Sphingomonas Strains Producing Greatly Increased Yield Of PHB-Deficient Sphingan (Diutan)

Abstract

PHB-deficient Sphingomonas strains having improved sphingan yield are provided. Certain of the Sphingomonas strains are diutan-producing strains that exhibit a dramatic improvement in productivity and yield due to a combination of certain genetic modifications that affect PHB and sphingan synthesis. Moreover, the sphingans produced from such strains have superior characteristics including improved filterability, clarity, and improved rheology-modifying characteristics. The sphingans provided are, thus, highly desirable in a variety of commercial and industrial uses, including personal care items, cement applications, and oilfield applications.

1 . A mutant strain of the genus  Sphingomonas , containing:
 at least one genetic modification that substantially or entirely eliminates the production of polyhydroxybutyrate (PHB);   at least one genetic modification that results in increased production of a sphingan, comprising a genetic modification that increases the expression of at least one gene involved in sphingan synthesis, wherein said at least one gene involved in sphingan synthesis is selected from the group consisting of the genes contained in the insert in plasmids pS8 (SEQ ID NO: 1), pX6 (SEQ ID NO: 54), the plasmid contained in strain ATCC PTA-10102, the plasmid contained in strain ATCC PTA-10103, and  Sphingomonas  homologs thereof;   whereby the mutant strain of the genus  Sphingomonas  can produce an increased production of a sphingan that is essentially free of PHB relative to a congenic strain containing the at least one genetic modification that substantially or entirely eliminates the production of PHB and lacking the at least one genetic modification that results in increased production of a sphingan.   
     
     
         2 . The mutant strain of the genus  Sphingomonas  of  claim 1 , wherein the sphingan is selected from the group consisting of diutan, S-7, gellan, S-88, welan, rhamsan, S-198, NW-11, and alcalan. 
     
     
         3 . The mutant strain of the genus  Sphingomonas  of  claim 2 , wherein the sphingan is diutan. 
     
     
         4 . The mutant strain of the genus  Sphingomonas  of  claim 1 , wherein said at least one gene involved in sphingan synthesis is selected from the group consisting of  Sphingomonas  sp. ATCC 53159 genes dpsS, dpsG, dpsR, dpsQ, dpsl, dpsK, dpsL, dpsJ, dpsF, dpsD, dpsC, dpsE, dpsM, dpsN, atrD, atrB, dpsB, rmlA, rmlC, rmlB, rmlD, orf7, orf6, orfs, and Sphingomonas homologs thereof. 
     
     
         5 . The mutant strain of the genus  Sphingomonas  of  claim 1 , wherein said  Sphingomonas  homolog of at least one gene involved in sphingan synthesis is derived from a  Sphingomonas  species selected from the group consisting of  Sphingomonas elodea  ATCC 31461,  Sphingomonas  sp. ATCC 31555,  Sphingomonas  sp. ATCC 31961,  Sphingomonas  sp. ATCC 53159,  Sphingomonas  sp. ATCC 31554,  Sphingomonas  sp. ATCC 31853,  Sphingomonas  sp. ATCC 21423,  Sphingomonas  sp. ATCC 53272, and  Sphingomonas  sp. FERM-BP2015. 
     
     
         6 . The mutant strain of the genus  Sphingomonas  of  claim 1 , wherein the at least one genetic modification that results in increased production of a sphingan is selected from the group consisting of:
 (i) an operable linkage of at least one gene involved in sphingan synthesis to an ectopic promoter;   (ii) an increased number of copies per bacterial chromosome of at least one gene involved in sphingan synthesis; and   (iii) any combination thereof,   
       wherein each of said at least one gene involved in sphingan synthesis are contained in a bacterial chromosome or extrachromosomal element. 
     
     
         7 . The mutant strain of the genus  Sphingomonas  of  claim 1 , wherein the at least one genetic modification that substantially or entirely eliminates the production of PHB is a mutation that constitutively or conditionally inactivates or deletes a gene selected from the group consisting the phaA gene, the phaB gene, and the phaC gene or a combination thereof. 
     
