Isolation, Metabolism And Breeding Of A Strain Producing Succinic Acid With Crop Straw Hydrolytes Including Pentose And Hexose

As a member of the C4-dicarboxylic acid family, succinic acid can be utilized not only as end product, but also as a precursor for a variety of fine chemicals, including 1,4-butanediol, tetrahydrofuran,γ-butyrolactone, phenyl-products, biodegradable polymers PBS and so on. Although succinic acid is currently produced predominantly from petrochemical precursors through reduction of maleic anhydride, once succinic acid could be produced from biomass, the lower cost would allow it to compete with more chemicals currently produced from petroleum-based feedstocks. The result would be a significant reduction in the use of petroleum resources. Comparison with the bioalcohol production, the succinic acid fermentation has prominent advantage, considering its substrate conversion yield over 100% theoretically and its character of fixing CO₂, even though the current research of bioalcohol was hotter than that of succinic acid. Once the fermentation technics was scaled-up successfully, we would set up a green platform by converting those crop straw to succinic acid. The study was focused on the Screening, breeding and metabolic modulating of a strain producing succinic acid with corn straw hydrolyte, and the main conclusions were as follows:(1) Many of the isolated strains were obligate or facultative anaerobes that produced primarily succinic acid and shared other similarities with well-known rumen species. About 300 strains producing succinic acid were isolated, and the strain S.JST was considered as the most potential strain through the primary fermentation in this experiment. By the basic physiological characterization, we found that the strain S.JST was close to the genus of Actinobacillus. Further API test showed that this strain had the similar character with the species of A. succinogenes. Then the 16S r RNA genes identification was carried out to confirm the results of physiological characterizations. The sequence of isolated strain had been submitted to the GenBank, and the accession number was EF044771.1. Based on the 99% high homology between strain S.JST and strain 130Z, we can confirm that the strain S.JST belongs to the A. succinogenes species, so the obtained strain S.JST was named A, succinogenes S.JST.(2) By the analysis of intermediate and its inhibitor, we found that the strain had the character of utilizing glucose and xylose simultaneously and that the succinic acid was hydrogenated from OAA by two steps, and the further analysis of the correlative enzyme and its gene cleared up the whole metabolic pathway and its correlative branch. Basing on the pathway analysis we set up the metabolic network from the metabolic matrix by which the fluxes were calculated. Flux distribution showed that succinic acid was the main product whose flux was also the highest among those end products, and from the AC-CoA acetic acid and alcohol were the main byproducts and the node analysis indicated that the node arrounding pyruvic acid was flexible which was suit for alteration.(3) The parent strain could not grow on the fluoroacetate plate because of its high phosphotransacetylase enzyme activity who played a very important role in transferring fluoroacetate to fluoro-acetyl coenzyme A, and the fluoro-acetyl coenzyme A could be further transferred to lethal Intermediate fluoro-citrate. So the interdiction surrounding phosphotransacetylase might occur in those strains who can grow well on the fluoroacetate plate. By this method we got a pta mutant strain M.JSTP. We analyzed the phosphotransacetylase enzyme activity and its gene sequence from the parent strain S.JST and the pta mutant strain M.JSTP. The result showed that the enzymic specific activity of phosphotransacetylase decreased. The change of enzyme activity led to the changed fluxes and thus led to the changed end products concentration. The end products concentration and intermediate fluxes all showed that the metabolism of acetic acid and succinic acid were decreased simultaneously. The decreasing of acetic acid did not increase the flux of succinic acid but decreased the flux of succinic acid, which seemed unreasonable. But the analysis of metabolic mechanism explained this phenomenon. We can find that if 100% glucose were converted to succinic acid through EMP the formation of [H] reducing power could not satisfy the requirement of the consumption of [H] reducing power, that is to say, in theory, there is contradiction between the formation and consumption of [H] reducing power. However this lacking could be made up by the redundant NADH produced by the metabolism of acetic acid because 2 mol net NADH were produced when 1 mol acetic acid was formatted. So, for improving the balance of [H] reducing power, other method must be imported. At the same time other mutagenesis for decreasing the metabolism of alcohol was also needed.(4) By site-directed mutagenesis we got a pta-adh coupled mutant strain with the pta mutant strain as parent strain. Due to a "TAA" codon introduced to the middle of the adh gene, the alcohol dehydrogenase enzymic activity decreased abviously. Flux analysis showed that alcohol flux was decreased and that succinic acid flux was increased to a certain extant. Due to the condition that cell was still in short of [H] reducing power, the extent of ascending of succinic acid was obviously weaker than that of descending of alcohol. For the coupled mutant strain, the potential of producing succinic acid was not exerted entirety because the [H] donor balance was still not maintained very well, so further metabolic modulating was needed.(5) By the modulating of H₂ and ORP and the ratio of EMP to HMP, the metabolism surrounding H electronid donor was well balanced, and so the flux of succinic acid was increased markedly at last. At the same time, xylulokinase which was the key enzyme for the metabolism of xylose was further activated by the replacing CaCO₃ with MgCO₃ as the neutralization for the fermentation course, which led to the fermentation of glucose and xylose simutaniously. (6) On the base of single factor experiment for primary culture medium ingredient, orthogonal design experiment showed that good succinic acid yield was gained when the concentration of metal ion and vitamin were as following (mmol/L) : Mg²⁺ 6.0 and Mn²⁺ 0.9 and Fe²⁺ 0.6 and Zn²⁺ 1.5 and V_B2 0.005 and V_B5 0.012 and V_B14 0.003 and V_B12 0.01, respectively. Further analysis with response surface methodology and neural network showed that better succinic acid yield was gained when the condition was as following: 67% CO₂ and 4.8 H₂ and 5.9 mmol/L biotin. Comparison with the regressive analysis of the response surface methodology, the neural network has the better ability of simulating and predicting.(7) By the simulation of line model and no-line model, we found the no-line model through MATLAB soft could reflect the fermentation kinetics of this strain, and the growth model and the producing model obtained was as following, respectively:...