Lipid Body Storage In Sorangium Cellulosum: Accumulation, Consumption And Its Relationships With Epothilone Production

Myxobacteria are Gram-negative gliding bacteria. They are noted for features that are characteristic of their complex life cycle, culminating in the formation of fruiting bodies and myxospores and the production of an extensive battery of bioactive compounds. Each of these properties plays an important role in establishing the particular ecological function of the myxobacteria.In addition to these defining characteristics, myxobacterial cells have also been shown to efficiently degrade biomacromolecules or living microbes. Myxobacteria are thus divided into bacteriolytic and cellulolytic groups based on their degradation abilities. Members of the bacteriolytic group prey on living microbes but are unable to digest the cellulosic materials. By contrast, members of the cellulolytic group degrade cellulosic materials, but not living microbes. When myxobacteria face starvation or suffer unfavorable environmental conditions, cells form a macroscopic fruiting body containing thousands of resting myxospores. Those myxospores are sufficiently resistant to desiccation, heat, and UV light. Therefore, myxobacteria are considered social bacteria.Sorangium cellulosum is a cellulolytic myxobacterial species. Strains of Sorangium cellulosum are able to produce abundant, diverse and novel secondary metabolites, accounting for nearly half of the total metabolites discovered in myxobacteria. Some Sorangium metabolites have been shown to be potentially important in clinical therapy. For example, epothilones are 16-membered polyketide macrolides that are produced by some strains of Sorangium cellulosum. The compounds are biosynthesized by Sorangium cells using a polyketide synthase hybridized with a non-ribosomal peptide synthetase. Epothilones are the only known natural microbial compounds that mimic the action of the mitotic inhibitor taxol on cancer cells (i.e., stabilizing the polymerized microtubules). Several epothilones or their derivatives are currently being tested in clinical trials. In 2007, ixabepilone was authorized for clinical use by the U.S. Food and Drug Administration. Although Sorangium strains are appreciated as an important resource that can be used for drug screenings, there are few studies on their metabolic characteristics, mainly due to the difficulties in manipulating Sorangium cells.Sorangium strains ecologically prefer to live in habitats with ample organic carbon debris, such as soil, bark, rotting plant material, and herbivore dung. Sorangium cells produce many enzymes that efficiently degrade cellulosic macromolecules and normally grow quickly in medium containing mineral salts plus cellulose as the only organic carbon source.Myxobacteria are famous for their excellent ability to produce various bioactive compounds. In the past two decades, about 100 basic structures and 500 structural variants have been discovered in myxobacteria and have been fully characterized chemically, which account for about 3.5% of the presently known secondary metabolites of microbial origin. Nearly all Sorangium strains produce some type of compounds with diverse biological activities.The Sorangium So0157-2 strain was isolated from a soil sample collected on the ChengHai Lake bank. Our previous bioactivity screening analysis confirmed that So0157-2 is a promising strain for the production of bioactive secondary metabolites. Sorangium cellulosum So0157-2 is an epothilone producer with good fermentation characteristics.Epothilone production is a very interesting topic and has substantial economic value. In contrast to the biomass and substrate in the medium, the epothilone production is very low. Improving the utilization rate of the carbon and nitrogen sources and enhancing the transformation efficiency are the expected methods.We observed that the Sorangium cells were able to form a number of intracellular lipid body-like inclusion particles during the growth stage under various culture conditions. The particles gradually disappeared after growth, concurrent with the production of the secondary metabolite epothilones. We were interested in investigating the formation and roles of the lipid body-like inclusions in Sorangium cells. In this paper, we determined that the inclusion particles were a triglyceride mixture of different types of fatty acids. In addition to the saturated straight-chain fatty acids 13:0,15:0,17:0, and 18:0, we also detected monounsaturated fatty acids 16:lAllc and 18:1△9c, the polyunsaturated fatty acid with conjugated double bonds 18:2A9c,llc, and iso-branched fatty acids i14:0 and i16:0. From the ESI/MS, the major component of triglycerides was i 16:0/i16:0/i16:0 triglycerides.The formation and consumption of the particles were investigated. The lipid body inclusions were produced mainly during the exponential growth stage of the organism. Once the environmental carbon sources were exhausted (determined by the level of reducing sugars), the lipid body inclusions did not continue to accumulate. Instead, these inclusion particles were gradually consumed, as indicated by a decrease in both number and size of the lipid body inclusions. Furthermore, cell biomass was also observed to be sustained for approximately 10 days following the conclusion of the exponential growth stage. However, cell biomass quickly decreased once the lipid inclusion was almost completely consumed. These results suggest that abundant nutrient conditions allow the lipid body inclusions to store both carbon and energy in cells that are in