 14, that encodes the ammo acid sequence set forth in SEQ ID NO: 13, a sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO 13, a sequence that is at least 90% identical to the ammo acid sequence set forth in SEQ ID NO: 13, and a sequence that is at least 85% identical to the ammo acid sequence set forth in SEQ ID NO: 13. [0019] it is a further object of this invention to have a polypeptide that is encoded by a

polynucleotide having the sequence of SEQ ID NO: 14, the full-length complement of SEQ ID NO: 14, the reverse complement of SEQ ID NO: 14, a sequence that is at least 95% identical to SEQ ID NO: 14, a sequence that is at least 90% identical to SEQ ID NO: 14, and a sequence thai is at least 85% identical to SEQ ID NO: 14.

[0020] it is an object of this invention to have a rhamnosyltransferase B polypeptide having the annuo acid sequence set forth in SEQ ID NO: 13, an amino acid sequence that is at least 95% identical to SEQ ID NO: 13, an amino acid sequence that is at least 90% identical to SEQ ID NO: 13, and an amino acid sequence that is at least 85% identical to SEQ ID NO: 13.

[0021] It is another object of this invention to have an expression vector containing a polynucleotide that encodes for the amino acid sequence set forth in SEQ ID NO: 13, an amino acid sequence that is at least 95% identical to SEQ ID NO: 13, an amino acid sequence that is at least 90% identical to SEQ ID NO: 13, and an amino acid sequence that is at least 85% identical to SEQ ID NO: 13.

[0022] It is a further object of this invention to have a recombinant ceil containing an expression vector containing a polynucleotide that encodes for the amino acid sequence set forth in SEQ ID NO: 13, an amino acid sequence that is at least 95% identical to SEQ ID NO: 13, an amino acid sequence that is at least 90% identical to SEQ ID NO: 13, and an amino acid sequence that is at least 85% identical to SEQ ID NO: 13.

[0023] it is an object of this invention to have a polynucleotide encoding an N-acyi-homoserine lactone-dependent transcription regulator. It is a further object of this invention that the

polynucleotide has the sequence set forth in SEQ ID NO: 16, the full length complement of SEQ ID NO: 16, and the reverse complement of SEQ ID NO: 16. It is also an object of this invention thai the polynucleotide has a sequence that is at least 95 % identical to the sequence in SEQ ID NO: 16, at least 90% identical to the sequence in SEQ ID NO: 16, and at least 85% identical to the sequence in SEQ ID NO: 16. [0024] it is an object of this invention to have a polynucleotide encoding N-acyl-homoserine iactone-dependent transcription regulator. It is a further object of this invention that the polynucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO: 15, a sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 15. a sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 15, and a sequence that is at least 85% ideniicai to the ammo acid sequence set forth in SEQ ID NO: 15.

[0025] It is another object of t is invention to have an expression vector containing a

polynucleotide that encodes for N-acyl-homoserine iactone-dependent transcription regulator, the nucleotide sequence of SEQ ID NO: 16, the full-length complement of SEQ ID NO: 16, the reverse complement of SEQ ID NO: 16, a sequence that is at least 95% identical to SEQ ID NO: 16, a sequence that is at least 90% identical to SEQ ID NO; 16, a sequence that is at least 85% identical to SEQ ID NO; 16, that encodes the ammo acid sequence set forth in SEQ ID NO: 15, a sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 15, a sequence that is at least 90% identical to the ammo acid sequence set forth in SEQ ID NO: 15, and a sequence that is at least 85% identical to the ammo acid sequence set forth in SEQ ID NO: 15.

[0026] It is another object of this invention to have a recombinant cell that contains an expression vector which contains a polynucleotide that encodes for N-acyl-homoserine iactone- dependent transcription regulator, the nucleotide sequence of SEQ ID NO: 16, the frill-length complement of SEQ ID NO; 16, the reverse complement of SEQ ID NO: 16, a sequence that is at least 95% identical to SEQ ID NO: 16, a sequence that is at least 90% identical to SEQ ID NO: 16, a sequence that is at least 85% identical to SEQ ID NO: 16, that encodes the amino acid sequence set forth in SEQ ID NO; 15, a sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 15, a sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO; 15, and a sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 15.

S [0027] it is a further object of this invention to have a polypeptide that is encoded by a

polynucleotide having the sequence of SEQ ID NO: 16, the full-length complement of SEQ ID NO: 16, the reverse complement of SEQ ID NO: 16, a sequence that is at least 95% identical to SEQ ID NO: 16, a sequence that is at least 90% identical to SEQ ID NO: 16, and a sequence that is at least 85% identical to SEQ ID NO: 16.

[0028] it is an object of this invention to have a N-acy -homoserine lactone-dependent transcription regulator polypeptide having the amino acid sequence set forth in SEQ ID NO: 15, an amino acid sequence that is at least 95% identical to SEQ ID NO: 15, an amino acid sequence that is at least 90% identical to SEQ ID NO: 15, and an amino acid sequence that is at least 85% identical to SEQ ID NO: 15.

[0029] It is another object of this invention to have an expression vector containing a

polynucleotide that encodes for the amino acid sequence set forth in SEQ ID NO: 15, an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 , an amino acid sequence that is at least 90% identical to SEQ ID NO: 15, and an amino acid sequence that is at least 85% identical to SEQ ID NO: 15.

[0030] It is a further object of this invention to have a recombinant cell containing an expression vector containing a polynucleotide that encodes for the amino acid sequence set forth in SEQ ID NO: 15, an amino acid sequence that is at least 95% identical to SEQ ID NO: 15, an amino acid sequence that is at least 90% identical to SEQ ID NO: 15, and an amino acid sequence that is at least 85% identical to SEQ ID NO: 15.

[0031] It is an object of this invention to have a recombinant cell containing an expression vec tor which contains a polynucleotide sequence encoding rhamnosyltransferase C. It is a further object of this invention to have a recombinant Pseudomanas ch!ororap is containing an expression vector which contains a polynucleotide sequence encoding rhamnosyltransferase C. such that the wild-type P. chlororaphis lacks the gene encoding rhamnosyltransferase C and such that the recombinant P. chlororaphis is capable of producing dirhamnose-lipid. [0032] it is a further object of this invention to have a recombinant P. chlororaphis containing an expression vector that encodes a rharnnosyltransf erase C operably linked to a promoter such that the recombinant/*, chlororaphis is capable of producing dirhamnose- lipid.

[0033] it is an object of this invention to have a novel method of producing dirhamnose-lipid by transfecting Pseudomonas chlororaphis with an expression vector which contains a

polynucleotide that encodes rhamnosyltransferase C to produce a recombinant P. chlororaphis,, and growing the recombinant P. chlororaphis in appropriate media for the production of monoihamnose-lipid and dirhamnose-lipid.

[0034] It is another object of this invention to have a novel method of producing dirhamnose- lipid by growing a recombinant P. chlororaphis in appropriate media, it is a further object of this invention that the recombinant P. chlororaphis contains an expression vector which contains a polynucleotide that encodes rfiamnosyltransferase C operably linked to a promoter and such that the media is appropriate for the production of monorhamnose-lipid and dirhamnose-lipid.

[0035] It is an object of this invention to have a recombinant Pseudomonas chlororaphis which contains an expression vector which contains one or more polynucleotides that encode a promoter, a polynucleotide that encodes rhamnosyltransferase A, and a polynucleotide that encodes rhamnosyltransferase B . It is a further object of this invention thai the polynucleotide encoding rhamnosyltransferase A and the polynucleotide encoding rhamnosyltransferase B are operably linked to one of the promoters in the expression vector.

[0036] it is another object of this invention to have a recombinant Pseudomonas chlororaphis which contains an expression vector which contains one or more polynucleotides that encode a promoter, a polynucleotide that encodes rhamnosyltransferase A, and a polynucleotide that encodes rhamnosyltransferase B. It is a further object of this invention that the polynucleotide encoding rhamnosyltransferase A and the polynucleotide encoding rhamnosyltransferase B are operably linked to one of the promoters in the expression vector and that the promoters are P2 promoters. [0037] it is further object of this invention to have a recombinant Pseudomonas cMoror phis which contains an expression vector which contains one or more polynucleotides that encode a promoter (one of which is a P2 promoter), a polynucleotide thai encodes l hainnosyltransferase A, and a polynucleotide that encodes rhamnosyliraiisferase B. It is a further object of tins invention that the polynucleotide encoding rhanmosyitransfera.se A and the polynucleotide encoding rhamnosyltiansferase B are contiguous and are operably linked to the same P2

promoter.

[0038] It is an object of this invention to have a recombinant Pseudomonas cMororaphis which contains an expression vector which contains one or more polynucleotides that encode a promoter, a polynucleotide that encodes rhamiiosyltransferase A, and a polynucleotide that encodes rhamnosyltransferase B . It is a further object of this invention that the polynucleotide encodmg rhamnosyltransferase A is operably linked to a first promoter in the expression vector, and the polynucleotide encoding rhamnosyltransferase B is operably linked to a second promoter in the expression vector, such that the first promoter and the second promoter can be the same promoter or different promoters.

[0039] It is a further object of this invention to have a novel method for producing

monorhamnose-lipid by P. cMororaphis by transfecting P. cMororaphis with an expression vector which contains at least one polynucleotide encoding a promoter, a polynucleotide

encoding rhamnosyltiansferase A. and a polynucleotide encoding rhamnosyltransferase B„ such thai the polynucleotide encoding rhamnosyltransferase A and the polynucleotide encoding rhamnosyltransferase B are operably linked to at least one promoter contained within the expression vector (winch generates a recombinant P. cMororaphis) and growing the recombinant P. cMororaphis hi an appropriate media (one appropriate for production of monorhamnose lipid by the recombinant P. cMororaphis).

[0040] it is a further object of this invention to have a novel method for producing

monorhamnose-lipid by P. cMororaphis by transfecting P. cMororaphis with an expression vector which contains at least one polynucleotide encoding a promoter, a polynucleotide encoding rhamnosyltransferase A. and a polynucleotide encoding rhamnosyltransferase B, such thai the polynucleotide encoding rhaniiiosyitransferase A and the polynucleotide encoding rhairmosyitransierase B are operabiy linked to at least one promoter contained within the expression vector (which generates a recombinant P. chlororaphis) and growing the recombinant P. chlororaphis hi an appropriate media (one appropriate for production of nionorhanmose lipid by the recombinant P. chlororaphis) under stirring conditions or non-stirring conditions.

[0041] it is an object of this invention to have a recombinant cell that contains an expression vector that contains a first promoter operabiy linked to a first polynucleotide sequence encoding rhanmosyltransferase A, and a second promoter operabiy linked to a second polynucleotide sequence encoding rhanmosyltransferase C, but the recombmaiit cell lacks a functioning wild- type rhlA.

