Succinic acid
Succinic acid
110-15-6
disodium succinate
Phthalocyanine pigment
Compound dyes
Compound green
Composite blue
METHOD FOR PREPARING SUCCINIC ACID(cas:110-15-6) USING SUCROSE AS A CARBON SOURCE
Release time:2016/8/10 17:29:47

The present invention relates to a method for preparing succinic acid, which comprises culturing a succinic acid(cas:110-15-6)-producing microorganism in a medium containing sucrose as a carbon source, and more particularly, a method for preparing succinic acid(cas:110-15-6), which comprises culturing a succinic acid(cas:110-15-6)-producing microorganism under batch or fed-batch culture conditions in a medium containing sucrose as a carbon source at a high concentration. According to the present invention, when inexpensive sucrose, whose price corresponds to 28.9% of glucose price, is used as a carbon source, microorganisms have improved resistance against organic acids including succinic acid(cas:110-15-6) compared to the case using other carbon sources such as glucose to significantly increase succinic acid productivity as well as final succinic acid concentration, thus making it possible to reduce costs required for separating and purifying succinic acid.

METHOD FOR PREPARING SUCCINIC ACID USING SUCROSE AS A CARBON SOURCE
TECHNICAL FIELD
The present invention relates to a method for preparing succinic acid(cas:110-15-6), which comprises culturing a succinic acid(cas:110-15-6)-producing microorganism in a medium containing sucrose as a carbon source, and more particularly, a method for preparing succinic acid, which comprises culturing a succinic acid-producing microorganism under batch or fed-batch culture conditions in a medium containing sucrose as a carbon source.


BACKGROUND ART

Succinic acid(cas:110-15-6), which is widely known as an amber acid, has been produced by chemical synthesis and microbial fermentation, and can be used as a precursor of various chemical products and thus has high utility for economic and industrial applications (Zeikus et al, Appl. Microbiol. Biotechnol., 51 :545, 1999; Song et al, Enzyme Microbial Technol., 39: 352, 2006). Particularly, the demand for succinic acid is expected to be dramatically increased due to the finding that succinic acid can be used as a main source of biodegradable polymers (Willke et al., Appl. Microbiol. Biotechnol., 66:131, 2004). Most succinic acid for industrial use is currently produced using petroleum or liquefied natural gas as a raw material by chemical companies. When considering the latest sharp increase in petroleum prices, limited fossil resources and environmental concerns, there is an urgent need to develop a process for efficiently producing succinic acid by microbial fermentation, which can replace chemical synthesis process. With microbial culture technology, virtual cell technology and strain improvement through metabolic engineering, technology for succinic acid production has been remarkably progressed (Kim et al., Biotech. Bioeng., 97:657, 2007; Song et al., Enzyme Microbial Technol, 39:352, 2006).


Succinic acid(cas:110-15-6) production in a medium containing glucose as a culture material using Fusarium martii strain by Lockwood et al in 1938, triggered studies on succinic acid(cas:110-15-6) production using microorganisms. Since then, studies on the development of a process for effectively producing succinic acid(cas:110-15-6) through microbial fermentation, have been continuously carried out in various aspects, and various kinds of microorganisms (Actinobacillus, Anaerobiospirillum, Bacteroides, Mannheimia, Succinimonas, Succinivibrio etc.), which can efficiently produce succinic acid(cas:110-15-6), were identified. Meanwhile, studies on the development of succinic acid-producing mutant strain through genetic manipulation, have been mainly carried out using Escherichia? coli, and various kinds of succinic acid-producing E. coli mutants such as strains capable of producing succinic acid in an aerobic condition, etc., have been developed (US 5,770,435; US 6,648,061; Hong et al, Biotechnol. Bioeng., 74:89, 2001; Vemuri et al., J. Ind. Microbiol. Biotechnol., 28:325, 2001; Lin et al, Metabol. Eng., 7:116, 2005; Lin et al, Biotechnol. Bioeng., 90:775, 2005).


The present inventors isolated an excellent Mannheimia succiniciproducens MBEL55E (KCTC 0769BP) producing succinic acid(cas:110-15-6) with high efficiency from the rumen of Korean native cattle, and completed its full genome sequence and characterized metabolic properties thereof (Hong et al, Nature Biotechnol., 22:1275, 2004). Also, the present inventors have constructed its mutants, M. succiniciproducens LPK (KCTC 10558BP) by disrupting a gene encoding lactate dehydrogenase^/^) and a gene encoding pyruvate formate-lyase(#/7) from M. succiniciproducens MBEL 55E and M. succiniciproducens LPK7 (KCTC 10626BP) by disrupting a phosphotransacetylase gene(/??α) and an acetate kinase gene(αcfc4) in the mutant strain, M. succiniciproducens LPK (WO 2005/052135 Al; Lee et al, Appl Environ. Microbiol, 72:1939, 2006). In addition to that, the present inventors have constructed a mutant strain, M. succiniciproducens PALK
(KCTC 10973BP) (PCT/KR2007/003574) by disrupting a lactate dehydrogenase gene(ldhA), a phosphotransacetylase gene (pta) and an acetate kinase gene (ackA) in the M. succiniciproducens MBEL55E strain, M. succiniciproducens ALKt (PCT/KR2008/000012) by overexpressing a phosphotransacetylase gene (pta) in PALK strain, and a mutant strain, M. succiniciproducens ALK (PCT/KR2008/000012) by disrupting a lactate dehydrogenase gene(ldhA) and an acetate kinase gene (ackA) in the M. succiniciproducens MBEL55E strain.

