Abstract
In this study, after the expression of a pyruvate carboxylase gene (PYC) cloned from Meyerozyma guilliermondii in a marine-derived yeast Yarrowia lipolytica SWJ-1b, a transformant PG86 obtained had much higher PYC activity than Y. lipolytica SWJ-1b. At the same time, the PYC gene expression and citric acid (CA) production by the transformant PG86 were also greatly enhanced. When glucose concentration in the medium was 60.0 g L−1, CA concentration formed by the transformant PG86 was 34.02 g L−1, leading to a CA yield of 0.57 g g−1 of glucose. During a 10-L fed-batch fermentation, the final concentration of CA was 101.0 ± 1.3 g L−1, the yield was 0.89 g g−1 of glucose, the productivity was 0.42 g L−1 h−1 and only 5.93 g L−1 reducing sugar was left in the fermented medium within 240 h of the fed-batch fermentation. HPLC analysis showed that most of the fermentation products were CA.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Karaffa L, Kubicek CP (2003) Aspergillus niger citric acid accumulation: do we understand this well working black box? Appl Microbiol Biotechnol 61:189–196
Förster A, Aurich A, Mauersberger S, Barth G (2007) Citric acid production from sucrose using a recombinant strain of the yeast Yarrowia lipolytica. Appl Microbiol Biotechnol 75(6):1409–1417
Förster A, Jacobs K, Juretzek T, Mauersberger S, Barth G (2007) Overexpression of the ICL1 gene changes the product ratio of citric acid production by Yarrowia lipolytica. Appl Microbiol Biotechnol 77(4):861–869
Liu XY, Chi Z, Liu GL, Wang F, Madzak C, Chi ZM (2010) Inulin hydrolysis and citric acid production from inulin using the surface-engineered Yarrowia lipolytica displaying inulinase. Metab Eng 12:469–476
Stottmeister U, Hoppe K, Ruttloff H (1991) Organische Genußsäuren. Lebensm Biotechnol Entwickl Asp 1:516–547
Liu XY, Chi Z, Liu GL, Madzak C, Chi ZM (2013) Both decrease in ACL1 gene expression and increase in ICL1 gene expression in marine-derived yeast Yarrowia lipolytica expressing INU1 gene enhance citric acid production from inulin. Mar Biotechnol 15(1):26–36
Chi Z, Wang ZP, Wang GY, Khan I, Chi ZM (2015) Microbial biosynthesis and secretion of l-malic acid and its applications. Crit Rev Biotechnol 41(2):228–237
Jitrapakdee S, St Maurice M, Rayment I, Wallace Cleland WW, John C, Wallace JC, Attwood PV (2008) Structure, mechanism and regulation of pyruvate carboxylase. Biochem J 413:369–387
Li Y, Chi Z, Wang GY, Wang ZP, Liu GL, Lee CF, Ma ZC, Chi ZM (2015) Taxonomy of Aureobasidium spp. and biosynthesis and regulation of their extracellular polymers. Crit Rev Microbiol 41(2):228–237
Brown SH, Bashkirova L, Berka R, Chandler T, Doty T, McCall K, McCulloch M, McFarland S, Thompson S, Yaver D, Berry A (2013) Metabolic engineering of Aspergillus oryzae NRRL 3488 for increased production of l-malic acid. Appl Microbiol Biotechnol 97(20):8903–8912
Yin X, Madzak C, Du G, Zhou J, Chen J (2012) Enhanced alpha-ketoglutaric acid production in Yarrowia lipolytica WSH-Z06 by regulation of the pyruvate carboxylation pathway. Appl Microbiol Biotechnol 96(6):1527–1537
Peleg Y, Battat E, Scrutton MC, Goldberg I (1989) Isoenzyme pattern and subcellular localisation of enzymes involved in fumaric acid accumulation by Rhizopus oryzae. Appl Microbiol Biotechnol 32(3):334–339
Lin H, San KY, Bennett GN (2005) Effect of Sorghum vulgare phosphoenolpyruvate carboxylase and Lactococcus lactis pyruvate carboxylase coexpression on succinate production in mutant strains of Escherichia coli. Appl Microbiol Biotechnol 67(4):515–523
Peters-Wendisch PG, Schiel B, Wendisch VF, Katsoulidis E, Mockel B, Sahm H, Eikmanns BJ (2001) Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum. J Mole Microbiol Biotechnol 3(2):295–300