[0042] It is an object of this invention to have a recombinant cell that contains an expression vector that contains a first promoter operabiy linked to a first polynucleotide sequence encoding rhanmosyltransferase A, and a second promoter operabiy linked to a second polynucleotide sequence encoding rhanmosyltransferase C, but the recombmaiit cell lacks a functioning wild- type rhlA. It is a further object of this invention that the first promoter is an inducible promoter and the second promoter is a constitutive promoter.

[0043] It is an object of this invention to have a recombinant cell that contains an expression vector that contams a first promoter operabiy linked to a first polynucleotide sequence encoding rhanmosyltransferase A, and a second promoter operabiy linked to a second polynucleotide sequence encoding rhanmosyltransferase C, but the recombinant cell lacks a functioning wild- type rhlA. It is a further object of this invention that the first promoter and the second promoter are different inducible promoters and that different compounds control the expression of the first inducible promoter and the second inducible promoter.

[0044] it is an object of this invention to have a recombinant cell that contains a first expression vector that contams a first promoter operabiy linked to a first polynucleotide sequence encoding rhanmosyltransferase A and a second expression vector that contams a second promoter operabiy linked to a second polynucleotide sequence encoding ilianrnosyltraiisferase C, but the recombinant cell lacks a functioning wild-type rhiA.

[0045] It is an object of this invention to have a recombinant cell that contains a first expression vector that contains a first promoter operably linked to a first polynucleotide sequence encoding rhanniosyltransfera.se A and a second expression vector that contains a second promoter operably linked to a second polynucleotide sequence encoding rhamnosyliraiisferase C, but the recombinant cell lacks a functioning wild-type rhiA. It is a further object of this invention that the first promoter is an inducible promoter and the second promoter is a constitutive promoter.

[0046] It is another object of this invention to have a recombinant cell that contains a first expression vector that contains a first promoter operably linked to a first polynucleotide sequence encoding rhamnosyltransferase A and a second expression vector that contains a second promoter operably linked to a second polynucleotide sequence encoding

rhamnosyltransferase C, but the recombinant cell lacks a functioning wild-type rhiA. It is a further object of this invention that the first promoter and the second promoter are different inducible promoters and that different compounds control the expression of the first inducible promoter and the second inducible promoter.

[0047] It is an object of this invention to have a recombinant cell that contains an expression vector that contains a first promoter operably linked to a first polynucleotide sequence encoding rhamnosyltransferase B, and a second promoter operably linked to a second polynucleotide sequence encoding rhamnosyltransferase C, but the recombinant cell lacks a functioning wild- type r l ^' B.

[0048] it is an object of this invention to have a recombinant cell that contains an expression vector that contains a first promoter operably linked to a first polynucleotide sequence encoding rhamnosyltransferase B, and a second promoter operably linked to a second polynucleotide sequence encoding rhamnosyltransferase C, but the recombinant cell lacks a functioning wild- type rhiB. It is a further object of this invention that the first promoter is an inducible promoter and the second promoter is a constitutive promoter. [0049] It is an object of this invention to have a recombinant cell that contains an expression vector that contains a first promoter operably linked to a first polynucleotide sequence encoding rhanmosyltransfera.se B, and a second promoter operably linked to a second polynucleotide sequence encoding rhamnosyltransferase C, but the recombinant cell lacks a functioning wild- type rhlB. It is a further object of this invention that the first promoter and the second promoter are different inducible promoters and that different compounds control the expression of the first inducible promoter and the second inducible promoter.

[0050] It is an object of this invention to have a recombinant cell that contains a first expression vector that contains a first promoter operably linked to a first polynucleotide sequence encoding rhamnosyltransferase B and a second expression vector that contains a second promoter operably linked to a second polynucleotide sequence encoding rhamnosyltransferase C, but the recombinant cell lacks a functioning wild-type rhlB.

[0051] It is an object of this invention to have a recombinant cell that contains a first expression vector that contains a first promoter operably linked to a first polynucleotide sequence encoding rhamnosyltransferase B and a second expression vector that contains a second promoter operably linked to a second polynucleotide sequence encoding rhamnosyltransferase C, but the recombinant cell lacks a functioning wild-type rhlB. It is a further object of this invention that the first promoter is an inducible promoter and the second promoter is a constitutive promoter.

[0052] It is another object of this invention to have a recombinant cell that contains a first expression vector that contains a first promoter operably linked to a first polynucleotide sequence encoding rhamnosyltransferase B a d a second expression vector that contains a second promotei- operably linked to a second polynucleotide sequence encoding

rhamnosyltransferase C, but the recombinant cell lacks a functioning wild-type rhlB. It is a further object of this invention that the first promoter and the second promoter are different inducible promoters and tha t different compounds control the expression of the first inducible promoter and the second inducible promoter. [0053] it is an object of this invention to have a recombinant cell that contains a first expression vector that contains a first promoter operabiy linked to both a first polynucleotide sequence encoding rhainnosyliraiisferase B and a third polynucleotide sequence encoding

rhanmosyltransfer A, and a second expression vec tor that contains a second promoter operabiy linked to a second polynucleotide sequence encoding rhamnosyliraiisierase C, but the recombinant cell lacks a functioning wild-type rhlA and rhlB.

[0054] It is an object of this invention to have a recombinant cell that contains a first expression vector that contains a first promoter operabiy linked to both a first polynucleotide sequence encoding rhamnosyliraiisferase B and a third polynucleotide sequence encoding

rhanmosyltransfer A. and a second expression vector that contains a second promoter operabiy linked to a second polynucleotide sequence encoding rhamnosyliraiisferase C, but the

recombinant cell lacks a functioning wild-type rhlA and rhlB. It is a further object of this invention that the first promoter is an inducible promoter and the second promoter is a constitutive promoter.

[0055] It is an object of this invention to have a recombinant cell that contains a first expression vector that contains a first promoter operabiy linked to both a first polynucleotide sequence encoding rhaiimosyliransferase B and a third polynucleotide sequence encoding

rhanmosyltransfer A, and a second expression vector that contains a second promoter operabiy linked to a second polynucleotide sequence encoding rhamnosyltransferase C, but the recombinant cell lacks a functioning wild-type rhlA and rhlB. It is a further object of this invention thai the first promoter and the second promoter are different inducible promoters and thai different compounds control the expression of the first inducible promoter and the second inducible promoter.

[0056] It is an object of this invention to have a recombinant cell that contains an expression vector that contains a first promoter operabiy linked to a first polynucleotide sequence encoding rhamnosyltransferase A and rhamnosyltransferase B, and a second promoter operabiy linked to a second polynucleotide sequence encoding rhamnosyltransferase C. but the recombinant cell lacks a functioning wild-type rhlA and rh!B. [0057] It is an object of this invention to have a recombinant cell that contains an expression vector that contains a first promoter operabiy linked to a first polynucleotide sequence encoding rhamnosyitransfera.se A and rhanmosyltransferase B, and a second promoter operabiy linked to a second polynucleotide sequence encoding rhamnosyltraiisferase C. but the recombinant cell lacks a functioning wild-type rh!A and rhlB. It is a further object of this invention that the first promoter is an inducible promoter and the second promoter is a constitutive promoter.

[0058] It is an object of this invention to have a recombinant cell that contains an expression vector thai contains a first promoier operabiy linked to a first polynucleotide sequence encoding rhamnosyltransferase A and ihamnosyltiansferase B, and a second promoter operabiy linked to a second polynucleotide sequence encoding rhamnosyliransferase C, but the recombinant cell lacks a functioning wild-type rhlA and rh!B, It is a further object of this invention that the first promoter and the second promoter are different inducible promoters and that different compounds control the expression of the first inducible promoier and the second inducible promoter.

[0059] It is a farther object of this invention to have a method for controlling the ratio of monorhamnose lipid to dirhainnose lipid produced by a recombinant cell. In particular the recombinant cell contains a expression vector that contains an inducible promoter operabiy linked to a first polynucleotide encoding rhamnosyiiransfera.se A and a constitutive promoter operabiy linked to a second polynucleotide encoding rhamnosyliransferase C. but lacks a functioning wild-type rhlA. The method involves culturing the recombinant cell in a media appropriate for production of rhamnolipids, adding a compound that controls the expression of the inducible promoter, and removing the compound that controls the expression of the inducible promoter at an appropriate time such that the production of nionorhamnose-lipid decreases or stops but production of dirhamnose-lipid is able to continue. It is another object of this invention that the recombinant cell has a functioning wild-type rblB.

[0060] It is another object of this invention to have a method for controlling the ratio of monorhamnose lipid to dirhainnose lipid produced by a recombinant cell. In particular the recombinant cell contains an expression vector that contains an inducible promoter operably linked to a first polynucleotide encoding rhamnosyitransfera.se B and a constitutive promoter operably linked to a second polynucleotide encoding rliarnnosyltransferase C. but lacks a functioning wild-type rhlB. The method involves eulhiriiig the recombinant cell in a media appropriate for production of rhamnolipids. adding a compound that controls the expression of the inducible promoter, and removing the compound that controls the expression of the inducible promoter at an appropriate time such that the produc tion of monorhamnose-lipid decreases or stops but production of dirhamnose-lipid is able to continue. It is another object of this invention that the recombmant cell has a functioning wild-type rh!A.

[00 1] It is a further object of this invention to have a method for controlling the ratio of monorhamnose lipid to dirhamnose lipid produced by a recombmant cell. I particular the recombmant cell contains an expression vector thai contains an inducible promoter operably linked to a first polynucleotide encoding rhamnosyltransf erase A and rhanmosyltransferase B and a constitutive promoter operably linked to a second polynucleotide encoding

riiarimosylhansferase C, but lacks a functioning wild-type rh!A and rhlB. The method mvolves culturing the recombmant cell in a media appropriate for production of rhamnolipids, adding a compound that controls the expression of the inducible promoter, and removing the compound that controls the expression of the inducible promoter at an appropria te time such that the production of monorhamnose-lipid decreases or stops but production of dirhamnose-lipid is able to continue.

[0062] It is a further object of this invention to have a method for controlling the ratio of monorhanmose lipid to dirhamnose lipid produced by a recombinant cell In particular the recombinant cell contains a first expression vector that contains an inducible promoter operably linked to a first polynucleotide encoding rliarnnosyltransferase A and a second expression vector that contains a constitutive promoter operably linked to a second polynucleotide encoding rhamnosyitiansferase C, but lacks a functioning wild-type rhlA. The method involves culturing the recombinant cell in a media appropriate for production of rhamnolipids, adding a compound that controls the expression of the inducible promoter, and removing the compound that controls the expression of the inducible promoter at an appropriate time such that the production of monorhamnose-lipid decreases or slops but production of dkha nnose-lipid is able to continue, it is another object of this invention that the recombinant cell has a functioning wild-type rhIB.

