In order to explore the feasibility of yeast in producing D-arabitol using xylose as a substrate, based on Candida tropicalis CU-208, an engineered strain for D-arabitol production was constructed by means of metabolic engineering in this study. Based on the verification of the endogenous D-arabitol dehydrogenase(ARD)function of C. tropicalis, the following metabolic modifications were conducted. Firstly, xks gene was knocked out to block xylose to enter the pentose-phosphate pathway, and xdh gene was overexpressed to construct strain FYM01. Then, ard gene was knocked out to block D-arabitol consumption to construct strain FYM02. Next, adh gene was overexpressed to construct strain FYM04. Finally, zwf gene was overexpressed to enhance the NADPH coenzyme supply, and strain FYM05 was construct. The results showed that ard gene was involved in the catabolism of D-arabitol. Besides, the strain FYM05 obtained via metabolic transformation could not only utilize glucose as an auxiliary carbon source to provide growth energy and reducing power, but also use xylose to produce D-arabitol. The yield of D-arabitol in shake flask by strain FYM05 reached 11.35 g/L, which is 11.7 times higher than that of the parental strain. In brief, in this study, a recombinant C. tropicalis that is capable of producing D-arabitol from xylose was successfully constructed through metabolic engineering, which laid the foundation for the industrial production of D-arabitol by C. tropicalis.
[1] KORDOWSKA-WIATER M. Production of arabitol by yeasts: Current status and future prospects[J].Journal of Applied Microbiology,2015,119(2):303-314.
[2] QI X H, LUO Y, WANG, X et al.Enhanced D-arabitol production by Zygosaccharomyces rouxii JM-C46:Isolation of strains and process of repeated-batch fermentation[J].Journal of Industrial Microbiology & Biotechnology, 2015, 42(5):807-812.
[3] 黄魏, 王晓丹, 李豆南, 等.微生物发酵产D-阿拉伯糖醇的研究进展[J].中国酿造, 2017, 36(9):6-10.
HUANG W, WANG X D, LI D N, et al.Research progress of D-arabitol production by microbial fermentation[J].China Brewing, 2017, 36(9):6-10.
[4] YOUNG H.Sweeteners and Sugar Alternatives in Food Technology[M].London:Blackwell Publishing, 2006:205-218.
[5] TOYODA T, OHTAGUCHI K.Selection of Kluyveromyces yeasts for the production of D-arabitol from lactose[J].Journal of Chemical Engineering of Japan, 2009, 42(7):508-511.
[6] 宋卫斌, 林燕青, 胡海燕, 等.产D-阿拉伯醇菌株的筛选、鉴定及其产D-阿拉伯醇条件的优化[J].微生物学报, 2011, 51(3):332-339.
SONG W B, LIN Y Q, HU H Y, et al.Isolation and identification of a novel Candida sp.H2 producing D-arabitol and optimization of D-arabitol production[J].Acta Microbiologica Sinica, 2011, 51(3):332-339.
[7] 张丽丽.高产D-阿拉伯糖醇酵母菌株的筛选及其发酵条件的研究[D].无锡:江南大学, 2009.
ZHANG L L.Screening of yeast strains and fermentation conditions for hight yield production of D-arabitol[D].Wuxi:Jiangnan Univer- sity, 2009.
[8] 蔡莉, 张扬, 朱宏阳, 等.1株产D-阿拉伯糖醇的菌株的分离筛选及鉴定[J].食品与发酵工业, 2009, 35(1):23-26.
CAI L, ZHANG Y, ZHU H Y, et al.Isolation and identification of a novel D-arabitol producing strain[J].Food and Fermentation Industries, 2009, 35(1):23-26.
[9] 钱卫东, 宁肖肖, 赵德志, 等.利用组合策略提高汉逊酵母发酵生产D-阿拉伯糖醇[J].安徽农业科学, 2014, 42(23):7 726-7 728.
QIAN W D, NING X X, ZHAO D Z, et al.An optimal combination strategy to increase D-arabitol production by Hansenula polymorpha[J].Journal of Anhui Agricultural Sciences, 2014, 42(23):7 726-7 728.
[10] 王惠莲, 杨廉婉, 那淑敏, 等.耐高渗透压酵母生产甘油及阿拉伯糖醇的研究——Ⅲ.Zygosaccharomyces chevalieri Guill.2.309产生甘油的条件[J].微生物学报, 1963, 9(2):200-201.
