Optimization of trichloroethylene degradation using soluble methane monooxygenase ofMethylosinus trichosporium OB3b expressed in recombinant bacteria

Abstract

By complementing cell-free extracts of fseudomonas putida Fl/pSMM020 with purified soluble methane monooxygenase (sMMO) components of Methylosinus trichosporium OB3b, the low cloned-gene sMMO activity in the recombinant strain was found to be due to incomplete activity of the hydroxylase component. To address this incomplete activity, additional sMMO-expressing strains were formed by transferring mmo-containing pSMM020 and pSMM050 into various bacterial species including pseudomonads and a-2 subdivision strains such as methanotrophs, methylotrophs, Agrobacterium tumefaciens A114, and Rhizobium meliloti 102F34 (11 new strains screened); sMMO activity was detected in the last two strains. To increase plasmid segregational stability, the hok/sok locus originally from Escherichia coli plasmid R1 was inserted downstream of the mmo locus of pSMMO2O (resulting in pSMM040) and found to enhance plasmid stability in f . putida F1 and R. meliloti 102F34 (first report of hok/sokin Rhizobium). To further increase sMMO activity, a modified Whittenbury minimal medium was selected from various minimal and complex media based on trichloroethylene (TCE) degradation and growth rates and was improved by removing the sMMO-inhibiting metal ions [Cu(ll), Ni(ll), and Zn(ll)l and chloramphenicol from the medium and by supplementing with an iron source (3.6 pM of ferrous ammonium sulfate). Using chemostat-grown f . putida Fl/pSMM040, it was found that sMMO activity was higher for cells grown at higher dilution rates. These optimization efforts resulted in a twofold increase in the extent of TCE degradation and more consistent sMMO activity.

SmoXYB1C1Z of Mycobacterium sp. Strain NBB4: a Soluble Methane Monooxygenase (sMMO)-Like Enzyme, Active on C 2 to C 4 Alkanes and Alkenes

“…sMMO can be expressed at high levels in heterologous methanotrophs (24,25) and at very low levels in a few other hosts (28,29), but to date, sMMO has not been functionally expressed in E. coli. This limitation has hindered research on this important enzyme.…”

“…sMMO can be expressed in methanotrophs that normally lack sMMO (24,25), which has allowed site-directed mutagenesis (26), but the system has limitations; e.g., the particulate methane MO (pMMO) is also present, and the system is under complex regulatory control (27). sMMO can also be ex-pressed, albeit with very low activity, in Pseudomonas (28) and Rhizobium (29). In the case of sBMO, genetic work has been enabled via homologous recombination in the natural host (Thauera butanivorans) (22), but this approach has some limitations similar to those of the homologous sMMO expression systems; e.g., it requires a gaseous inducer.…”

Improved System for Protein Engineering of the Hydroxylase Component of Soluble Methane Monooxygenase

“…Previous studies found that this component was inactive when expressed in Escherichia coli (50). The first reports of expression of the genes encoding the sMMO gene cluster of M. trichosporium in a heterologous host were by Wood and colleagues, who demonstrated the degradation of trichloroethylene by sMMO expressed in Pseudomonas putida, Agrobacterium tumefaciens, and Rhizobium meliloti (19,20). We subsequently described a homologous expression system for the wild-type sMMO hydroxylase, in which recombinant (wild-type) sMMO genes were expressed, yielding highly active sMMO in a derivative of M. trichosporium known as mutant F (26).…”

Mutagenesis of the “Leucine Gate” To Explore the Basis of Catalytic Versatility in Soluble Methane Monooxygenase

“…Mutagenesis of the hydroxylase component of sMMO requires the use of an especially developed expression system because attempts to date to obtain expression of active hydroxylase in E. coli have been unsuccessful (44). Expression of active sMMO has been reported to occur in various heterologous systems (16,17,24) as well as the homologous system used here. The previous work that we performed using the homologous expression system for sMMO, which enabled the first mutagenesis of the active site of the enzyme (38), utilized a homologous expression host (M. trichosporium mutant F [26]) from which only part of the mmoX gene was deleted, thus limiting the region of the hydroxylase that could be mutagenized.…”