Lactic acid is a kind of hydroxy acid, which can be divided into D-lactic acid and L-lactic acid due to its combination site of hydroxyl. It is widely applied in food, medicine and biomaterial industries. As the growing demand for biodegradable plastics, researches on producing lactic acid by biotechnology have drawn wide attentions. This study investigated the impact of pdc gene knock out and gene overexpression of LDH and 2-HAD on cell growth and D-lactic acid production, based on the diploid Candida tropicalis. pdc1 gene knock out strain AK01, pdc2 gene knock out strain AK02, pdc1/pdc2 gene knock out strain AK03, 2-had gene overexpression strain AK04, AK06 and d-ldh gene overexpression strain AK05, AK07 were obtained by homologous recombination. The results showed that there was growth inhibition and notably ethanol reduction in pdc1 gene knock out strain AK01 and AK03. The growth inhibition was also found in d-ldh gene overexpression strain AK05 and AK07. pdc1 gene knock out and d-ldh gene overexpression strain AK05 produced 6.1 g/L D- lactic acid, which was 28 times more than that produced by the parental strain.
[1] LIAUD N, ROSSO M N, FABRE N, et al.L-lactic acid production by Aspergillus brasiliensis overexpressing the heterologous ldhA gene from Rhizopus oryzae[J].Microbial Cell Factories, 2015, 14(1):66-75.
[2] RYU A J, KIM T Y, YANG D S, et al.Engineering of Saccharomyces cerevisiae for enhanced production of L-lactic acid by co-expression of acid-stable glycolytic enzymes from Picrophilus torridus[J].Korean Journal of Chemical Engineering, 2018, 35(8):1 673-1 679.
[3] PARK H J, BAE J H, KO H J, et al.Low-pH production of D-lactic acid using newly isolated acid tolerant yeast Pichia kudriavzevii NG7[J].Biotechnology and Bioengineering, 2018, 115(9):2 232-2 242.
[4] WANG Y, TASHIRO Y, SONOMOTO K.Fermentative production of lactic acid from renewable materials:Recent achievements, prospects, and limits[J].Journal of Bioscience & Bioengineering, 2015, 119(1):10-18.
[5] 黄国昌, 熊大维, 张婷, 等.发酵法生产乳酸的研究进展[J].江西科学,2014, 32(5):660-666.
HUANG G C, XIONG D W, ZHANG T, et al.Advances in fermentation methods for lactic acid production[J].Jiangxi Science, 2014, 32(5):660-666.
[6] 钟伟. 酿酒酵母产D-乳酸的代谢工程研究[D].北京:中国科学院大学(中国科学院过程工程研究所),2019.
ZHONG W.Metabolic engineering of Saccharomyces cerevisiae for D-lactic acid production[D].Beijing:University of Chinese Academy of Sciences,2019.
[7] NAKANO S, UGWU C U, TOKIWA Y.Efficient production of D-(-)-lactic acid from broken rice by Lactobacillus delbrueckii using Ca(OH)2 as a neutralizing agent[J].Bioresource Technology, 2012, 104(1):791-794.
[8] BAEK S H, KWON E Y, BAE S J, et al.Improvement of D-lactic acid production in Saccharomyces cerevisiae under acidic conditions by evolutionary and rational metabolic engineering[J].Biotechnology Journal, 2017,12(10):1700015.
[9] ILMÉN M, KOIVURANTA K, RUOHONEN L, et al.Production of L-lactic acid by the yeast Candida sonorensis expressing heterologous bacterial and fungal lactate dehydrogenases[J].Microbial Cell Factories, 2013, 12(1):53.
[10] LEE J W, IN J H, PARK J B, et al.Co-expression of two heterologous lactate dehydrogenases genes in Kluyveromyces marxianus for L-lactic acid production[J].Journal of Biotechnology, 2017, 241(1):81-86.
[11] HOU Q, HE Q, LIU G, et al.Identification and application of novel low pH-inducible promoters for lactic acid production in the tolerant yeast Candida glycerinogenes[J].Journal of Bioscience and Bioengineering, 2019, 128(1):8-12.
[12] KOIVURANTA K T, ILMÉN M, WIEBE M G, et al.L-lactic acid production from D-xylose with Candida sonorensis expressing a heterologous lactate dehydrogenase encoding gene[J].Microbial Cell Factories, 2014, 13(1):1-14.
[13] ZHANG L H, CHEN X Z, CHEN Z, et al.Development of an efficient genetic manipulation strategy for sequential gene disruption and expression of different heterologous GFP genes in Candida tropicalis[J].Applied Microbiology and Biotechnology, 2016, 100(22):9 567-9 580.
[14] 张志禹, 朱涛, 林荣胜,等.热带假丝酵母的二元酸分泌现象及其动力学[J].清华大学学报, 1999, 39(12):27-30.
ZHANG Z Y, ZHU T, LIN R S, et al.Kinetics of secretion and acid production by Candida tropicalis cells in dicarbonxylic acid fermentation system[J].Journal of Tsinghua University, 1999, 39(12):27-30.
[15] 刘树臣, 焦鹏, 曹竹安.热带假丝酵母代谢烷烃过程中的β-氧化和代谢调控[J].微生物学报, 2002, 42(1):125-128.
LIU S C, JIAO P, CAO Z A.Metabolism and β-oxidation of alkane-utilizing Candida tropicalis[J].Acta Microbiologica Sinica, 2002, 42(1):125-128.
[16] ZHONG W, YANG M H, MU T Z, et al.Systematically redesigning and optimizing the expression of D-lactate dehydrogenase efficiently produces high-optical-purity D-lactic acid in Saccharomyces cerevisiae[J].Biochemical Engineering Journal, 2019, 144(1):217-226.
[17] BONETE M J, FERRER J, PIRE C, et al.2-Hydroxyacid dehydrogenase from Haloferax mediterranei, a D-isomer-specific member of the 2-hydroxyacid dehydrogenase family[J].Biochimie, 2000, 82(12):1 143-1 150.
[18] BAEK S H, KWON E Y, KIM Y H, et al.Metabolic engineering and adaptive evolution for efficient production of D-lactic acid in Saccharomyces cerevisiae[J].Applied Microbiology & Biotechnology, 2016, 100(6):2 737-2 748.
[19] RODRÍGUEZ L, CHÁVEZ F P, BASABE L, et al.Development of an integrative DNA transformation system for the yeast Candida utilis[J].FEMS Microbiology Letters, 1998, 165(2):335-340.
[20] 项峥, 陈献忠, 张利华,等.利用可重复使用的URA3标记基因建立热带假丝酵母基因敲除系统[J].遗传, 2014, 36(10):1 053-1 061.
XIANG Z, CHEN X Z, ZHANG L H, et al.Development of a genetic transformation system for Candida tropicalis based on a reusable selection marker of URA3 gene[J].Hereditas, 2014, 36(10):1 053-1 061.
[21] 马瑞, 欧阳嘉, 李鑫, 等.高效液相色谱法同时测定生物质乳酸发酵液中有机酸及糖类化合物[J].色谱,2012, 30(1):62-66.
MA R,OUYANG J, LI X, et al.Simultaneous determination of organic acids and saccharides in lactic acid fermentation broth from biomass using high performance liquid chromatography[J].Chinese Journal of Chromatography, 2012,30(1):62-66.
[22] KONG X, ZHANG B, HUA Y, et al.Efficient L-lactic acid production from corncob residue using metabolically engineered thermo-tolerant yeast[J].Bioresource Technology, 2019, 273(2):220-230.