[0063] It is another object of this invention to have a method for controlling the ratio of

monorhamnose lipid to dirhainnose lipid produced by a recombinant cell, hi particular the recombinant cell contains a first expression vector that contains an inducible promoter operably linked to a first polynucleotide encoding rhamnosyltransferase B and a second expression vector that contains a constitutive promoter operably linked to a second polynucleotide encoding rhamnosyltransferase C, but lacks a functioning wild-type rhIB. The method involves eu! taring the recombinant cell in a media appropriate for production of rhamnolipids, adding a compound that controls the expression of the inducible promoter, and removing the compound thai controls the expression of the inducible promoter at an appropriate time such that the production of monorhamnose-lipid decreases or stops but productio of dirhamnose-iipkl is able to continue. It is another object of this invention thai the recombinant cell has a functioning wild-type rhlA.

[0064] It is a further object of this invention to have a method for controlling the ratio of monorhamnose lipid to dirhainnose lipid produced by a recombinant cell. In particular the recombinant cell contains a first expression vector that contains an inducible promoter operably linked to a first polynucleotide encoding rhamnosyltransferase A and rhanmosyltransferase B and a second expression vector containing a constitutive promoter operably linked to a second polynucleotide encoding rhamnosyltransferase C, but lacks a functioning wild-type rhlA and rhIB. The method involves culturing the recombinant cell in a media appropriate for production of rhamnolipids, addin a compound that controls the expression of the inducible promoter, and removing the compoimd that controls the expression of the inducible promoter at an appropriate time such that the production of monorhamnose-lipid decreases or stops but production of dkhamnose-iipid is able to continue.

[0065] it is a further object of this invention to have a metiiod for controlling the ratio of monorhamnose lipid to dirhainnose lipid produced by a recombinant cell. In particular the recombmant cell contains an expression vector that contains a first inducible promoter operably linked to a first polynucleotide encoding rhamnosyltransferase A and a second inducible

IS promoter operably linked to a second polynucleotide encoding rhamnosyltraiisferase C, but lacks a functioning wild-type rhlA . The method involves cuiturin the recombinant ceil in a media appropriate for production of rhamnolipids. adding a first compound that controls the expression of the first inducible promoter, adding a second compound that controls the expression of the second inducible promoter at a first appropriate time, and removing the first compound that controls the expression of the first inducible promoter at a second appropriate time suc that the production of monorhaninose- lipid decreases or stops but production of dirhamnose-iipid is able to continue and such that the first appropriate time and the second appropriate time and either may precede or be subsequent to each other. It is another object of this invention that the recombinant cell has a functioning wild-type rhlB.

[0066] It is a further object of this invention to have a method for controlling the ratio of monorhamnose lipid to dirhamnose lipid produced by a recombinant cell. In particular the recombinant cell contains an expression vector that contains a first inducible promoter operably linked to a first polynucleotide encoding rhamnosyitransferase B and a second inducible promoter operably linked to a second polynucleotide encoding rhamnosyitransferase C, but lacks a fimctioiiing wild-type rhlB. The method involves cultiuing the recombinant cell in a media appropriate for production of rhamnolipids, adding a first compound tha t controls the expression of the first inducible promoter, adding a second compound that controls the expression of the second inducible promoter at a first appropriate time, and removing the first compound that controls the expression of the first inducible promoter at a second appropriate time such that the production of monorhanmose- lipid decreases or stops but production of dirhamnose-iipid is able to continue and such that the first appropriate time and the second appropriate time and either may precede or be subsequent to each other. It is another object of this invention that the recombinant cell has a functioning wild-type rhlA.

[0067] It is a further object of this invention to have a method for controlling the ratio of monorhamnose lipid to diihamnose lipid produced by a recombinant cell In particular the recombinant cell contains an expression vector that contains a first inducible promoter operably linked to a first polynucleotide encoding rhamnosyitransferase A and rhamnosyitransferase B, and a second inducible promoter operably linked to a second polynucleotide encoding rhairmosyltransferase C, but lacks a functioning wild-type rhlA and rhlB. The method involves culturing the recombinant cell in a media appropriate for production of rhaiiinolipids. adding a fust compound that controls the expression of the first inducible promoter, adding a second compound that controls the expression of the second inducible promoter at a first appropriate time, and removing the first compound that controls the expression of the first inducible promoter at a second appropriate time such that the production of monorhamnose-lipid decreases or stops but production of dkharnnose-lipid is able to continue and such that the first appropriate time and the second appropriate time and either may precede or be subsequent to each other.

[0068] It is a further object of this invention to have a method for controlling the ratio of monorhamnose lipid to dirhairtnose lipid produced by a recombinant cell. In particular the recombinant cell contains a first expression vector that contains a first inducible promoter operably linked to a first polynucleotide encoding ihamnosyltiansferase A and a second expressio vector that contains a second inducible promoter operably linked to a second polynucleotide encoding ihamnosyltiansferase C, but lacks a functioning wild-type rhlA. The method involves culturing the recombinant cell in a media appropriate for production of rhamnolipids, adding to the media a first compoimd thai controls the expressio of the first inducible promoter, adding a second compound that controls the expressio of the second inducible promoter to the media at a first appropriate time, and removing the first compound that controls the expressio of the first inducible promoter from the media at second appropriate time such that the first appropriate time is independent of and may precede or be subsequent to the second appropriate time. It is another object of this invention that the recombmant cell has a functioning wild-type rhlB.

[0069] It is a further object of this invention to have a method for controlling the ratio of monorhamnose lipid to dirhamnose lipid produced by a recombinant cell hi particular the recombmant cell contains a first expression vector that contains a first inducible promoter operably linked to a first polynucleotide encoding lhamnosyltransferase B and a second expression vector that contains a second inducible promoter operably linked to a second polynucleotide encoding rhanniosyitransfera.se C, but lacks a functioning wild-type rhlB. The method involves culturing the recombinant ceil in a media appropriate for production of rharnnolipids, adding to the media a first compound that controls tlie expression of tlie first inducible promoter, adding a second compound that controls the expression of the second inducible promoter to tlie media at a first appropriate time, and removing the first compound that controls the expression of the first inducible promoter fiom tlie media at second appropriate time such that the first appropriate time is independent of and may precede or be subsequent to the second appropriate time. It is another object of this invention that the recombinant cell has a functioning wild-type ;·///..1

[0070] It is a further object of this invention to have a method for controlling the ratio of monorhamiiose lipid to dirhamnose lipid produced by a recombinant cell. In particular the recombinant cell contains a first expression vector that contains a first inducible promoter operably linked to a first polynucleotide encoding both riiarimosyltraiisfeiase A and

rhamnosyltransferase B, and a second expression vector that contains a second inducible promoter operably linked to a second polynucleotide encoding rhamnosyltransferase C, but lacks a functioning wild-type rhLi and rhlB. The method involves culturing the recombinant cell in a media appropiiate for production of rhamnolipids. adding to the media a first compound that controls the expression of the first inducible promoter, adding a second compound that controls the expression of the second inducible promoter to the media at a first appropriate time, and removing the fir st compound that controls the expression of the first inducible promoter from the media at second appropriate time such that the first appropriate time is independent of and may precede or be subsequent to the second appropriate time.

[0071] It is an object of this invention to have a recombinant Pseudomonas chloror phis containing an expression vector which contains a promoter operably linked to a polynucleotide that encodes an -acyl-homoserine lactone dependent transcription regulator from P.

chororaphis subsp. aureofacie , such that the recombinant P. chlororaphis produces monorhamnose-iipid under stirring conditions. It is a further object of this invention that the recombinant P. chlororaphis contains functioning rhlA and rhlB.

[0072] it is an object of this invention to have a recombinant Pseudomonas chlororaphis containing an expression vector which contains a promoter operably linked to a polynucleotide thai encodes an N-acyl-homoserine lactone dependent transcription regulator from P.

chororaphis subsp. aureofaciem and contains a promoter operably linked to a polynucleotide encoding rhainnosyliraiisferase C, such that the recombinant P. chiororaphis produces monorhaninose-lipid and dirhamnose-lipid under stirring conditions. It is a further object of this invention that the recombinant P. chiororaphis contains functioning rh!A and rhlB.

[0073] it is an object of this invention to have a recombinant Pseudomonas chiororaphis containing a fir st expression vector which contains a promoter operably linked to a

polynucleotide that encodes an N-acyl-homoserine lactone dependent transcription regulator from P. chororaphis subsp. aureofaciem and contains a second expression vector which contains a promoter operably linked to a polynucleotide encoding rhamnosy!transferase C, such that the recombinant P. chiororaphis produces monorhamnose-lipid and dirhamnose-lipid under stirring conditions. It. is a further object of this inventio that the recombinant. P. chiororaphis contains functioning rhlA and rhlB.

[0074] A method for producing monorhamnose-lipid under stirring conditions comprising cuituring a recombinant Pseudamonas chiororaphis in appropriate media under stirring

conditions. The recombinant .P. chiororaphis contains an expression vector which contains a promoter operably linked to a polynucleotide thai encodes an N-acyl-homoserine lactone dependent transcription regulator from P. chororaphis subsp. aureofaciem, and contains functioning rhlA and rhIS.

[0075] A method for producing monorhamnose-lipid and dirhamnose-lipid under stirring conditions comprising cuituring a recombinant Pseudotnonas chiororaphis in appropriate media under stirring conditions. The recombinant P. chiororaphis contains an expression vector which contains a promoter operably linked to a polynucleotide that encodes an N-acyl-homoserine lactone dependent transcription regulator from P. chororaphis subsp. aureofaciem and a promoter operably linked to polynucleotide encoding rhamnosyltransferase C, and contains functioning rhiA and rhlB, [0076] A method for producing monorhamriose- lipid and dirhamnose-lipid under stirring conditions comprising culmrmg a recombinant Pseiido onas chlororaphis in appropriate media under stirring conditions. The recombinant P. chlororaphis contains a first expression vector which contains a promoter operably linked to a polynucleotide that encodes an N-acyi- homoserine lactone dependent transcription regulator from P. chororaphis subsp. aareofaciens, a second expression vector which contains a promoter operably iinked to polynucleotide encoding rhamnosyltransferase C, and functioning rhlA and rhlB.

BRIEF DESCRIPTION OF THE FIGURES

[0077] Figure 1 is the putative metabolic pathway of rhamiiolipid biosynthesis.

[0078] Figure 2 is the gene locus of . chlororaphis NRRL B-30761 showing the relative positions of amplicons A. B, C, and D. In Figure 2, acnB is a (3 '-partial) aconitate hydra ase gene; rhtAp _d , is rharnnosyltransferase A (where Pch = P, chlororaphis); rhlBp _d , is

rharimosyltiansfeiase B: r/?/j¾¾¾ is N-acyl-homoserine iactone-dependent transcription regulator; and RBS is iibosomal-biading-site.

[0079] Figure 3 is a liquid chromatography mass spectroscopy (LC-MS) spectrum of silica cohmin-pmified iL from the control strain P. chlororaphis ί[ρΒ829-Ρ2-¾ρ] (top) and R?L from P. chlororaphis [pBS29-P2-r/?/Cp _ae ] (bottom). Letters A-F refer to the compositions of the alkyl chains: A=C8/C10, B=C10/C10, C=C10 C12: 1, D=C10/C12:0, E=C12:1/C12:0, F=C12:0/C12:0.

