Genetic engineering for trichloropropane degradation

PhD ceremony: Ms. G. Samin, 11.00 uur, Academiegebouw, Broerstraat 5, Groningen

Dissertation: Genetic engineering for trichloropropane degradation

Promotor(s): prof. D.B. Janssen

Faculty: Mathematics and Natural Sciences

A genetically modified organism is constructed by Ghufrana Samin for the degradation of a toxic compound and applied successfully in a lab-scale bioreactor to remove 1,2,3-trichlororpropane (TCP)-contaminated groundwater.

TCP is a toxic compound that has caused serious groundwater pollution at chemical waste disposal sites. The availability of a bacterium that can degrade this compound would allow the development of bioreactors for TCP removal from contaminated groundwater. A possible degradation pathway would start with hydrolytic dehalogenation of TCP to 2,3-dichloro-1-propanol (DCP), followed by further oxidative metabolism. In the first part of her study, Samin isolated a strain of Pseudomonas putida from contaminated soil on basis of its capacity to utilize DCP as growth substrate. To transform TCP into DCP, she expressed an engineered haloalkane dehalogenase (DhaA31) constitutively under control of the dhlA promoter and introduced into the genome of the DCP-degrading bacterium by using a transposon delivery system. The transposon-located antibiotic marker was subsequently removed. Growth of the engineered bacterium on TCP was indeed observed. The genetically engineered TCP-degrading strain is stable, free of any additional plasmid-encoded antibiotic resistance marker and has the ability to completely mineralize TCP with quantitative stoichiometric release of inorganic chloride.

The application of the strain in lab-scale bio-reactors was also investigated by Samin. Her results indicate that with the constructed strains, the reactor removed TCP (80-98%) at various residence times (116 h and 23 h). During the construction of the genetically engineered bacterium, she found that haloalkane dehalogenase was exported to the periplasm even though no signal sequence was present. The results indicate the potential use of DhaA as a tag for the periplasmic export of heterologously expressed proteins.