Gene Cloning Of Enzymes Related To The Myxobacterial TAG Lipid Metabolism And Preliminary Studies On Their Flunctions

Myxobacteria are gram-negative gliding bacteria that have complicated social lives and excellent ability to produce various bioactive compounds. At present, the research found that several types of myxobacterial cells were able to form a number of intracellular lipid body inclusions during the growth stage under various culture conditions. For example, lipid body formation plays a central role in cell fate determination during developmental differentiation of M. xanthus DK1622and the lipid bodies are filled with neutral lipids (mostly triacylglycerols, TAGs). We also observed that the Sorangium cellulosum So0157-2cells were able to form lipid bodies of which the major components were a triglyceride mixture of different types of fatty acids. Stored both carbon and energy, the lipid body inclusions are consumed to sustain cell survival and epothilone production when nutrients become deficient. However, there are few reports on the myxobacteria TAG metabolic enzymes. The study based on the physiological and metabolic characteristics of myxobacteria has great theoretical significance and economic value for development and utilization of myxobacterial resources.Among different myxobacterial species, the cellulolytic myxobacterium Sorangium cellulosum is extremely interesting in drug screening, and almost half of the discovered myxobacterial secondary metabolites are produced by different strains of this species. In addition to these defined characteristics, S. cellulosum also has excellent and extensive degradation ability on various macromolecules, including many different kinds of polysaccharide and lipid, of which the involved enzymes have been less investigated. S. cellulosum has the known largest bacterial genome, reaching13.1Mb in the sequenced So ce56strain. Consistent with the extensive degradation ability on a wide range of macromolecules, the S. cellulosum genome contains many open-reading frames (ORFs) predicted encoding different hydrolytic enzymes. For instance,32ORFs on So ce56genome were predicted to encode various lipases/esterases (EC3.1.1.). The predicted products of these ORFs are all in low similarity to those studied lipases/esterases and none of them has been characterized, suggesting their potentially specific characteristics.Our work based on the genome sequencing of S. cellulosum So0157-2and the published genomes of S. cellulosum So ce56and M. xanthus DK1622. We analyzed the key enzymes of triglyceride synthesis and degradation from these three strains in order to conjecture the characteristics of the myxobacteria TAG metabolic enzymes and expand the useful gene resources. Diacylglycerol acyltransferase (DGAT)(EC2.3.1.20) catalyzes the final step of triglyceride biosyntheses. Triacylglycerol lipase (EC3.1.1.3) is the main enzyme of triacylglycerol degradation and usage. In the present study, to explore characteristics and commercial significance of S. cellulosum degradation enzymes, we cloned, expressed and characterized the lipase genes lip A, lipB from strain So0157-2. In the mean time, to explore the physiological functions of the genes in myxobacterial cells, we constructed DGAT and lipase gene targeting vectors, knocked out some genes related to TAG metabolism, and analyzed the in vivo physiological characteristics of the mutants.First of all, we analyzed the key enzymes of triglyceride synthesis and degradation from S. cellulosum So ce56, So0157-2and M. xanthus DK1622. Consistent with the extensive degradation ability on a wide range of macromolecules, the myxobacterial genome contains many open-reading frames (ORFs) predicted encoding TAGs hydrolytic enzymes (lipases). The predicted products of these ORFs are all in low similarity to those studied lipases/esterases and none of them has been characterized. It provides a basis for subsequent gene expression and function analysis.Two genes, coding lipases from the genome of S. cellulosum So0157-2, were cloned. We named them lipA and lipB. They were expressed and purified. The biochemical feature was characterized using the active protein.The lipA-containing plasmid, pET22b-LipA, was expressed in E. coli BL21(DE3). The recombinant LipA (r-LipA) was in a soluble form, which was further purified using Ni-NTA affinity chromatography. SDS-PAGE analysis of the purified r-LipA showed a single band corresponding to approximate35.6kDa. Searching in the GenBank database shows that LipA has low similarity to those studied lipases with a maximum identity of30%to a human monoglyceride lipase (3JW8_A), suggesting their potentially specific characteristics. The protein exhibited the maximum activity at30℃and the relative activity still reached to35.2%even at0℃and almost completely lost at60℃. It lost nearly80%of activity after30-min pre-incubation at55℃and the half-life was just10min. According to the presently prevalent definition, we define our protein a cold-adapted enzyme. In contrast to the cold-adaptation characteristics, the lipase retained high activity in a broad pH range (3.0-10.5), and reached the maximum at pH8.0. The r-LipA exhibited high activity against short-chain fatty acids, and the activity significantly decreased with the increase of the acyl chain length. It indicated that LipA was not the key enzyme to degradate lipids body of So0157-2. Most of the metal ions detected could inhibit the activity of r-LipA. The enzyme activity was enhanced by the presence of Ca2+and Mg2+at a concentration of10mM, approximately1.4-,1.26-fold, respectively. The r-LipA was found to be able to retain a high level of activity in the presence of many commercially available detergents and organic solvents. That r-LipA demonstrated exhibited wide tolerances against various condition such suggests that it might be a useful catalysts in the organic synthesis of chiral intermediates, allowing relatively unstable compounds to be produced at low temperatures.Searching in the GenBank database shows that LipB has high similarity to those studied lipases. Prediction of transmembrane region of LipB sequence has three distinct hydrophobic regions. That would be the main reason that contributed to the subsequent heterologous expression difficult to achieve soluble expression and protein refolding lower activity. LipB was cloned into pET-28a to construct pET28-LipB. E. coli BL21(DE3) was used as the host of the expression. The recombinant protein was expressed as inclusion body. The inclusion body was resolved and purified with Ni affinity chromatography. Dialysis was done to recover the activity. Due to lower activity, the the analysis of the enzymatic properties of r-LipB use a sensitive GENMED bacteria lipase activity colorimetric quantitative assay kit. The results indicated that the optimal temperature of r-LipB was30℃, similar to r-LipA. It is known that myxobacterial strains normally apt to live in<30℃ environments. These enzymes were probably for their essential for the survival and ecological adaptative survivals.Finally, based on the conjugation method we constructed DGAT and lipase genes targeting vectors and knock out genes for mutants in M. xanthus DK1622. However, no difference in lipid body formation, growth, social motility, and EPS forming ability could be observed when these strains were compared with the wild type. The fruiting body formation and sporulation of the YL1801and YL1803mutants were enhanced compared to DK1622. According to the heat treatment results of the fruiting bodies, we speculated that MXAN4638, MXAN5522and MXAN6874may have some relationship with the composition of membrane lipid synthesis. The mechanism needs further experimental proof. In the mean time, based on the conjugation method with low dosages of dual selecting antibiotics established by early laboratory work, we constructed dgat and lipB gene targeting vectors (pCC4D and pCC4T, respectively) in S. cellulosum So0157-2. However, the screening and the purification of mutants are in progress.