Faculty Opinions recommendation of Biodegradation of 1,2,3-trichloropropane through directed evolution and heterologous expression of a haloalkane dehalogenase gene.

Author(s):  
Peter Williams
Microbiology ◽  
1997 ◽  
Vol 143 (1) ◽  
pp. 109-115 ◽  
Author(s):  
A. N. Kulakova ◽  
M. J. Larkin ◽  
L. A. Kulakov

1999 ◽  
Vol 181 (7) ◽  
pp. 2050-2058 ◽  
Author(s):  
Gerrit J. Poelarends ◽  
Johan E. T. van Hylckama Vlieg ◽  
Julian R. Marchesi ◽  
Luisa M. Freitas Dos Santos ◽  
Dick B. Janssen

ABSTRACT The newly isolated bacterial strain GP1 can utilize 1,2-dibromoethane as the sole carbon and energy source. On the basis of 16S rRNA gene sequence analysis, the organism was identified as a member of the subgroup which contains the fast-growing mycobacteria. The first step in 1,2-dibromoethane metabolism is catalyzed by a hydrolytic haloalkane dehalogenase. The resulting 2-bromoethanol is rapidly converted to ethylene oxide by a haloalcohol dehalogenase, in this way preventing the accumulation of 2-bromoethanol and 2-bromoacetaldehyde as toxic intermediates. Ethylene oxide can serve as a growth substrate for strain GP1, but the pathway(s) by which it is further metabolized is still unclear. Strain GP1 can also utilize 1-chloropropane, 1-bromopropane, 2-bromoethanol, and 2-chloroethanol as growth substrates. 2-Chloroethanol and 2-bromoethanol are metabolized via ethylene oxide, which for both haloalcohols is a novel way to remove the halide without going through the corresponding acetaldehyde intermediate. The haloalkane dehalogenase gene was cloned and sequenced. The dehalogenase (DhaAf) encoded by this gene is identical to the haloalkane dehalogenase (DhaA) of Rhodococcus rhodochrous NCIMB 13064, except for three amino acid substitutions and a 14-amino-acid extension at the C terminus. Alignments of the complete dehalogenase gene region of strain GP1 with DNA sequences in different databases showed that a large part of adhaA gene region, which is also present in R. rhodochrous NCIMB 13064, was fused to a fragment of a haloalcohol dehalogenase gene that was identical to the last 42 nucleotides of thehheB gene found in Corynebacterium sp. strain N-1074.


2002 ◽  
Vol 68 (7) ◽  
pp. 3582-3587 ◽  
Author(s):  
Tjibbe Bosma ◽  
Jirí Damborský ◽  
Gerhard Stucki ◽  
Dick B. Janssen

ABSTRACT Using a combined strategy of random mutagenesis of haloalkane dehalogenase and genetic engineering of a chloropropanol-utilizing bacterium, we constructed an organism that is capable of growth on 1,2,3-trichloropropane (TCP). This highly toxic and recalcitrant compound is a waste product generated from the manufacture of the industrial chemical epichlorohydrin. Attempts to select and enrich bacterial cultures that can degrade TCP from environmental samples have repeatedly been unsuccessful, prohibiting the development of a biological process for groundwater treatment. The critical step in the aerobic degradation of TCP is the initial dehalogenation to 2,3-dichloro-1-propanol. We used random mutagenesis and screening on eosin-methylene blue agar plates to improve the activity on TCP of the haloalkane dehalogenase from Rhodococcus sp. m15-3 (DhaA). A second-generation mutant containing two amino acid substitutions, Cys176Tyr and Tyr273Phe, was nearly eight times more efficient in dehalogenating TCP than wild-type dehalogenase. Molecular modeling of the mutant dehalogenase indicated that the Cys176Tyr mutation has a global effect on the active-site structure, allowing a more productive binding of TCP within the active site, which was further fine tuned by Tyr273Phe. The evolved haloalkane dehalogenase was expressed under control of a constitutive promoter in the 2,3-dichloro-1-propanol-utilizing bacterium Agrobacterium radiobacter AD1, and the resulting strain was able to utilize TCP as the sole carbon and energy source. These results demonstrated that directed evolution of a key catabolic enzyme and its subsequent recruitment by a suitable host organism can be used for the construction of bacteria for the degradation of a toxic and environmentally recalcitrant chemical.


1999 ◽  
Vol 18 (5) ◽  
pp. 571-576 ◽  
Author(s):  
Henrik Naested ◽  
Marko Fennema ◽  
Lin Hao ◽  
Mathias Andersen ◽  
Dick B. Janssen ◽  
...  

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