Food and Fermentation Industries >

2025 , Vol. 51 >Issue 13: 8 - 16

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.040883

Effects of C4-dicarboxylate transporters overexpression on malic acid fermentation in Aspergillus niger

  • SHI Bingbing ,
  • TULUHONG·Tuohuti ,
  • HAN Tianqi ,
  • XU Shijie ,
  • XUE Xianli ,
  • CHEN Yue ,
  • WANG Depei
Expand
  • 1(College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China)
    2(Key Laboratory of Industrial Fermentation Microbiology (Ministry of Education), Tianjin 300457, China)
    3(Tianjin Engineering Center for Microbial Metabolism and Fermentation Process Control Technology, Tianjin 300457, China)

Received date: 2024-08-29

  Revised date: 2024-09-25

  Online published: 2025-08-04

Fold

Abstract

L-malic acid serves as an important platform chemical with diverse applications in food, medicine, and agriculture.Its microbial fermentation is notable for environmental friendliness and sustainability.This study investigated the effects of five C4-dicarboxylate transporters (DCT1-5) in Aspergillus niger strain CGMCC 10142 on extracellular L-malic acid secretion.Genetic engineering techniques were applied to construct overexpressing strains:C-pDCT1, C-pDCT2, C-pDCT3, C-pDCT4, and C-pDCT5.L-malic acid production and key enzyme transcript levels were analyzed at various time points through shake flask fermentation.The results indicate that all transformed strains significantly increased L-malic acid production compared to the original strain, with the C-pDCT2 strain achieving the highest production of 14.48 g/L after 60 hours of fermentation, a 22.09% increase over the original strain.The significant rise in dct2 gene transcript levels promotes the extracellular transport of L-malic acid, driving the expression of key glycolytic enzymes and citrate synthase.Consequently, transcript levels of isocitrate lyase and malate synthase increase.It is inferred that the increased L-malic acid production accumulates via the glyoxylate cycle pathway.This study establishes a foundation for optimizing the metabolic pathway of malic acid in Aspergillus niger.

Key words： Aspergillus niger; L-malic acid; C4-dicarboxylate transporters; transcriptional analysis

Cite this article

SHI Bingbing , TULUHONG·Tuohuti , HAN Tianqi , XU Shijie , XUE Xianli , CHEN Yue , WANG Depei . Effects of C4-dicarboxylate transporters overexpression on malic acid fermentation in Aspergillus niger[J]. Food and Fermentation Industries, 2025 , 51(13) : 8 -16 . DOI: 10.13995/j.cnki.11-1802/ts.040883

