Genetic Modification of Pseudomonas fluorescens ATCC 13525 for oxalate secretion by
incorporation of artificial oxalate gene cluster
Phosphorus deficiency is a major problem for plant growth and crop yield. This problem
persists in whole world over the century but very little progress has been done towards meeting
the demand of food supply required into next 50 years. Multiple factors are responsible for this
problem which includes depletion of natural rock phosphate (RP) ore reserve, high refixation of
applied P in alfisols, pollution generated during the chemical fertilizers production and high cost
of chemical fertilizers limit the use of chemical fertilizers. Thus, use of biofertilizer could be an
effective approach. In alfisol soils, the crop yield is affected by many factors including very low
amount of available P, Al toxicity and acidic properties of soli. Fluorescent pseudomonads are
well-known plant-growth promoting rhizobacteria with root colonization and efficient biocontrol
property. Oxalic acid is a very strong organic acid with excellent chelating properties. It is
required in low amount (~ 5mM) and being a two carbon molecule low amount of glucose (2
oxalate molecule from 1 molecule of glucose) is required. Mineral phosphate can be solubilized
in both alkaline vertisol (via acidification) and acidic alfisols (via chelation) by genetic
manipulation of rhizobacteria for oxalate secretion. Targeted genetic manipulation of central
carbon metabolic pathway of Pseudomonas fluorescens 13525 was done to make it enable to
secrete out higher amount of oxalic acid. Oxalate secretion was achieved in Pseudomonas
fluorescens ATCC 13525 by incorporation of gene encoding oxaloacetate acetyl hydrolase (oah)
of Aspergillus niger, Fomitopsis plaustris oxalate transporter (FpOAR) and Vitreoscilla
hemoglobin (vgb) genes. P. fluorescens (pKCN2) containing oah gene alone accumulated 19
mM intracellular oxalic acid but secreted 1.6 mM. However, in presence of artificial oxalate
operon containing oah and FpOAR gene under lac promoter in plasmid pKCN4, P. fluorescens
secreted 15 mM oxalate in the medium while 3.6 mM remained inside. This transformant
solubilized 509 µM P from rock phosphate in acidic alfisol which is 4.5 fold higher than the Pf
(pKCN2) transformant. Genomic integrant of artificial oxalate gene cluster, containing oah gene
of A. niger, FpOAR gene of F. plaustris under lac promoter, vgb gene of Vitreoscilla under
natural oxygen sensitive promoter and egfp under growth dependent promoter, were created to
nullify the plasmid load on metabolism of host. P. fluorescens int1, a genomic integrant of the
artificial oxalate operon (plac-FpOAR-oah), secreted 4.8 mM oxalic acid, released 329 µM P
while another genomic integrant, Pf int2, containing artificial oxalate gene cluster (plac- FpOARoah, vgb, egfp) secreted 5.4 mM oxalic acid and released 351 µM P, in acidic alfisol. Genomic
integrants, Pf int1 and Pf int2 showed enhanced root colonization, improved growth and
increased P content of Vigna radiata plants. Thus, this study demonstrates oxalic acid secretion
in P. fluorescens ATCC 13525 by incorporation of artificial oxalate operon and artificial oxalate
gene cluster imparts mineral phosphate solubilizing ability leading to enhanced growth and P
content of Vigna radiata in acidic alfisol soil. Aditionally, incorporation of vgb gene along with
artificial oxalate operon could help in better survival and colonization of organism in soil
environment.