Zhou J, Zhou H, Du G, Liu L, Chen J (2010) Screening of a thiamine-auxotrophic yeast for α-ketoglutaric acid overproduction. Lett Appl Microbiol 51(3):264–271
Peksel A, Torres N, Liu J, Juneau G (2002) 13C-NMR analysis of glucose metabolism during citric acid production by Aspergillus niger. Appl Microbiol Biotechnol 58(2):157–163
Wang GY, Chi Z, Song B, Wang ZP, Chi ZM (2012) High level lipid production by a novel inulinase-producing yeast Pichia guilliermondii Pcla22. Bioresour Technol 124:77–82
Wang GY, Zhang Y, Chi Z, Liu GL, Wang ZP, Chi ZM (2015) Role of pyruvate carboxylase in accumulation of intracellular lipid of the oleaginous yeast Yarrowia lipolytica ACA-DC 50109. Appl Microbiol Biotechnol 99(4):1637–1645
Wang F, Yue L, Wang L, Madzak C, Li J, Wang XH, Chi ZM (2009) Genetic modification of the marine-derived yeast Yarrowia lipolytica with high-protein content using a GPI-anchor-fusion expression system. Biotechnol Prog 25(5):1297–1303
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Beijing, pp 367–370 (Chinese translated edn)
Xuan JW, Fournier P, Gaillardin C (1988) Cloning of the LYS5 gene encoding saccharopine dehydrogenase from the yeast Yarrowia lipolytica by target integration. Curr Genet 14(1):15–21
Zhao CH, Cui W, Liu XY, Chi ZM, Madzak C (2010) Expression of inulinase gene in the oleaginous yeast Yarrowia lipolytica and single cell oil production from inulin-containingmaterials. Metab Eng 12(6):510–517
Zhang F, Wang ZP, Chi Z, Raoufi Z, Abdollahi S, Chi ZM (2013) The changes in Tps1 activity, trehalose content and expression of TPS1 gene in the psychrotolerant yeast Guehomyces pullulans 17-1 grown at different temperatures. Extremophiles 17(2):241–249
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1):248–254
Bologna FP, Andreo CS, Drincovich MF (2007) Escherichia coli malic enzymes: two isoforms with substantial differences in kinetic properties, metabolic regulation, and structure. J Bacteriol 189(16):5937–5946
Liu GL, Wang DS, Wang LF, Zhao SF, Chi ZM (2011) Mig1 is involved in mycelial formation and expression of the genes encoding extracellular enzymes in Saccharomycopsis fibuligera A11. Fungal Genet Biol 48(9):904–913
Camp BJ, Farmer L (1967) A rapid spectrophotometric method for the determination of citric acid in blood. Clin Chem 13(6):501–505
Spiro RG (2001) Analysis of sugars found in glycoproteins. Methods Enzymol 8:3–26
Chi Z, Liu J, Zhang W (2001) Trehalose accumulation from soluble starch by Saccharomycopsis fibuligera sdu. Enzyme Microb Technol 28(2):240–245
Otto C, Yovkova V, Aurich A, Mauersberger S, Barth G (2012) Variation of the by-product spectrum during α-ketoglutaric acid production from raw glycerol by overexpression of fumarase and pyruvate carboxylase genes in Yarrowia lipolytica. Appl Microbiol Biotechnol 95(4):905–917
Zelle RM, de Hulster E, van Winden WA (2008) Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carboxylation, oxaloacetate reduction, and malate export. Appl Environ Microbiol 74(9):2766–2777
de Jongh WA, Nielsen J (2008) Enhanced citrate production through gene insertion in Aspergillus niger. Metab Eng 10:87–96
Acknowledgments
This study was supported by Natural Science Foundation of Shandong Province, the Grant Number is ZR2014CZ001.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
449_2016_1607_MOESM2_ESM.doc
Supplementary material 2: Analysis of the fermentation products (C), standard CA (B) and l-malic acid (A) by using HPLC. (DOC 291 kb)
Rights and permissions
About this article
Cite this article
Tan, MJ., Chen, X., Wang, YK. et al. Enhanced citric acid production by a yeast Yarrowia lipolytica over-expressing a pyruvate carboxylase gene. Bioprocess Biosyst Eng 39, 1289–1296 (2016). https://doi.org/10.1007/s00449-016-1607-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00449-016-1607-8