     
         8 . The mutant strain of the genus  Sphingomonas  of  claim 1 , wherein the at least one genetic modification that substantially or entirely eliminates the production of PHB is an insertion or deletion that inactivates the phaC gene. 
     
     
         9 . The mutant strain of the genus  Sphingomonas  of  claim 3 , wherein the mutant strain of the genus  Sphingomonas  is able to produce diutan at a rate of at least about 0.15 g/L/hr or a yield of diutan of at least about 12 g/L. 
     
     
         10 . The mutant strain of the genus  Sphingomonas  of  claim 3 , wherein the mutant strain of the genus  Sphingomonas  is able to produce diutan at a rate of at least about 0.2 g/L/hr or a yield of diutan of at least about 15 g/L. 
     
     
         11 . The mutant strain of the genus  Sphingomonas  of  claim 3 , wherein the mutant strain of the genus  Sphingomonas  increases the rate of production or yield of diutan by at least about 50% relative to a congenic strain containing the at least one genetic modification that substantially or entirely eliminates the production of PHB and lacking the at least one genetic modification that results in increased production of a sphingan. 
     
     
         12 . The mutant strain of the genus  Sphingomonas  of  claim 3 , wherein a clarified diutan produced from the mutant strain of the genus  Sphingomonas  yields less than 0.5% residue in a 15% HCl solubility and residue test, or less than 0.1 wt % PHB when measured using gas chromatography. 
     
     
         13 . The mutant strain of the genus  Sphingomonas  of  claim 3 , wherein a clarified diutan produced from the mutant strain of the genus  Sphingomonas  and rehydrated as one liter of 0.04% diutan in seawater can pass through a Nuclepore filter in less than five minutes at a flow pressure of approximately 20 psi;
 wherein the Nuclepore filter is approximately 47 mm in diameter and has a pore size of approximately 3 microns.   
     
     
         14 . The mutant strain of the genus  Sphingomonas  of  claim 3 , wherein diutan produced from the mutant strain of the genus  Sphingomonas  is essentially free from PHB, and wherein the diutan exhibits a sea water 3 rpm viscosity of at least about 40 dial reading, a sea water 0.3 rpm viscosity of at least about 37,000 cp, or a low shear rate viscosity in the presence of polyethylene glycol dispersant of at least about 3,500 cp. 
     
     
         15 . The mutant strain of the genus  Sphingomonas  of  claim 3  that is a strain of  Sphingomonas  sp. ATCC 53159 selected from the group consisting of strains PDD3/pS8, PDD3/pX6, PDD6/pS8, and PDD6/pX6. 
     
     
         16 . A composition comprising diutan, wherein the composition is essentially free from polyhydroxybutyrate (PHB), and wherein the diutan exhibits a sea water 3 rpm viscosity of at least about 32 dial reading, a sea water 0.3 rpm viscosity of at least about 24,000 cp, or a low shear rate viscosity in the presence of polyethylene glycol dispersant of at least about 3,000 cp. 
     
     
         17 . The composition of  claim 16  wherein the diutan exhibits a sea water 3 rpm viscosity of at least about 40 dial reading, a sea water 0.3 rpm viscosity of at least about 37,000 cp, or a low shear rate viscosity in the presence of polyethylene glycol dispersant of at least about 3,500 cp. 
     
     
         18 . The composition of  claim 16  wherein the diutan exhibits a sea water 3 rpm viscosity of at least about 45 dial reading, a sea water 0.3 rpm viscosity of at least about 40,000 cp, or a low shear rate viscosity in the presence of polyethylene glycol dispersant of at least about 5,500 cp. 
     
     
         19 . A method of making a sphingan that is essentially free from polyhydroxybutyrate (PHB), comprising:
 growing a mutant strain of the genus  Sphingomonas  under conditions that facilitate the production of sphingan; and   optionally, isolating the sphingan from the resulting culture, wherein said mutant strain of the genus  Sphingomonas  contains:   at least one genetic modification that results in increased production of a sphingan; and   at least one genetic modification that substantially or entirely eliminates the production of PHB.   
     
     
         20 . The method of  claim 19 , wherein the sphingan is diutan.