[0080] Figure 4A is a HPLC chromatogram of crude rhamnolipids produced by P. chlororaphis NRRL B-30761 under non-stirring conditions. Figure 4B is a HPLC chromatogram of crude rhamnolipids produced by P. chlororaphis :[pBS29-P2-r 4Z?] clone 1 unde non-stirring conditions. Figure 4C is a HPLC chromatogram of crude rhamnolipids produced by P.

chlororaphis [pBS29-P2-r 42?] clone 2 under non-stirring conditions.

[0081] Figure 5 shows integrated areas of rhamnolipid peaks of P. chlororaphis NRRL B-30761 and its pBS29-P2-r ? 5 recombinant strains (clones 1 and 2) cultured under non-stirring conditions . Monorhanmolipids (Rh 1 -C 10-C 12: 1 and Rh 1 -C 10-C 12} eluted at retention times of about 36.5 and 37.8 iiiin, respectively.

[0082] Figure 6 shows the production of monorhanmolipids by . chlororaphis [pBS29-P2- rhiAB] recombinant strain under static growth and at 200 rpni rotary shaking.

DETAILED DESCRIPTION OF THE INVENTION

[0083] A "protein" or "polypeptide" is a chain of amnio acids arranged in a specific order determined by the coding sequence in a polynucleotide encoding the protein or polypeptide. Each protein or polypeptide lias a unique function.

[0084] The expression "heterologous nucleic acid sequence", "heterologous polynucleotide ^" " or "heterologous gene" as used herein, refers to a gene or polynucleotide or nucleic acid sequence that is not in its natural environment (in other words, has been altered by the hand of man), hi an exemplary embodiment, a heterologous polynucleotide is a polynucleotide from one species that is introduced into another species. In another exemplary embodiment, a heterologous polynucleotide can be a nucleic acid sequence joined to a regulatory elenient(s) e.g., a promoter, that is not found naturally associated with the polynucleotide. Heterologous genes, heterologous polynucleotides, heterologous nucleic acid sequences are typically produced using recombinant DNA techniques.

[0085] The terms "isolated", "purified", or "biologically pur e" as used herein, refer to material thai is substantially or essentially free from components that normally accompany the material in its native state. In an exemplary embodiment, purity and homogeneity are determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A nucleic acid that is the predominant species present in a preparation is substantially purified, hi an exemplary embodiment, the term "purified" denotes that a nucleic acid or protein gives rise to essentially one band in an eiectrophoretic gel. Typically, isolated nucleic acids or proteins ha ve a level of purity expressed as a range. The lower end of the range of purity for the component is about 60%, about 70% or about 80% and the upper end of the range of purity is about 70%, about 80%, about 90% or more than about 90%.

[0086] The term "nucleic acid" as used herein, refers to a polymer of ribonucleotides or deoxyribonucleotides. Typically,, "nucleic acid" polymers occur in either single- or double- stranded form, but are also known to form structures comprising three or more strands. The term "nucleic acid" includes naturally occurring nucleic acid polymers as well as nucleic acids comprising known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, whicli have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Exemplary analogs include, without limitation, phosphorothioaies, phosphoramidates, methyl phosphonates, chiial-methyl phosphonates, 2-O-methyl

ribonucleotides, pepfide-nucleic acids (PNAs). " DNA", "RNA", "polynucleotides",

"polynucleotide sequence", "oligonucleotide", "nucleotide", "nucleic acid", "nucleic acid molecule", "nucleic acid sequence", "nucleic acid fragment ^" ", and "isolated nucleic acid fragment" are used interchangeably herein.

[0087] Unless otherwise indicated, a particular nucleic acid sequence also implicitly

encompasses conservatively modified variants thereof (e.g.. degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generatin sequences in which the third position of one or more selected (or ail) codons is substituted with mixed-base and/or deoxyinosine residues (see e.g., Batzer <?? «/. 1991. Nucleic Acid Res. 19:5081 ; Ohtsuka et at 1985. J. Biol. Chem.

260:2605-2608: and Rosso!ini et al. 1994. Mol. Cell. Probes 8:91-98).

[0088] in addition to the degenerate nature of the nucleotide codons which encode amino acids, alterations in a polynucleotide that result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded polypeptide, are well known in the art. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine or histidine, can also be expected to produce a functionally equivalent protein or polypeptide.

[0089] The term "label" as used herein, refers to a composition detectable by spectroscopic, photochemical, biochemical, nmimochemicaL or chemical means. Exemplary labels include ^" , fluorescent dyes, electron-dense reagents, enzymes (e.g... as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available.

[0090] As used herein a nucleic acid "probe", oligonucleotide ''probe", or simply a "probe" refers to a nucleic acid capable of binding to a target nucleic acid of complementar sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe may include nainral (i.e., A, G, C, or T) or modified bases (e.g., 7-deazaguanosine, inosiiie, etc.). In addition, the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization. Thus, for example, probes may be peptide nucleic acids in which the constituent bases are j ined by peptide bonds rather than phosphodiester linkages. It will be understood by one of skill in the art that probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. In one exemplary embodiment, probes are directly labeled as with isotopes, cln omophores, lumiphores, cliromogens etc . In other exemplary embodiments probes are indirectly labeled e.g., with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence.

[0091] Thus, the term "probe" as used herein refers to a probe that is bound, either eovalently, through a linker or a chemical bond, or noncovalentiy, through ionic, van der Waals,

electrostatic, or hydr ogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe. [0092] The term "primer" as used herein, refers to short nucleic acids, typically a DNA oligonucleotide of at least about 15 nucleotides in length. In an exemplary embodiment, primers are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand. Annealed primers are then extended along the target DNA strand by a DNA polymerase enzyme. Primer pahs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PGR) or other nucleic-acid amplification methods known in the art.

[0093] PGR primer pairs are typically derived from a known sequence, for example, by using computer programs intended for thai purpose such as Piimer (Version 0.5 (01991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). One of ordinary skill in the art will appreciate that the specificity of a particular probe or primer increases with its length. Thus, for example, a primer comprising 20 consecutive nucleotides of a promoter complex sequence will anneal io a related target sequence with a higher specificity than a corresponding piimer of only 15 nucleotides. Thus, in an exemplar embodiment, greater specificity of a nucleic acid primer or probe is attained with probes and primers selected to comprise 20, 25, 30, 35, 40, 50 or more consecutive nucleotides of a selected sequence.

[0094] Nucleic acid probes and primers are readily prepared based on the nucleic acid sequences disclosed herein. Methods for preparing and using probes and primers and for labeling and guidance in the choice of labels appropriate for various purposes are discussed, e.g.., in Sambrook e.f at, Molecular Cloning, A Laboratory Manual 2nd ed. 1989, Cold Spring Harbor Laboratory; and Current Protocols in Molecular Biology, Ausubel et i, eds., 1994, John Wiley & Sons). The term "recombinant" when used with reference, e.g., to a ceil, or nucleic acid, protein, or vector, indicates that the cell, organism, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the ceil is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinaiit or wild-type) form of the cell or express native genes that are otherwise abnormally expressed, over expressed, under expressed or not expressed a t ail. [0095] The terms "transgenic", "transformed' \ "transfomiation", "transformed" and "transfeetion" are similar- in meaning to "recombinant". "Transformation", "transgenic", and "transfection" refers to the transfer of a poiymicleotide into the genome of a host organism or into a cell. Such a transfer of polynucleotides can result in genetically stable inheritance of the polynucleotides or in the polynucleotides remaining exte-ehrornosonially (not integrated into the chromosome of the cell). Genetically stable inheritance may potentially require the transgenic organism or cell to be subj ect for a period of time to one or more conditions which require the transcription of some or ail of transferred polynucleotide in order for the transgenic organism or cell to live and/or grow. Polynucleotides that are transformed into a cell but are not integrated into the host's chromosome remain as an expression vector within the cell. One may need to grow the cell under certai conditions in order for the expression vector to remain i the cell or the cell's progeny. Further, for expressio to occur the organism or ceil may need to be kept under certain conditions. Host organisms or cells containing the recombinant polynucleotide ca be referred to as "transgenic" or "transformed" organisms or cells or simply as "transformants", as well as recombinant organisms or cells.

[0096] A method of selecting an isolated polynucleotide that affects the level of expression of a polypeptide in a vims or in a host ceil (eukaryotic, such as plant, yeast, fungi, or algae;

prokaryotic, such as bacteria) may include the steps of: constructing an isolated polynucleotide of the present invention; introducing the isoiated polynucleotide into a host ceil; measuring the level of a polypeptide in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide in the host cell containing the isolated polynucleotide with the level of a polypeptide in a host cell that does not contain the isolated polynucleotide.

[0097] An "expression cassette" as used herein, refers to a nucleic acid construct typically generated recombinantly or synthetically, which comprises a series of specified nucleic acid elements that permit tr anscription of a particular nucleic acid hi a host cell

[0098] Typically, an "expression cassette" is part of an "expression vector". An expression vector or simply a "vector", as used herein, refers to nucleic acid capable of replicating in a selected host cell or organism. An expression vector can replicate as an autonomous structure, or alternatively can integrate into the host cell chromosomes or the nucleic acids of an organelle, and thus replicate along with the host cell genome. Thus, an expression vector are

polynucleotides capable of replicating in a selected host cell, organelle, or organism, e.g., a plasniid, virus, artificial chromosome, nucleic acid fragment, and for which certain genes on the expression vector are transcribed and tr anslated into a polypeptide or protein within the cell, organelle or organism; or any suitable construct known in the art, which comprises an

"expression cassette".

[0099] The term "capable of hybridizing under stringent hybridization conditions" as used herein, refers to annealing a first nucleic acid to a second nucleic acid under stringent hybridization conditions (defined below), hi an exemplary embodiment, the first nucleic acid is a test sample, and the second nucleic acid is the sense or antisense strand of a nucleic acid of interest. Hybridiza tion of the first and second nucleic acids is conducted under standard stringent conditions, e.g., high temperature and/or low salt content, which tend to disfavor hybridization of dissimilar nucleotide sequences.

[00100] Any expression vector containing the polynucleotides described herein operabiy linked to a promoter is also covered by this inve tion. A polynucleotide sequence is operabiy linked to an expression control sequence(s) (e.g., a promoter and, optionally, an enhancer) when the expression control sequence controls and regulates the tiansciiption and translation of that polynucleotide sequence. An expression vector is a replicon, such as plasniid, phage or cosmid, and which contains the desired polynucleotide sequence operabiy linked to the expression control sequence(s). The promoter may be, or is identical to, a viral, phage, bacterial, yeast, insect, plant, or mammalian promoter. Similarly, the enhancer may be the sequences of an enhancer from virus, phage, bacteria, yeast, insects, plants, or mammals.