References

[1] CHEN X L, WANG Y C, DONG X X, et al.Engineering rTCA pathway and C4-dicarboxylate transporter for L-malic acid production[J].Applied Microbiology and Biotechnology, 2017, 101(10):4041-4052.
[2] 袁恺, 周卫强, 唐堂, 等.微生物制备L-苹果酸的研究进展[J].生物加工过程, 2021, 19(3):227-233.
YUAN K, ZHOU W Q, TANG T, et al.Microbial synthesis of L-malic acid:A review[J].Chinese Journal of Bioprocess Engineering, 2021, 19(3):227-233.
[3] CHI Z, WANG Z P, WANG G Y, et al.Microbial biosynthesis and secretion of L-malic acid and its applications[J].Critical Reviews in Biotechnology, 2016, 36(1):99-107.
[4] WEI Z, XU Y X, XU Q, et al.Microbial biosynthesis of L-malic acid and related metabolic engineering strategies:Advances and prospects[J].Frontiers in Bioengineering and Biotechnology, 2021, 9:765685.
[5] 王颖珊, 郭峰, 严伟, 等.四碳有机酸生物合成的代谢工程研究进展[J].生物工程学报, 2021, 37(5):1697-1720.
WANG Y S, GUO F, YAN W, et al.Advances in the metabolic engineering for the production of tetracarbon organic acids[J].Chinese Journal of Biotechnology, 2021, 37(5):1697-1720.
[6] IYYAPPAN J, BHARATHIRAJA B, BASKAR G, et al.Process optimization and kinetic analysis of malic acid production from crude glycerol using Aspergillus niger[J].Bioresource Technology, 2019, 281:18-25.
[7] KÖVILEIN A, KUBISCH C, CAI L Y, et al.Malic acid production from renewables:A review[J].Journal of Chemical Technology & Biotechnology, 2020, 95(3):513-526.
[8] BATTAT E, PELEG Y, BERCOVITZ A, et al.Optimization of L-malic acid production by Aspergillus flavus in a stirred fermentor[J].Biotechnology and Bioengineering, 1991, 37(11):1108-1116.
[9] BRINK H G, GEYER-JOHNSON M, SWART R M, et al.Malic acid production by Aspergillus oryzae:The immobilized fungal fermentation route[J].Biofuels, Bioproducts and Biorefining, 2023, 17(2):363-379.
[10] 吴军林, 吴清平, 张菊梅, 等.L-苹果酸生物合成研究进展[J].食品科学, 2014, 35(3):238-242.
WU J L, WU Q P, ZHANG J M, et al.Progress in L-malic acid biosynthesis[J].Food Science, 2014, 35(3):238-242.
[11] 金利群, 李晓菡, 赵贝贝, 等.微生物有机酸转运蛋白的研究进展[J].微生物学报, 2023, 63(9):3386-3408.
JIN L Q, LI X H, ZHAO B B, et al.Organic acid transporters in microorganisms[J].Acta Microbiologica Sinica, 2023, 63(9):3386-3408.
[12] DARBANI B, STOVICEK V, VAN DER HOEK S A, et al.Engineering energetically efficient transport of dicarboxylic acids in yeast Saccharomyces cerevisiae[J].Proceedings of the National Academy of Sciences of the United States of America, 2019, 116(39):19415-19420.
[13] JANAUSCH I G, ZIENTZ E, TRAN Q H, et al.C4-dicarboxylate carriers and sensors in bacteria[J].Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2002, 1553(1-2):39-56.
[14] 姜巨全, 张正来, 徐桐, 等.大肠杆菌中dctA基因敲除及其苹果酸摄取功能鉴定[J].东北农业大学学报, 2018, 49(5):45-52.
JIANG J Q, ZHANG Z L, XU T, et al.Knock-out of dctA gene from Escherichia coli K12 and characterization of its function for malic acid transportation[J].Journal of Northeast Agricultural University, 2018, 49(5):45-52.
[15] ASAI K, BAIK S H, KASAHARA Y, et al.Regulation of the transport system for C4-dicarboxylic acids in Bacillus subtilis[J].Microbiology, 2000, 146 (Pt 2):263-271.
[16] LIU J J, XIE Z P, SHIN H D, et al.Rewiring the reductive tricarboxylic acid pathway and L-malate transport pathway of Aspergillus oryzae for overproduction of L-malate[J].Journal of Biotechnology, 2017, 253:1-9.
[17] MICHIELSE C B, HOOYKAAS P J J, VAN DEN HONDEL C A M J J, et al.Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori[J].Nature Protocols, 2008, 3(10):1671-1678.
[18] 曹张磊, 王德培, 张岚.提高根癌农杆菌介导黑曲霉转化效率的研究[J].天津科技大学学报, 2016, 31(2):20-25.
CAO Z L, WANG D P, ZHANG L.Improvement of transformation efficiency of Aspergillus niger mediated by Agrobacterium tumefaciens[J].Journal of Tianjin University of Science & Technology, 2016, 31(2):20-25.
[19] PELEG Y, STIEGLITZ B, GOLDBERG I.Malic acid accumulation by Aspergillus flavus[J].Applied Microbiology and Biotechnology, 1988, 28(1):69-75.
[20] KANG N K, LEE J W, ORT D R, et al.L-malic acid production from xylose by engineered Saccharomyces cerevisiae[J].Biotechnology Journal, 2022, 17(3):2000431.
[21] JIANG Y M, ZHENG T W, YE X H, et al.Metabolic engineering of Escherichia coli for L-malate production anaerobically[J].Microbial Cell Factories, 2020, 19(1):165.
[22] 张文芝, 郭坚华.微生物发酵工艺优化研究进展[J].广东农业科学, 2013, 40(6):114-117.
ZHANG W Z, GUO J H.Research advances in microbial fermentation process optimization[J].Guangdong Agricultural Sciences, 2013, 40(6):114-117.
[23] ZHOU S H, DING N N, HAN R H, et al.Metabolic engineering and fermentation optimization strategies for producing organic acids of the tricarboxylic acid cycle by microbial cell factories[J].Bioresource Technology, 2023, 379:128986.
Options
Outlines

/

〈 〉

Powered by Beijing Magtech Co. Ltd,.