the exponential growth stage. When nutrients become deficient, the lipid body inclusions are consumed to sustain cell survival.To investigate the biosynthesis and consumption of lipid body inclusions, inhibitors of fatty acids were used. Fatty acid synthases are required for the generation of lipid body inclusion particles. Isoniazid is an inhibitor of the enoyl-ACP reductase, which is a type II fatty acid synthase composed of a series of individual enzymes responsible for the biosynthesis of fatty acids. Without the inhibitor, the inclusion particles disappeared after more than 2 weeks. Interestingly, the inclusion particles almost completely disappeared in most cells after 4-day incubation in the presence of isoniazid. Interestingly, the addition of 1%(w/v) sodium acetate, which is a biosynthetic component of lipids, along with isoniazid, negated the effects observed by the addition of isoniazid alone. These results indicate that the presence of the inclusion particles is important for the survival of Sorangium cells. Specific inhibitor of fatty acid biosynthesis blocked the synthesis of fatty acids and thus accelerated the disappearance of the lipid body inclusions, which could be rescued by the presence of the subunits. It is likely that there is a dynamic balance between the formation and consumption of lipid body inclusions.For further confirmation, the effects of isoniazid and acetate on the inclusions were further tested in a nutrient-free HEPES buffer. When the inclusion-containing Sorangium cells were transferred into the HEPES buffer, the consumption of lipid body inclusions was accelerated, such that they were nearly eliminated after an additional 4 days of incubation, and the Sorangium cells were gradually disrupted. Similar to that in normal cultures, the addition of 1%(w/v) sodium acetate prevented the loss of the lipid inclusions and sustained the size of the inclusions for an extended period of time. Interestingly, if 1%(w/v) sodium acetate was further added into the HEPES buffer containing cells after 4-day incubation, the cells that had lost the lipid body inclusions reformed them.The addition of 1%(w/v) sodium propionate or sodium acetate limited the consumption of the lipid body inclusions, but the production of epothilones was concurrently increased. Upon the addition of 1%(w/v) sodium propionate, total epothilone production was found to increase 1.5-fold. In addition to the increase in total epothilone production, the yield of epothilone B exceeded the yield of epothilone A with an A:B ratio of 1:1.4, whereas the normal ratio of epothilone A:B in So0157-2 is approximately 1.4:1. The addition of high sodium propionate concentrations to solid CNST medium at the beginning of cultivation even resulted in an epothilone A.B ratio of 1.7.6, although total epothilone production was very low.Inhibitors of fatty acid synthase enzymes were also used to determine their effects on the production of epothilones. The addition of 10 mg/L of isoniazid at the time of inoculation resulted in a 2.9-fold increase in total epothilone production, but increasing the isoniazid concentration led to toxicity in cells, with a consequent decrease in total epothilone production, and also affected the epothilone A:B ratio (e.g. 1000 mg/L of isoniazid led to a ratio of epothilone A:B of 4.2:1). Other inhibitors of fatty acid synthase enzymes had the same effect. The addition of 100 mg/L of triclosan (which inhibits the same enzymes as isoniazid) at the time of inoculation resulted in a 1.8-fold increase in total epothilone production; the addition of 100 mg/L of sesamol, which blocks NADPH production by inhibiting the malic enzyme, increased total epothilone production by 1.7-fold.We further investigated the lipid body inclusions at the molecular level. The major component of inclusions are triglycerides, so we probed the key enzymes of triglyceride synthesis and degradation. Diacylglycerol acyltransferase (DGAT) (EC 2.3.1.20) catalyzes the final step of triglyceride biosyntheses. The CoA thioesters of alkanoic acids are used as substrates for the esterification of DAGs, with the concomitant release of CoA. DGAT is the unique enzyme that separates TAG from the biosynthesis of other lipids. Triacylglycerol lipase (TAGL) (EC 3.1.1.3) and monoglyceride lipase (MAGL) (EC 3.1.1.23) are the important enzymes of triacylglycerol degradation and usage. TAGL catalyzes the hydrolysis of a variety of acylglycerols at the interface of lipid and water. MAGL catalyzes the hydrolysis of monoglyceride.The recently finished genome sequencing project for So ce56 provided a reference for our gene search. Based on the alignment of their homologous genes, we found the potential genes of DGAT, TAGL, and MAGL. The genes were heterologously expressed in E. coli BL21(DE3) by use of the T7 promoter-and polymerase-based pET and pGEX systems.We expressed the recombinant proteins in E. coli hosts. MAGL was soluble and biologically active, but TAGL and DGAT expression produced inactive inclusion bodies under the control of a strong T7 promoter despite the use of five different expression vectors. TAGL inclusion bodies were subsequently dissolved in 8 M urea and purified using a Ni-nitrilotriacetic acid column followed by dialysis. The refolded protein was soluble and biologically active by this denaturation-renaturation procedure. We further investigated the enzymatic reaction parameters.Based on the conjugation method, we constructed DGAT and TAGL gene targeting vectors (pCC4D and pCC4T, respectively). However, the screening and the purification of mutants are in progress.