[00101] The term "operabiy linked" refers to the association of two or more nucleic acid fragments on a single polynucleotide so that t e function of one is affected by the other. For example, a promoter is operabiy linked with a coding sequence so that the promoter is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operabiy linked to regulatory sequences in sense or antisense orientation. When a promoter is operabiy linked to a polynucleotide sequence encoding a protein or polypeptide, the polynucleotide sequence should have an appropriate start signal (e.g.. ATG) in front of the polynucleotide sequence to be expressed. Further, the sequences should be in the correct reading frame to permit transcription of the polynucleotide sequence under the control of the expression control sequence and.

translation of the desired polypeptide or protein encoded by the polynucleotide sequence. If a gene or polynucleotide sequence that one desires to insert into an expression vector does not contain an appropriate start signal, such a start signal can be inserted in front of the gene or polynucleotide sequence. In addition, a promoter can be operably linked to a RNA gene encoding a functional RNA.

[00102] The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity". A reference sequence is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, or gene sequence given in a sequence listing.

[00103] The terms "identical" or percent "identity", in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 85% identity, 90% identity, 99%. or 100% identity), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[00104] The phrase "substantially identical in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least about 85%, identity, at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or 100% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In an exemplary embodiment, the substantial identity exists over a region of the sequences that is at least about 50 residues in lengtli. In another exemplary embodiment, the substantial identity exists over a region of the sequences that is at least about 100 residues in length. In still another exemplary embodiment, the substantial identity exists over a region of the sequences that is at least about 150 residues or more, in length, hi one exemplary embodiment, the sequences are substantially identical over the entire length of nucleic acid or protein sequence.

[00105] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the progr am parameters.

[00106] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from about 20 to about 600. usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g.. by the local homology algorithm of Smith & Waterman, Adv. Appl. Math, 2:482 (1981), by the homology alignment algorithm of Needleman & Wuiiseh, J. Mol. Biol. 48:443 ( 1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g.. Current Protocols in Molecular Biology (Ausubel et !., eds. 1995 supplement)).

[00107] An exemplary algorithm for sequence comparison is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity'. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle. J. Mol EvoL 35:351-360 (1987). The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153 (1989). The program can align up to 300 sequences, each of a maximum length of 5.000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. Using PILEUP, a reference sequence is compared to other test sequences to determine the percent sequence identity relationshi using the following parameters: default gap weight (3.00). default gap length weight (0.10), and weighted end gaps. PILEIJP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux ei «?/., 1984. Nuc. Acids Res. 12:387-395.

[00108] Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms _. , which are described in Altschul ei ai, Nuc. Acids Res. 25:3389-3402 (1977) and Alischul ei l, J. Mol. Biol.

215:403-410 (1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http:/ www.ncbi.nlm.n-h.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et l, 1990). These initial neighborhood word hits act as seeds for initiating searches to find kaiger HSPs containing them. The word hits are extended hi both directions along each sequence for as far' as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, clue to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T _; and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults; a wordiength (W) of 11, an expectation (E) or 10, M=5, N4 arid a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordiength of 3, and expectation (E) of 10, and the

BLOSUM62 scoring matrix (see Henifcoff & Henikof 1989. Proc. Natl. Acad. Set USA

89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a compariso of both strands.

[00109] The BLAST algorithm also perforins a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, 1993. Proc. Natl. Acad. Sci. USA

90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01 , and most preferably less than about 0.001.

[00110] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence. [001 1 1 ] The phrase "selectively hybridizes to" or "specifically hybridizes to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., iota! cellular or library DNA or RNA). In general, two nucleic acid sequences are said to be

"substantially identical" when the two moiecuies or their complements selectively or specifically hybridize to each either under stringent conditions.

[00112] The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Trjssen, Techniques in

Biochemistry and Molecular Biology-Hybridization with Nucleic. Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1 93). Generally, stringent conditions are selected to be about 5-1 (fC lower than the thermal melting point (T _m ) for the specific sequence at a defined ionic strength pH. The T _m is the temperature (under defined ionic str ength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T _m , 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to i .0 M sodium ion concentration (or other salts) at pH 7 0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g. , greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as fonnaniide. For high stringency hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary high stringency or stringent hybridization conditions include: 50% formaniide, SxSSC and 1% SDS incubated at 42°C or 5xSSC and 1% SDS incubated at 65 °C, with a wash in 0.2xSSC and 0.1% SDS at 65°C. However, other high stringency hybridization conditions known in the art can be used.

[001 13] Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical This situation can occur, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary "moderately stringent hybridization conditions" include hybridization in a buffer of 40% foimaiinde, 1 M NaCl, 1% SDS at 37°C ₅ and a wash in IxSSC at 45°C. A positive hybridization is at least twice

background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.

[00114] This invention utilizes routine techniques in the field of molecular biology. Basic texts disclosing the general methods of use in tins invention include Sambrook et ah. Molecular Cloning— A Laboratory Manual (2nd Ed.). Vol. 1-3. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1 89; Kriegler, Gene Transfer and Expression; A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausuhel et al . eds., 1 94). Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular" biology maybe found in e.g.,. Benjamin Lewin, Genes FX. published by Oxford

University Press, 2007 (ISBN 0763740632): Krebs, et al. (eds.). The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.). Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[00115] For nucleic acids, sizes are given in either kilobases (kb) or base pairs (bp).

Estimates are typically derived from agarose or acryiarnide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kiiodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.

[00116] Oligonucleotides and polynucleotides that are not commercially available can be chemically synthesized e.g. , accordin to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), or using an automated synthesizer, as described in Van Devanter et ah, Nucleic Acids. Res. 12:6159-6168 (1984). Other methods for synthesizing oligonucleotides and polynucleotides are known hi the art. Purification of oligonucleotides is by either native aerylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Reamer, J. Chrom. 255: 137-149 (1983).

[00117] The sequenc e of the cloned genes and synthetic oligonucleotides can be verified after cloning using, e.g., the chain termination method for sequencing double-stranded templates of Wallace ei at. Gene 16:21-26 (1981). Using of machines for sequencing DNA or RNA is known in the art field.

[00118] A "cell" includes prokaryotic cells, eukaryotic cells, viruses, fungi, and other similar organisms. Bacteria are an example of prokaryotic cells. Plants, algae, mammals, birds, fish, reptiles, amphibians ar e examples of eukaryotic organisms that have cells.

[00119] Turning now to the invention described herein, genes encoding

riiaiimosylhansferase A and rhamnosyltransferase B (rhlA and rh!B, respectively) present in Pseudomanas chlororaphis NRRL B-30761 are isolated and sequenced. In addition, the gene encoding N-acyl-homoserine lacioiie-dependeiit transcription regulator (rhlR) is also isolated and sequenced. Ii is discovered that P. chlororaphis NRRL B-307 1 lacks the gene encoding riiaiimosylhansferase C which could possibly explain why P. chlororaphis NRRL B-30761 does not produce dirhamnose-lipid. The gene for rhamnosyltransferase C (rhIC) is isolated from another bacteria, is sequenced, and is placed in an expression vector which is subsequently transfected into P. chlororaphis NRRL B-30761. The recombinant P. chlororaphis NRRL B- 30761 is then able to produce dnha nnose-lipid.

Example 1 Cloning and characterization of rh!A, rh!B, and rh!R

[00120] Pseudomonas chlororaphis NRRL B-30761 is a non-pathogenic organism that produces monor arnnolipids (RiL) (U.S. Patent 7,202,063). Genetic characterization of this strain is undertaken to facilitate metabolic engineering effort to improve its rhamnolipid biosynthesis potential both in terms of product yield and structural variety. The genes responsible for RjL synthesis, rhamnosyltransferase A and B {rhlA and rh!B, respectively) are cloned and characterized using a PCR approach described previously (Solaiman 2000.

Biotechnol. Lett, 22:789-794; Solaiman, et al. 2008, J. of Industrial Microbiology and Biotechnology? 35:111-120). Prior to initiating cloning of rhlA and rhlB, sequence alignment analysis is performed on the reported sequences oirhlA and rhlB oi Bur kh older ia pseudomallei K96243 (gi 53721039), Burkhok ria mallei ATCC 23344 (gi 53715870), Psetidomonas aeruginosa PAOl (gi 15595198), P. aeruginosa PAOl (gi 9949611) and P. aeruginosa DSM 2659 (gi 452502). From the highly conserved regions identified in the aligned sequences, a set of degenerative PGR primers (Al . A2, A3 and A4; Table 1) is generated to perform nested PGR amplification.

Table 1

[00121] Primers Al and A2 are used in the first-round PGR using Taq DNA polymerase

(New England Biolabs, Ipswich. MA) per supplier's instructions with the following thermal- cycling program: 94°C, 5 minutes; 42 ^'3 C, 1 minute; 72°C, 2 minutes; then 30 cycles of [94°C, 40 seconds; 55°C, 40 seconds; 72*C, 1 minute]; and finally 72 ^,: 'C. 7 minutes. The resultant reaction product mixtme is used as templates for the ensuing nested PGR using primers A3 and A4 and performed under the same conditions as described.

[00122] The PGR amplification generated amplicon A which is 1.33-kb and spans the 3 '- end and the 5 ' -end of rhamnosyltrartsferase B ( /?/¾¾¾) genes of P. chlororaphis. See Figure 2. The nucleotide sequence of amplicon A is determined on Applied Biosystems 3730 DNA Analyzer (Life Technologies Corp., Carlsbad, CA) using manufacturer's instructions. Two sets of target-specific primers are generated based on the sequence of amplicon A. These target-specific primers are used to amplify the flanking genomic sequences by a chromosomal gene walking approach using Seegene's DNA Walking Speedup Kit (Roekviile, MD) per manufacturer's instructions. One set of the target-specific primers. TSP-B1 (5'-CCAGGCGCAAACGACATCAC-3' (SEQ ID NO: 5)) and TSP-B2 (5'-

CCC AGGAC ACGG AAACC AAG-3 ' (SEQ ID NO: 6)), are used to obtain arnplieon B which is 1.1 kb long (see Figure 2). Amplicon B is sequenced on Applied Biosystems 3730 DNA

Analyzer (Life Technologies Corp., Carlsbad, CA) using manufacturer's instructions to obtain its nucleotide sequence. Another set of target-specific primers, TSP-C1 (5'- GGCGCTTGCC ATTGACTCTG-3 ^* (SEQ ID NO: 7)) and TSP-C2 (5 ^* -

CAACGCACTACGCC ACAAAC-3 ' (SEQ ID NO: 8)), are used to obtain amplicon C which is 0.7 kb long (see Figure 2). Amplicon C is also sequenced on Applied Biosystems 3730 DNA Analyzer (Life Technologies Corp., Carlsbad, CA) using manirfaeiurer s instructions. A third set of target-specific primers, TSP-D1 (5 ' -CTGG ACG ATGC ^' G ATC AC A ACG-3 ' (SEQ ID NO: 9» and TSP-D2 (5'- CTGCGACGCTGCCTCTTGTGAA-3 ' (SEQ ID NO: 10)) are designed based on the sequence of amplicon C and are used to generate amplicon D (1.02 kb) which is also sequenced on Applied Biosystems 3730 DNA Analyzer (Life Technologies Corp., Carlsbad, CA) using manufacturer's instructions. The sequences of amplicons A, B, C and D are assembled into a contiguous 3,900 bp-long sequence (GenBank accession number JN415770). Four potential gene sequences are identified through an open-reading- frame search using PC-based Clone Manager 9 (Scientific and Educational Software, Gary, NC). Based on similarity analyses of these DNA sequences using BLAST, the contiguous sequence is annotated with a partial (i.e., C-terminal portion) aconitate hydratase gene (acnB), and the complete rhL4. _Pck (894 bps, SEQ ID NO: 12), rhW _Pch (1,272 bps, SEQ ID NO: 14), and rhlR _Pc (726 bps, SEQ ID NO: 16) (see f igure 2).

