The Research On Citric Acid Production By Marine-derived Yeast Yarrowia Lipolytica

Citric acid (CA) is an important organic acid used widely in pharmaceutical,food and chemical cosmetics industries, which is mainly obtained by the fermentationof starchy grains. Recently, it is well regarded that the starchy grains can’t be used asthe materials for the industrial production of CA because of their shortage and highprice. Therefore, cassava starch and inulin, which can be hydrolyzed enzymatically toglucose and fructose respectively, may be good materials for CA production.CA is mainly obtained from the fermentation using Aspergillus niger. However,this process usually causes environmental pollution, and the mycelium of A.niger isadverse to genetic operation. Recently, it has been found that Y. lipoltica is a betterproducer of CA, A bioprocess for CA production using Y. lipolyica has severaladvantages compared to that using A. niger. In Recent year, it has been found that themarine environments are also rich in yeast resources, over120strains of Y. lipolyicaisolated from different marine environments were screened for CA production, and Y.lipolyica SWJ-1b (Collection number:2E00068at Marine Microorganisms CultureCollection of China) was found could produce more CA than other yeasts tested inthis study. In the batch cultivation with Y. lipolytica SWJ-1b,24.0±0.7g/l (CAproductivity0.20g/l h) of CA was yielded from40.0g/l of glycerol. The best carbonproducing CA was glucose and its appropriate concentration in culture medium forCA production was40.0g/l,25.5±1.2g/l (0.21g/l h) of CA was yielded from40.0g/l glucose with the inoculation of2.0×108cells/l medium.We found that cassava starch couldn’t be hydrolyzed and used by Y. lipolyticaSWJ-1b and amylase gene couldn’t be express in this strain either. So amylasesproduced by S. fibuligera IFO0111was used to hydrolyze cassava starch, and thehydrolysate was used to produce CA by Y. lipolytica SWJ-1b. In the batchcultivation,29.4±0.3g/l (0.25g/l h) of CA was yielded from40.0g/l hydrolysate ofcassava starch. In the fed-batch cultivation in2-l fermenter, CA production was increased to60.2±1.8g/l (0.21g/l h).It was found that strain Y. lipolytica SWJ-1b couldn’t synthesize exo-inulinase.In this study, the inulinase gene isolated from Kluyveromyces marxianus CBS6556was transformed in to Y. lipolytica SWJ-1b. The expressed inulinase was displayedon the yeast cells and was used to hydrolyze inulin, and CA was produced frominulin directly by the engineered yeast INU-87. The activity of the immobilizedinulinase was22.6±0.5U/mg cell dry weight, and it was found the displayedinulinase was an exo-inulinase and had the same characters as those of nativeinulinase produced by the wild type strain K. marxianus and could be easily reused.With the transformant INU-87,31.2±1.6g/l (0.26g/l h) of CA was yielded from40.0g/l of inulin, while68.9±2.4g/l (0.22g/l h) of CA was yield from100.0g/l ofinulin in the2-l fermenter.A disadvantage of wild type strains of Y. lipolytica using in CA producting is thesecretion of ICA as a by-product and the accumulation of lipid in its cells, which willnegatively affect CA production. ICA production is controled by iso-citrate lyase (ICL)while lipid accumulation is governed by ATP-citrate lyase (ACL). In order to reduce ICAproduction and the accumulation lipid and further improve the CA production, the ACL1gene was partially deleted and the copy number of the ICL1gene was increased inINU-87(Fig5-1), and the genetically modified strain IAI-30was obtained. It was foundthat the ACL1gene expression and ACL activity in strain IAI-30were declined and theICL1gene expression and ICL activity were promoted. It was also found that lipidcontent and iso-citric acid production were greatly reduced and citric acid production wasgreatly enhanced (36.0±0.6g/l and0.30g/l h CA was yielded from40.0g/l of inulin) inthe fermentation of CA using IAI-30. In the2-l fermenter,84.0±1.6g/l (0.39g/l h) ofCA in the fermented medium was attained from100.0g/l of inulin with strain IAI-30.The CA was extracted from the fermented medium with calcium carbonate andwas purified, and the CA sample was analysed with HPLC, and its purity was95.0%.