Production of dirhamnose-lipid in recombinant nonpathogenic bacterium Pseudomonas chlororaphis
Inventors
Solaiman, Daniel • Ashby, Richard D. • Zerkowski, Jonathan A. • Gunther, Nereus W.
Assignees
US Department of Agriculture USDA
Publication Number
US-10190144-B2
Publication Date
2019-01-29
Expiration Date
2034-06-03
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Abstract
Pseudomonas chlororaphis NRRL B-30761 produces monorhamnolipids with predominantly 3-hydroxydodecenoyl-3-hydroxydecanoate (C12:1-C10) or 3-hydroxydodecanoyl-3-hydroxydecanoate (C12-C10) as the lipid moiety under static growth conditions. The cloning and sequencing of three genes and proteins involved in the biosynthesis of monorhamnose-lipid (R1L) is described. Expression of two of these genes, i.e., rhlA and rhlB, together in P. chlororaphis NRRL B-30761 increases R1L production by at least 10-fold. Also the generation of a recombinant P. chlororaphis NRRL B-30761 capable of synthesizing dirhamnose-lipid (R2L) is described. Characterization of R1L and R2L produced by the recombinant P. chlororaphis NRRL B-30761 is also described.
Core Innovation
This invention relates to the genes encoding enzymes and regulatory proteins involved in dirhamnose-lipid biosynthesis, particularly focusing on a recombinant, nonpathogenic Pseudomonas chlororaphis capable of producing monorhamnolipids (R1L) and dirhamnose-lipids (R2L). The work includes cloning and sequencing three genes and proteins involved in the biosynthesis of monorhamnose-lipid (rhlA, rhlB, and rhlR) from P. chlororaphis NRRL B-30761.
The problem addressed is the deficiency of dirhamnose-lipid production in P. chlororaphis NRRL B-30761 due to the absence of the rhlC gene, which encodes rhamnosyltransferase C responsible for converting monorhamnolipids to dirhamnose-lipids. This deficiency limits the bacterium's production capabilities and application potential. The invention overcomes this by introducing and expressing a heterologous rhlC gene from Pseudomonas aeruginosa into P. chlororaphis NRRL B-30761, thereby enabling dirhamnose-lipid synthesis.
Further improvements include genetically engineering P. chlororaphis to express rhlA and rhlB genes operably linked to constitutive promoters, resulting in at least a ten-fold increase in R1L production under both stirring and non-stirring conditions. Additionally, the invention describes methods to control the ratio of monorhamnose-lipid to dirhamnose-lipid production by using inducible promoters to regulate the expression of rhlA, rhlB, and rhlC genes. Expression of a heterologous N-acyl-homoserine lactone-dependent transcription regulator (rhlR) from P. chlororaphis subsp. aureofaciens further allows production of R1L and R2L under stirring conditions, overcoming oxygen level control limitations inherent in the native strain.
Claims Coverage
The patent discloses two independent claims encompassing recombinant bacterial strains and methods of producing dirhamnose-lipid.
Recombinant expression of heterologous rhamnosyltransferase C in Pseudomonas chlororaphis
A recombinant P. chlororaphis derived from NRRL B-30761 comprising an expression vector with a promoter operably linked to a heterologous polynucleotide encoding a heterologous Pseudomonas rhamnosyltransferase C, wherein endogenous rhlA and rhlB genes remain operably linked to native promoters.
Methods for producing dirhamnose-lipid using recombinant Pseudomonas chlororaphis
Methods comprising culturing or growing the recombinant P. chlororaphis described above in a medium capable of supporting rhamnose-lipid or dirhamnose-lipid biosynthesis, thereby producing dirhamnose-lipid.
The independent claims cover a recombinant nonpathogenic Pseudomonas chlororaphis genetically modified to express a heterologous rhlC gene enabling dirhamnose-lipid production, and methods utilizing this recombinant bacterium to produce dirhamnose-lipids in suitable culture media.
Stated Advantages
The recombinant P. chlororaphis exhibits at least a 10-fold increase in monorhamnose-lipid production compared to the parental strain.
The production of dirhamnose-lipid is enabled in a nonpathogenic bacterial host, broadening the application of rhamnolipids while lowering production costs.
Using inducible promoters allows controlled regulation of the ratio between monorhamnose- and dirhamnose-lipid production.
Expression of heterologous regulatory protein rhlR permits monorhamnose- and dirhamnose-lipid production under stirring conditions, overcoming oxygen-level dependent expression limitations in the native strain.
Documented Applications
Production of monorhamnose-lipids and dirhamnose-lipids by recombinant Pseudomonas chlororaphis for applications sensitive to pathogenic substances, such as food and medical uses.
Use of genetically engineered nonpathogenic bacteria to lower the costs and broaden the applications of rhamnolipid biosurfactants.
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