[00123] A comparison of the structural features of the gene-products of r p _c¾ (i.e. ,

RhlApt _h ). rhIBpc _h (i.e., RhlBp _th ), and is performed with proteins having some sequence homology and that are published in GenBank. BLASTP analysis reveals that the putative amino-acid sequence of Rhl p _ch (297 residues, SEQ ID NO; 1 1) is only 63% identical (in a 27S amino acid highly conserved segment) to its closest -matched RhlAp _ae sequence of P. aeruginosa (GenBank Accesion No. AAG06867). The highly conserved 278 amino acid region contains an alplia/beta-hydioiase domain likely to be important for enzyme activity. The C- terrninus (17 amino acids) of RhlA _¾h lacks appreciable similarity to RhlAp _asj C-terminus, and the BL ASTP analysis tails to identify a sequence having high similarity. RhlA catalyzes the

3S reaction of two molecules of 3 -hydroxy acyl-acyl carrier protein (ACP) to fomi 3- hydiOxyaIkanoyi-3-hydfoxyalkanoate (Zhu and Rock. 2008. J. of Bacteriology 190:3147-3154) thai is then incorporated into rhamnolipid. PJilA thus catalyzes the first committed step of r amnolipid biosynthesis (DezieL et at 2003. Microbiology 149:2005-2013; Van Gennip, et at 2009. Acta Pathologica, Microbiologica, et I mimologica Scandinavica 117:537-546).

[00124] Not wishing to be bound to any particular theory, the dissimilarity of the amino acid sequence to the corresponding enzyme in P. aeruginosa could explain the observation that P. cMororaphis synthesizes rhamnolipid having mostly 3-hydroxydodecanoyl- 3-hydroxydecanoate (Ci ₂ -Ci ) (30%) or 3-hydroxydodecenoyl-3-hydroxydodecanoate (Cn-.i-Ci©) (40%) as its lipid component (Gunrher, et at 2005), in contrast to the major rhamnolipids of , aeruginosa containing primarily 3-hydroxydecanoyl-3-hydroxydecanoate (Cio-Cio) (Zhu and Rock, 2008: Abdel-Mawgoud, et at 2010). This hypothesis provides support to an earlier hypothesis that the type of fatty acids incorporated into ihanmohpid is dictated in part by the specificity of RlilA, and not by the relative abundance of the fatty acid precursors (Cabrera- Valladares, et at 2006).

[00125] RhlB enzyme catalyzes the transfer of the first L-rhaninose m iety from dTDP-L- rhainnose onto the dimeric hydroxyfat y acid entity of rhamnolipid (Cabrera- Valladaies, et at 2006). A BLASTP analysis ofRhiBp* (423 amino acid; SEQ ID NO: 13) indicates that Rh!B _Pc!l has 63% identical (74% positives) amino acid residues to those of its closest-matched

counterpart, PJilBp _ae (426 amino acid) of P. aeruginosa (GenBank Accesion No.

YP OQl 347032.1 ). The entire length of RhiBp _cli has a conserved protein domain that belongs to the GTB-fype superfamily of glyeosyltransferases (Marehler-Bauer, et at 2011. Nucleic Acids Res. 39;D225-D229). These glyeosyltransferases are typically characterized by two structurally conserved (but not necessarily amino acid sequence homologous) domains separately located at the N- and C-temiini of the protein, with the cleft region between the two domains containing the catalytic site. Again, not wishing to be bound to any particular hypothesis, the degree of sequence dissimilarity between R iBp _d , and RhlBp _ae may partially account for then respective reactivity and Cio-C|<j in the RiL synthesis step of the pathway. [00126] RhiR is an N-aeyl-homoserine iactoiie-dependeiit transcription regulator protein thai controls the transcription of rhlA and rhlB genes via the rh! quorum-sensing circuitry (Chen, ef al. 2004. Biotechnology Progress, 20:1325-1331; Dekiinpe and Deziel 2009. Microbiology 155:712-723). Sequence comparison of R lRp _th (241 amino acid. SEQ ID NO: 15) arid its closest of .P. chlororaphis subsp. aureofaciens (241 amino acid; GenBank Accession No. AAK73190) by BLASTP demonstrates only 66% identical amino acid over the entire length of the protein. Not wishing to be bound by one particular hypothesis, this structural diversity may weii translate into functional difference that leads to the observation that

rhamnolipid is only synthesized by P. chlororaphis NRRL B -30761 grown under non-stirring conditions.

Example 2 Absence of rh!C in P. chlororaphis

[00127] It is hypothesized that the lack of R ₂ L in P. chlororaphis NRRL B-30761 is caused by the absence of rhlC coding for the enzyme that catalyzes the addition of rhanmose moiety onto RjL (Cabrera-Valladares, et at. 2006). Usin PCR, the presence of rhlC in strain P. chlororaphis NRRL B-30761 is examined. Degenerate, nested PCR primers (l ^¾ -round forward primer CL13-131-C8 (SEQ ID NO: 17) and reverse primer CL13-131-D36 (SEQ ID NO: 18), 2 ^lld -round (i.e.. nested) forward primer CL13-13I-C5 (SEQ ID NO: 19) and reverse primer CL13-131-D34 (SEQ ID NO: 20)) are designed based on alignment analysis of known rhlC genes in GenBank, and tested on P. aeruginosa PAOl (provided by Dr. P. Gastric, Duquesne University, Pittsburgh, PA) and PG201 strains. PCR is performed using Tag DNA polymerase (New England Biolabs. Ipswich, MA) with the following thermal-cycling program: 94 ^":i C, 5 minutes; 42°C, 1 minute; 72°C, 2 minutes; then 30 cycles of [94 ⁰ C, 40 seconds; 55°C, 40 seconds; 72°C, 1 minute]; and finally 72°C, 7 minutes. When using the two sets of primers, a prominent amplicoii at the expected size of 0.4-kb is observed in an agrose gel . The 0.4-kb DNA fragment is isolated by agarose-gel elu ion using a Rapid Gel Extraction System (Marligen Biosciences, Ijamsvil!e, MD). It is then blunt-end iigated into a linearized pT7Blue-3 vector using a Perfectly Blunt Cloning Kit (Novagen, Madison, WI) according to the manufacturer's instnic ions. The recombinant DNA is transfected into competent E. coli DH5a (Invitrogen, Carlsbad, CA) using manufacturer's instructions followed by isolatio of the amplicoii by using GenElute Plasmid Prep Kit (Sigma- Aidrich, St. Louis, MO) according to manufacturer's instructions and sequencing of the amplicon on Applied Biosystenis 3730 DNA Analyzer (Life Technologies Corp., Carlsbad, CA) using manufacturer's instructions. Nucleotide sequencing definitively confirms that a piece of rhlC gene was amplified vising the nested PCR protocol. When the same nested PCR protocol is applied to the genomic DNA of P. chlororaphis NRRL B-307 1 using the same four nested primers, the specific amplified DNA fragment of the anticipated size of 0.4-kb is not obtained. Nevertheless, nonspecific bands in the region of 0.4 kb are excised, and their nucleotide sequences are determined after siibcloning them into E.coli using pT7Biue-3 vector according to the manufacturer's instructions. None of these subcloned amplicoiis ( 13 randomly chosen cloned aniplicons ranging in size from 0.4- to 0.6-kb) contain nucleotide sequences over their entire length that matched rhlC on BLAST analyses. These results suggest that the lack of R L synthesis by P. chlororaphi NRRL B-30761 is likely caused by the absence of rhlC or a gene even slightly homologous to the sequences oirhlC found in RjL-producing organisms such as P. aeruginosa.

Example 3 Construction of recombinant P. chlororaphis expressing a heterologous

rhaninosyliransfera.se C

[00128] A goal of generating a genetically engineer nonpathogenic P. chlororaphis NRRL

B-307 1 which is capable of producing R?L is undertaken by first transfeciing and then expressing P. aeruginosa rhlC in P. chlororaphis. Such a transgenic bacterium would be valuable for use in the production of R L intended for food and medical applications that are sensitive to the potential occurrence of eve a trace amount of pathogenic subsiance(s) in the product. Using the nested PCR procedure described above in Example 2, rhlCp _a£ . in P.

aeruginosa PAOl is amplified. In the first-round PCR, primers CL-14-134UP (SEQ ID NO: 21) and CL-14-134DOWN (SEQ ID NO: 22) which are designed based on the rhlC and flanking sequences of P. aeruginosa PAO l genomes (GenelD; 877665) are used. In the second-round, primers RTTI-UP (SEQ ID NO; 23) and RTfl-DOWN (SEQ ID NO; 24) (see, Rahim, ei al. 2001. Molecular Microbiology 40:708-718) are used but with BamHl site built into the 5 '-terminus of RTTI-UP instead of-ScoRL and Hif/dlll site in RTTI-DOWN instead of BamHl. This nested PCR procedure results in the isolation of a 1.2-kb amplicon matching the expected size of the P.

aeruginosa rhlC. This amplicon is blunt-ended using Perfectly Blunt cloning kit (Novagen, Billerica, MA) per manufacturer's instructions. This 1.2-kb amplicon is spliced into the blunt- end Sspl site of a previously described expression vector pBS29-P2-gfp containing a P. svringae P2 promoter (Solaiman and Swingle, 2010. New Biotechnology 2-7:1-9) which is

dephosphoryiated using calf intestinal alkaline phosphatase enzyme (Invitrogen, Carlsbad. CA) per manufacturer's instructions. The P2 promoter is a constitutive promoter from P. syringae and is active in P. chlororaphis . Two recombinant piasmids, pBS29-P2-ri? C _ae (in which the rhlC is aligned with the promoter P2) and pBS29-P2-inv-r///Cp _fla (in winch tire orientation of rhiC is opposite to promoter P2), are first constructed and transfected hi competent E. coli DH5a (Invitrogen, Carlsbad, CA), The E. coli are grown and undergo a plasmi miniprep to isolate the pBS29 vector using the Zyppy Plasmid Miniprep Kit (Zymo Research, Irvine, CA), and the pertinent nucleotide sequences (especially rhlCp _ae ) ar verified by sequence determination using Applied Biosystems 3730 DNA Analyzer (Life Technologies, Caiisbad, CA). The nucleotide sequence of rMCp _ae is in SEQ ID NO: 25 and the amino acid sequence of RhlC¾e. is in SEQ ID NO: 26 (GenelD: 877665). The recombinant piasmids and the vector (i.e., pBS29-P2-g#> as negative control) are then individually electroporated into P. chlororaphis R L B-30761 using a previously described protocol (Soliaman, 1998. Biotechnol. Technique 12:829-832) to obtain 3 transforrnani strains separately containing the vector, the pBS29-P2-rMCp _a8 , and pBS29-P2-inv- rhiCp _as .

