In Vitro Directed Evolution Of Geobacillus Thermodenitrificans NG80-2LadA&Characterization Of NG80-2Facls

In vitro directed evolution of Geobacillus thermodenitrificans NG80-2LadAGeobacillus thermodenitrificans NG80-2, a described thermophilic bacterium（optimum65°C）, can degrade long-chain （C15to C36） alkanes. It is isolated from adeep-subsurface oil reservoir of Dagang oilfield, Northern China. It grows well in theanaerobic, deep subterranean oil-reservoir environment and can utilize crude oil asthe sole carbon source, thus it has the ability to enhance the fluidity of the crude oiland decrease heavy/light ratio of crude oils.LadA, a monooxygenase catalyzing the oxidation of n-alkanes to1-alkanols, isthe key enzyme for the degradation of long-chain alkanes （C15-C36） in G.thermodenitrificans NG80-2. LadA is a thermophilic monooxygenase that cancatalyze the longest chain of alkanes, to our knowledge. It is also one of the mostuseful enzymes that can utilize long chain alkanes up to now. Thus it can bepotentially used bioconvertions like pollutions and so on. During our previousresearch, the gene of LadA has been cloned, expressed and characterized. We havealso studied the enzyme characterization of LadA. What is more, the3-D structure ofLadA has also been analyzed by X-ray diffraction assays.In order to further improve its catalytic ability in hydroxylation of alkane andmake this enzyme more useful, the wild gene of LadA was aimed to further in vitrodirected evolution by error-prone PCR and saturation mutagenesis.9LadA mutantswith higher activity and higher catalytic efficiency had been found using a wellestablished high throughput screening method. Study of enzyme characteristics of themutant enzymes was researched. The molecular mechanism of the activity changeand the structure of LadA mutants was also analyzed. Directed evolution was done bycollecting the mutant libraries using E. coli DH5α,expressing LadA by E. coli BL21and screening the96-well plate based on spectroscopic assays of consumption ofNADPH. Ep-PCR was performed on the wild gene ladA. Optimized templateconcentration and cycle numbers were found and the mutant rate was controlledbetween2-4bp/gene.5similar libraries was established.7500mutants were screened. 3mutants （A102D, L320V and F146C/N376I） with higher activity were found.Site-directed mutagenesis was taken to recombine the four sites of the three mutantsand6more new enzymes were created. The6enzymes catalytic ability was analyzedto evaluate the interactions of the4sites. Saturation mutagenesis was used towardsthe four sites and another hotspot114revealed by the computer analysis. All the other19amino acids of the5sites were obtained by sequencing.6more new mutants（A102E, L320A, F146Q/N376I, F146E/N376I, F146R/N376I, F146N/N376I） wereobtained by analysis the activity of the all the mutants. Characterization of the9mutants （A102D, A102E, L320V， L320A, F146Q/N376I, F146E/N376I,F146R/N376I, F146N/N376I and F146C/N376I） was made. Comparing with thewildtype LadA, the hydroxylation activity(k_cat)of the purified LadA mutants onhexadecane was2–3.4-fold higher, with the activity of F146N/N376I being thehighest; the catalytic efficiency (k_cat/k_m) of the purified LadA mutants on hexadecanewas1.9–12.7-fold higher, with the activity of F146E/N376I and A102E being thehighest. Effects of the mutations on optimum temperature, pH and heat stability ofLadA were also investigated. A complementary study showed that Pseudomonasfluorescens KOB2Δ1strains expressing the LadA mutants grew more rapidly withhexadecane than the strain expressing wild type LadA, confirming the enhancedactivity of LadA mutants in vivo. Structural changes resulting from the mutationswere analyzed and the correlation between structural changes and enzyme activitywas discussed. The mutants generated in this study are potentially useful for thetreatment of environmental oil pollution and in other bioconversion processes.ⅡCharacterization of G. thermodenitrificans NG80-2FaclsThe functions of two long-chain fatty acid CoA ligase genes （facl） in crudeoil-degrading G. thermodenitrificans NG80-2were characterized. Facl1and Facl2encoded by GTNG₀892and GTNG₁447were expressed in Escherichia coli andpurified as His-tagged fusion proteins. Facl1was found to be a homology dimer in itsnative status while Facl2may act as a non-sphere monomer or a dimer. Both enzymesutilized a broad range of fatty acids ranging from acetic acid （C2） to melissic acid（C30）. The most preferred substrates were capric acid （C10） for Facl1and palmitic acid （C16） for Facl2, respectively. Both enzymes had an optimal temperature of60°C,an optimal pH of7.5, and required ATP as a cofactor. Thermostability of the enzymesand effects of metal ions, EDTA, SDS and Triton X-100on the enzyme activity werealso investigated. When NG80-2was cultured with crude oil rather than sucrose asthe sole carbon source, upregulation of facl1and facl2mRNA was observed by realtime RT-PCR. This is the first time that the activity of fatty acid CoA ligases towardslong-chain fatty acids up to at least C30has been demonstrated in bacteria.