In fact, costs required for a succinic acid production process using microorganisms are higher than that required for a succinic acid production process using chemical synthesis. Thus, in order to reduce the costs required for the process of producing succinic acid using microbial fermentation, various studies on the development of culture processes such as a batch culture, a fed-batch culture, a continuous culture etc., have been conducted (Lee et al, Appl. Microbiol. Biotechnol., 54:23, 2000; Lee et al., BioProc. Biosystems Eng., 26:63, 2003; Urbance et al, Appl. Microbiol. Biotechnol., 65:664, 2004). Together with the culture process development, studies on the development of inexpensive raw materials such as wood hydrolysate, glycerol, whey, corn steep liquor etc., have been also carried out by many researchers (Samuelov et al., Appl Environ. Microbiol. 65:2260, 1999; Lee et al., Appl Microbiol Biotechnol., 54:23, 2000; Lee et al., Biotechnol Bioeng., 72:41, 2001; Lee et al, Biotechnol Lett., 25:111, 2003; Lee et al, BioProc. Biosystems Eng., 26:63, 2003). However, studies reported to date suggest that when the above mentioned materials were used as a raw material instead of glucose, succinic acid productivity was far lower than that when glucose was used as a raw material and thus cost-effective succinic acid production could not be achieved.


Sucrose consisting of glucose and fructose is a disaccharide which is produced by green plants in the process of undergoing photosynthesis and is very abundant in nature. Especially, it accounts for 15-20%(w/v) of sugar cane and sugar beet juice, and is industrially produced by a very simple process of repeating evaporation/concentration and thus production cost thereof is very low. According to a report by Koutinas et al. (Ind. Crops and Products. 20:75, 2004), in which prices of various raw materials, used in the production of chemical materials using microorganisms, are calculated based on glucose content, sucrose is 26.1 cents based on lkg glucose, which is a very low price corresponding to 77%, 50% and 28.9% of the prices of wheat, molasses and glucose, respectively.


With respect to studies on useful material production using sucrose, biodegradable polymers by cell culture at high concentration (polyhydroxybutyrate, Lee et al., Biotechnol Lett., 15:971, 1993; Lee et al., Biotechnol. Techniques, 1 :59, 1997), citric acid (Forster et al., App. Microbiol. Biotechnol., 75: 1409, 2007), acetone, butanol, ethanol, isopropanol (George et al., Appl. Environ. Microbiol., 45:1160, 1983; Durre, Appl. Microbiol. Biotechnol., 49:639, 1998), itaconic acid (Kautola et al., Biotechnol. Lett., 11 :313, 1989), xanthan gum (Letisse et al., Appl. Microbiol. Biotechnol., 55:417, 2001) and the like have been reported. However, in case of sucrose, only basic studies on whether succinic acid-producing microorganism can grow using sucrose as a carbon source, and the kinds of final metabolites produced by sucrose consumption, and the like, have been conducted (Lee et al., Proc. Biochem., 35:49, 1999; Lee et al., Appl. Microbiol. Biotechnol, 58:663, 2002). Namely, there is no report of whether succinic acid can be produced using sucrose, a method for preparing succinic acid using sucrose and conditions thereof, and in addition to that, the development of technologies that can improve succinic acid- productivity and the final succinic acid concentration, and the like by efficiently utilizing sucrose.


For the economical production of succinic acid for industrial use by microbial fermentation, the following requirements should be satisfied; use of inexpensive raw materials such as sucrose, and an increase in succinic acid productivity and the final succinic acid concentration. Particularly, when considering the fact that the costs of raw materials account for about 50% of overall production cost of

chemicals by microbial fermentation, there is an urgent need to develop a method for preparing succinic acid using inexpensive raw materials.


Accordingly, the present inventors have made extensive efforts to develop an effective technology for producing succinic acid at a high concentration using sucrose, and as a result, developed a method for producing succinic acid using inexpensive sucrose instead of glucose, and confirmed that when succinic acid is produced in a medium containing sucrose as a carbon source, final succinic acid concentration is much higher than that in the conventional succinic acid production process, particularly, sucrose-containing medium prevents succinic acid from inhibiting cell growth resulted in the increase of final cell concentration, so that overall succinic acid productivity is significantly increased, thereby completing the present invention.

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