[00129] It is noted that instead of using pBS29-P2, one could use any expressio vector containing an inducible or constitutive promoter that is active in Pseudomonas spp. and which replicates in Pseudomonas spp. Further, the use of tetracycline resistance and kanamycln resistance genes within pBS29-P2 or any other expression vector is not necessary because other selection marker genes are known in the ait field and could be used. In addition, one could delete gfp from pBS2-9-P2 and still have an acceptable plasmid to use for expression of rhlC. Alternatively, r/i/C could be stably integrated into the genome of the Pseudomonas spp. using homologous recombination. See, e.g., Casey, et al. 1991 App . Environ. Microbiol. 57(9):2677- 2682; and Ravatn, et aL 1998 J Bacteriol. 180(17): 4360-4369.

Example 4 Production and characterization of R ₂ L from P. chlororaphis [pBS29-P2-r,¾/Ci _¾e ]. [00130] The next step is to test the ability of P. c lororaphis [pBS29-P2 -? ^* /?/€¾,] to biosynthesize 1¾L in defined medium. Cultures of . chlororaphis [pBS29-P2-r/i/Cj¾ _e ] or the control strain harboring plasmid [pBS29-P2-g$j] are grown in si 1-L Erlennieyer flasks each containing 200 niL of a Mineral Salts Medium (MSM) containing 2% glucose Mid 35 με/ηϊΤ· kanarnycin. (See Gunther, et l. 2005 and U.S. Patent 7.202,063.) Cultures are grown in a refrigerator-incubator at 25°C without shaking. At day 7, the cultures from all six flasks for each organism are pooled and lyophilized until dry. The weight of the dry culture is recorded as the cell-diy-weighf yield. The entire dried material (15-20 g, see Table 1} is successively extracted twice with 150 and 75 inL, respectively, of an ethanol/chloroform (1 :2 v/v) mixture. The extract is filtered through Whatman No. 2 paper. The solvent of the clarified extract is removed by evaporation using a Buchi Rotovapor R-124 (Brmkmann Instruments; Wesfbury, NY) imiil a syrupy inaieriai remained in the round-bottom flask. The flask is placed in a desiccator under vacuum for further drying. The weight of the dry syrupy material is recorded as crade rharimolipid yield.

[00131 ] After lyophiiizing the entire culture at the end of fermentation, the dried materials are subject to direct organic solvent-extraction. The solvent is removed by evaporation to obtain crade rharimolipid preparations, which has a syrupy consistency. The weight of the crude rhamnolipid syrup is recorded as product yield value. Results in Table 2 demonstrate that P. chlororaphis transforrnant containing pBS29-P2-r ?/ j _¾e produces crude rhamnolipid at a yield of 290 mg/1.2 L of culture, with a total cell-dry-weight yield of 18.3 g (per 1.2 L culture), hi comparison, P. chlororaphis jjpBS29-P2-g#>3 con ol strain yields 310 mg crade rharimolipid and 16 g of cell-dry- weight under similar fermentation conditions. No appreciable difference in terms of ceil growth and rhamnolipid synthesis is observed between the transforrnant expressing heterologous rklCp _ae and its control counterpart expressing gfp (Soiamian and Swingle, 2010) (see Table 2).

Table 2. Large-scale rhamnolipid yields

gfp]

P. c lororap is [pBS29-P2- 18.3 750; 290 55; 125 37; 89 a Materials were obtained from 1.2 L cultures

No R ₂ L was chromatographically isolatable

[00132] The total rhamnolipid yields, however, are lower than the values reported previously (Gunther, et ah 2005). Not wishing to be bound to any particular hypothesis, one possible explanation for this yield reduction is that the expression of a heterologous gene (i.e. , rhlC in the test strain or gfp in the control sample) is nietabohcally (e.g., reducing power of NADH and FADH?) and/or energetically (e.g., ATP or other molecules with high-energy bond) demanding, resulting in the diversion of resources from rhamnolipid syn thesis. Also, it is possible that the use of an antibiotic (i this case, kanamycin at 35 }ig. ^' mL) to ensure plasmid stability negatively affects the overall yields of cell biomass and metabolite (i.e., rhamnolipid) syntheses. Bacterial rhamnolipid synthesis has always been plagued by insufficient yield to achieve commercial viability. A literature survey on this subject generally shows that the reported yields for rhamnolipid produc tion are typically < 1 g /L culture under batch- fermentation conditions. (See, Dubey and Juwarkar 2001. World J. Microbiol. Biotedmol 17:61-9; and Santa Anna, et ah 2002. Bra:. J. Chem. Eng. 19: 159-66.) For valid comparison, only isolated rhamnolipid yields and not those colorirnetrically determined concentrations are considered here. More complex or sophisticated fermentation system, such as the recently reported 4-cyele bioprocess (Heyd, et a 2011. Biotechnology Progress 27:706-716), could in fact lead to an increased production of rhamnolipid resulting in higher product yield. In this method, bacteria are entrapped in magnetic alginate beads. After each cycle of fermentation, the beads are held by magnetic field while the culture broth is drained to harvest the rharrmolipids. Fresh culture broth is replenished, and another cycle of fermentation is performed with the retained immobilized bacteria. It is envisioned that this fermentation method with the P.

chlororaphis strains described herein could lead to high-yield production of rhamnolipid from nonpathogenic bacterial host. Example 5 Chromatographic separation of rharnnolipid, LC/MS determination, and tensionietric measurement.

[00133] To quantify the relative amounts of RiL and RiL that were produced, one must physically separate and weigh them. While a technique such as LC/MS can in principle provide equivalent information, if the sample is a c omplicated mixture of many compounds, as these are, baseline separation and integration of peaks may be difficult to achieve. Separation using silica gel chromatography is feasible to perform. The crude material obtained by etlianol/chioioibrm extraction (see Example 4 above) is dissolved in 90:10 chloroform methanoi and is applied to a column of 50 g silica (Fisher Scientific; Fairlawn, NJ) that is packed with the same solvent. Elation with this solvent affords a non-polar traction which is not characterized or identified. Monorhaniiiolipids. including RiL, are eluted next from the column by adding a more polar solvent mixture of 80/20/1 c oroform/mefhanol/water (solvent A) to the column. Finally, the dirhamno ipids, being more polar, are challenging to elate, and require a small amount of acetic acid in the elution solvent to remove them from the silica. Thus, 70:30:2:0.4

cWoroform meihanol/waier/acetic acid (solvent B) is added to the column, which is sufficient to elate both the R?L and an unidentified compound. The fractions containing these two compounds (i.e., R?L and the unknown contaminant) are pooled, dried, and re-applied to a new silica gel column (30 g) packed in solvent A. The unknown contaminant is removed with mis solvent, and then the R ₂ L is eluted with solvent B. For R]L and R ₂ L samples, f actions are pooled and dried, then stored under vacuum in a desiccator until co stant weight is attained. Two separate preparations from the tiansfomiant P. chlororophis [pBS29-P2-r/?/Cp _a J are treated in this manner.

[00134] The first crude rhamnolipid preparation yields 125 nig R ₂ L and 55 mg RiL, while the second contains 89 mg R ₂ L and 37 mg R ₅ L. Silica gel purification of the control preparation from . chloror phis [pBS29-¥2-gfp], on the other hand, gives 93 mg RiL, and no R ₂ L is observed. That there should be RiL present hi these preparations is unsurprising, because at the time of harvest some RiL will not have been operated on by the RliiC enzyme. The identity of RiL and R L is verified by LC/MS (see Figure 3). [00135] Surface tension measurements for ¾L and R ₂ L are performed as follows. The purified i½L samples from the two runs described above are redissolved in 1 :1

methanol'chtoroforni, are combined, are dried in vacuo, and are dissolved in 150 mL deionized water to give an approximately 2.1 mM solution. The R ₅ L samples from the two runs above are treated simiiaiiy to give an approximately 1.2 mM solution. These molar concentrations are only approximations, because each type of rhamno lipid is present with several different carbon chain lengths. The most abundant molecular weight species is used to calculate concentration, approximately 676 Da for R ₂ L and approximately 510 Da for RiL. Measurements are performed with the standard Willielmy plate method (see for example, Talom, ei al. 2012. Journal of Colloid and Interface Science 387:180-186) on a DaiaPhysics Instruments GmbH DC AT- 11 tensiometer (Filderstadt, Germany) at ambient temperature, 21-22°C. Solutions are filtered through a fine (4.5-5.0 micron) glass frit prior to use.

[00136] These measurements demonstrate thai the R _} L attains slightly lower surface tension than R ₂ L. The miniinmn surface tension of approximately 26 m /m is significantly lower than the range of 32-34 mN/m observed for another biosurfactani, sophorolipids (SL), and roughly comparable to that observed for mannosyleryfhritol lipid (MEL). Neither rhamnolipid is particularly efficient as a surfactant, however, their CMC values (in the range of 0.1 mM) are about an order of magnitude higher than those seen for SL or MEL .

Example 6 increased RjL and R ₂ L production under stirring conditions using rhlA and rhlB

[00137] To release the expression oirhlA and rhlB in P. chlororapkis NRRL B- 30761 from the control of oxygen resulted from shaking or stirring of culture, a recombinant expression vector pBS29-P2-r///-42? is constructed in which the contiguous rhIA _Pc }, (SEQ ID NO: 12) and rhIBp _c i, (SEQ ID NO: 14) genes are placed under the control of (/ ^" .<?. , operably linked to) the constitutive promoter P2 (Solaiman and Swingle, 2010. New Biotechnol. 27:1-9). pBS29-P2- rhlAB is then transfeeted into P. chlororapkis NRRL B-30761 by electroporation technique (Solaiman, 1998. Biotechnol. Techniques 12:829-932). In this recombinant P. chlororapkis, rhlA and rhlB previously in this bacterium remains unchanged. The resultant transformant, P. chlororapkis [pBS29-P2-rM4Zi], is grown in media as described supra. As shown in Figures 4A, 4B, and 4C, and in Figure 5, the recombinant strains produce, under non-stirring conditions, about 10-fold higher yield of rharnnolipids than the parental P. chlororaphis NRRL B-30761 strain. Monorhamnolipids (RhpC'io-Cr? _. ! and RI1 ₁ -C ₁ 0-C ₁₂ ) eluted at retention times of about 36.5 and 37.8 minutes, respectively as seen Figure 5. Figure 6 shows that P. chlororaphis recombinant strain can produce RjL at comparable yields regardless of whether or not the culture was shaken at a speed (rotary) of 200 rpm. This 10-fold higher yield of rhamnolipids compared to previously described yields of recombinant P. chlororaphis NRRL B -307 1 is surprising hi light of the complexity of producing rhamnolipids.

[00138] To obtain RiL production in stirring conditions, rhICp _ae is added to the expression vector pBS29-P2-r« 3 and transformed into P. chlororaphis NRRL B -30761. This new expression vector, pBS29-V2-rhlABC, contains rhL4 _Fch (SEQ ID NO; 12), rMB _Pch (SEQ ID NO. 14). and rMCp _ae (SEQ ID 25) which are all operationally linked to P2 promoter. To generate tin ^' s expression vector, the circular pBS29-P2-rM4.5 (supra) plasmid is cut with restriction enzyme Xbal located a short distance downstream from the end of rhlB. The tints linearized piasmid with 5 '-protuding ends is treated with Kle ow DNA polymerase enzyme to render it blunt- ended, then with calf intestinal alkaline phosphatase enzyme to remove the phosphate groups thereby preventing possible self-recircularization. Separately, rMCp _ae is cloned from pBS29-P2- !Cpa, (supra) by PGR using primers RTII-UP (SEQ ID NO: 23) and RTH-DQWN (SEQ ID NO: 24), and the resultant 1.2-kb amplicon is blunt-ended using Perfectly Blunt cloning kit (Novagen, Billerica, MA) per manufacturer's instructions (supra). This rhlC _Pae is ligate to the linearized pBS29-P2 -r .4.9 using T4 DNA ligase enzyme. The resultant circular pBS29-P2- rhlABC is transfected into P. chlororaphis NRRL B-30761, Production of RiL under shaking conditions is verified as described supra.

Example 7 Controlled production of RjL and R ₂ L by P. chlororaphis

[00139] It would be beneficial to be able to control the production of iL and RiL by P. chlororaphis so that one can produce approximately 100% RjL or approximately 100% R ₂ L or a desired propoilion ofRiL and R ₂ L. The key is to control the expression of rhIC and either rhlA or rhlB or both rhlA and rhlB using inducible promoters (henceforth LP ^' s). Because P. chlororaphis lacks rhlC, an expression vector containing r/?/Q _¾e , (or rh!C from another bacteria) under contr ol of an inducible promoter is transfected into P. chlororaphis so that the bacteria can convert RiL to R ₂ L. P. chlororaphis naturally expresses rhlA and rhlB so one can either delete one or both of rhlA and rhlB from P. chlororaphis by site-directed mutagenesis via a cross-over deletion and transfecthig the deleted gene (either rhlA or rh!B or both) back into the mutated P. chlororaphis via an expression vector containing one or both of these genes under control of (i. e. , operabiy linked to) an inducible promoter distinct from the inducible promoter controlling the expression of rhlC. in this maimer, one can prevent the expression of rhlC and produce only or primarily RiL. Then if one wants P. chlororaphis to produce R ₂ L, one suppresses the inducible promoter controlling rhL4 or rh 'B or both rhlA and rhlB, and activates the promoter that controls the expression of rhlC. The amount of time that one activates the inducible promoter controlling rhlC expression influence the ratio of RiL to R?L. Non-limiting examples of inducible promoters and their activators- ^' repressors include the follow ng:

heat shock promoters induced by heating the cells (U.S. Patent 4,710,473);

lacZ promoter induced by IPTG (isopropyi-P-D-thiogalactopyranoskie);

tetracycline promoter (ret) induced by tetracycline (Geisseiidoefer, et ah 1 90. Appl.

Micriobiol Biotechnol. 33:657-663);

araS promoter inducible by arabiiiose (Lubelska. etal. 2006. Extremophiles 10(5):383-

91);

arabmose-mducible P _BAD promoter from Escherichia colt (Guzman, et at. 1995. J.

Bacteriol 177:4121-4130);

pXyl-xy!R promoter induced by xylose {Kim, et ah 1996 Gene 181:71-76);

pSpac-Zae/ using lac operoii and IPTG (Yansura, et al. 1984 Proc. Natl. Acad. Set. USA 81 :439-443);

alkane-inducible promoter P _3l kB and alkS (Nieboer, et al. 1993. Mol. Microbiol. 8:1039- 1051);

P _a¾p / phoA phosphate-regulated promoters (Su, et al. 1990. Gene 90:129-133;

cadA regulated by pH and cadR

(http:/.% and

4S rhlR controlled by quorum-sensing and/or biofihn formation (Reis et al. 2001.

Bior source Tech. 102:6377-6384).

[00140] Knock-out strains of P. chlororophis NRRL B-30761 are constructed in which rhlA, rh!B, or rhlA-rh!B genes are inactivated through a gene-disruption phis homologous recombination mechanism using a method previously described and routinely practiced in this laboratory (see. Solaiman. et al. 2003. Appl. Microbiol. Biotee nol 62: 536—543). An alternative method for oligo-niediated allelic replacement procedure employs recombinase enzymes (see, Bryan and Swanson 2011. Mai Microbiol. 80:231-247; Swingle, et al. 2010. Mol. Microbiol. 75:138-148; Wang, et a!. 2009. Nature 460:894-898) to obtain the knock-out strains. Successful construction of rhlA(-), rhlB(-), or rhlA{-)-rhlB{-} knock-out . chlororaphis strains is confirmed by their inability to produce iL using productio and detection methods described supra. Next, expression vectors are constructed that express rhlA, rhlB, or rhlA-rhlB operably linked to one of the inducible promoters _. , IPi, described supra or any other suitable inducible promoter. Vector pCN51 (Nieto, et al. 1990. Gene 87: 145-149: Solaiman, et al 2002. Current Microbiology

44:189-195) is used to carry ΊΡχ-τΜΑ , IP i-rhlB, or Wi-rh!A-rhlB into the corresponding P.

chlororaphis knock-out strains. Successful complementation of the knock-outs is confirmed by production of RiL upon, and only upon, the addition of the appropriate inducer, INDi. Finally, pBS29-P2-rA/ p _a g is transfected into the complemented P. chlororaphis knock-outs {i.e., P. chlororaphis rhlA(-) [pCN51 -IPi-rh!A], P. chlororaphis rhlB(-) [pCNS ^' l-IPi-rMS], or P.

chlororaphis rhlA(-)-rhlB(-) [pCN51-IPi-rh A-rhlB]). To produce near-exclusively

(approximately 100%) the R ₂ L, the recombinant strain P. chlororaphis rhlA(-) [(pCN51-IPi- rM4)+(pBS29-P2-r/?/C )] _; P. chlororaphis rMB(-) [(pCN5l-IP _i -rhlB)+(pBS29-P2-rhlCp _oe )], or P. chlororaphis rhlA(-)-rhlB(-) [(pCN51 -IPi-rhL4-rMB)+(pBS29-P2-rhlCp _a )] is grown in the presence of the inducer, INDi. Production of RiL occurs, which is then acted on by the gene- product of rhlC to yield R ₂ L. INDi is then removed resulting in cessation of new R _\ L synthesis. Existing remaining R ₅ L is then completely converted to R ₂ L by the gene-product of rhlC.

[00141] Instead of transfecting the P. chlororaphis knock-outs (i.e., P. chlororaphis rhlAi- ) [pCN5 i-iP r/iZi], P. chlororaphis rhlB(-) [pCN5 i-IP r/i/¾ or P. chlororaphis rhlA(-)-rhlB(- ) (which uses an constitutive promoter), one can operably link rh C to a second inducible promoter (IP?) different from the inducible promoter used to control expression of rh!A, rhlB, or rhlA-rhlB (IPi) and which is control by a second inducer (TND?). Then pBS29-IP ₂ -r/i/Cp _ae is tansfecled into the complemented P. chlororaphis knock-outs (i.e., P. chlororaphis rhlA(-) [pCN51-iPi-r ;M], P. chlororaphis rh!B(-) [pCN51-IPi- rhlB], or . chlororaphis rh!A{-)-rhlB(-) [pCN51 -IPj -rhlA-rhlB]) to generate either P.

chlororaphis rMA(-) [(pCN51 -IPi-rALi )+(pBS29- lP ₂ -rhlC _Pos )], P. chlororaphis rhlB(-)

[(pCN51-IPi-i-i?/S)+(pBS29- IP ₂ -rMC _Pae )], or P. chlororaphis rhlA(-)-rhlBf-) [(pCN51-IP rA/^- rhlB)+ p S29- Tp2-rhlCp _ae )], respectively. These recombinant bacteria can be grown as described supra in the presence of I Di to produce ¾L. Then one can add ΓΝ¾ to the media which induces expression of rhlC which then converts R _{ L to R?L. Based on the amount of time INDi and IND? are present in the media, one can control the relative percentage of RjL to R?L produced by the recombinant bacteria.

Example 8 Production of RiL and R?L under stilling conditions using heterologous regulatory protei RhlR

[00142] To determine if one could overcome inability of P. chlororaphis NRRL B-30761 and pBS29-P2-r/.f/ p _ae transfected P. chlororaphis to produce RiL and R ₂ L, respectively, under stirring conditions, rhlR from another species (i.e.. heterologous) P. chlororaphis subsp.

aureofaci ns (rhl v_ _c h-au ^" > GenBank Accession No. AA 73190) is transfected into P.

chlororaphis NRRL B-30761 or P. chlororaphis { BS29-P2-r///<¾ _w ] . DNA encoding is obtained from commercial vendor and cloned into pCN51 (Nieto, et aL 1990. Gem 87: 145- 149; Soiaiman, et al. 2002. Current Microbiology 44:189-195) vector downstream of and operably linked to a constitutive promoter, and the resultant recombinant plasmid pCN51 - rhlRp ^-m _t is tansfecled by electroporation technique (Soiaiman 1998. Biotedmol Techniques 12:829-932) into P. chlororaphis NRRL B-30761 or P. chlororaphis [pBS29-P2-r/?/C _i¾e ]. The new species that produce heterologous regulatory protein RhlRp _.ch-aB which is not affected by oxygen level (i.e., stirring) can now produce R ₅ L (in the case of P. chlororaphis NRRL B-30761 host) or R ₂ L (in the case of P. chlororaphis [pBS29-P2-r/i C/¾ _g ]) under stirring conditions. [00143] It is within the scope of this invention to make expression vectors containing the polynucleotides encoding the genes disclosed herein operabiy linked to a variety of promoters (constitutive and/or inducible) that are active in various bacteria, fungi, algae, plant ceils, insect cells, and mammalian cells. These expression vectors can be used to transform the appropriate ceils (depending on the organism for which the promoter's are active) to generate recombinant cells which can produce monorhanniose-iipids and/or dirhamnose-lipids as described herein.

[00144] Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented hi the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and thai modifications and other embodiments ar e intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. All documents cited herein are incorporated by reference.