WANG H L, YANG L W, NA S M, et al.Studies on the production of glycerol and arabitol by osmophilic yeasts Ⅲ.Carbohydrate metabolism in osmophilic yeasts[J].Acta Microbiologica Sinica, 1963, 9(2):200-201.
[11] JAGTAP S S, RAO C V.Production of D-arabitol from D-xylose by the oleaginous yeast Rhodosporidium toruloides IFO0880[J].Applied Microbiology and Biotechnology, 2018, 102(1):143-151.
[12] GALL D L, RALPH J, DONOHUE T J, et al.Biochemical transformation of lignin for deriving valued commodities from lignocellulose[J].Current Opinion in Biotechnology, 2017, 45:120-126.
[13] 周平平.生物炼制发酵微生物的呋喃醛和酚醛抑制物脱毒机制和应用研究[D].上海:华东理工大学, 2019.
ZHOU P P.Analysis of detoxification mechanism of furan and phenolic aldehyde inhibitors of biorefinery fermenting strains and its extended applications[D].Shanghai:East China University of Science and Technology, 2019.
[14] 王旭.产D-阿拉伯糖醇酵母菌株的筛选、发酵与产木糖醇基因工程菌的构建[D].镇江:江苏大学, 2015.
WANG X.The isolation and fermentation of yeast strain producing D-arabitol and construction of genetic engineering strain producing xylitol[D].Zhenjiang:Jiangsu University, 2015.
[15] 周桂雄.代谢未脱毒木质纤维素稀酸水解物产乙醇耐毒酵母菌的筛选及特性研究[D].北京:首都师范大学, 2009.
ZHOU G X.Isolation and characterization of inhibitor-tolerant yeast strains for ethanol production from dilute-acid lignocellulosic hydrolysate without detoxification[D].Beijing:Capital Normal University, 2009.
[16] SOMANI A, SMITH D, HEGARTY M, et al.Draft genome assemblies of xylose-utilizing Candida tropicalis and Candida boidinii with potential application in biochemical and biofuel production[J].Genome Announcements, 2018, 6(7):e01594-e01517.
[17] ZHANG L, ZHANG H, LIU Y, et al.A CRISPR-Cas9 system for mul- tiple genome editing and pathway assembly in Candida tropicalis[J].Biotechnology and Bioengineering, 2020, 117(2):531-542.
[18] WONG B, LEESON S, GRINDLE S, et al.D-arabitol metabolism in Candida albicans:Construction and analysis of mutants lacking D-arabitol dehydrogenase[J].Journal of Bacteriology, 1995, 177(11):2 971-2 976.
[19] HAAS L O, CREGG J M, GLEESON M A.Development of an integrative DNA transformation system for the yeast Candida tropicalis[J].Journal of Bacteriology, 1990, 172(8):4 571-4 577.
[20] 王世杰, 张海冰, 沈微, 等.代谢工程改造热带假丝酵母高效合成中链ω-羟基脂肪酸[J].微生物学报, 2022, 62(1):77-89.
WANG S J, ZHANG H B, SHEN W, et al.Efficient synthesis of medium-chain ω-hydroxy fatty acids by metabolic engineering in Candida tropicalis[J].Acta Microbiologica Sinica, 2022, 62(1):77-89.
[21] 黄玉堃, 张利华, 张海冰, 等.代谢改造热带假丝酵母提高D-乳酸的产量[J].食品与发酵工业, 2021, 47(24):109-115.
HUANG Y K, ZHANG L H, ZHANG H B, et al.Metabolic engineer- ing of Candida tropicalis to improve D-lactic acid production[J].Food and Fermentation Industries, 2021, 47(24):109-115.
[22] ZHANG L H, CHEN Z, WANG J H, et al.Stepwise metabolic engineering of Candida tropicalis for efficient xylitol production from xylose mother liquor[J].Microbial Cell Factories, 2021, 20(1):105.
[23] 李敬知.辅酶合成对酿酒酵母异丁醇产量的影响[D].天津:河北工业大学, 2017.
LI J Z.Effect of cofactor biosynthesis on isobutanol yield in Saccha- romyces cerevisiae [D].Tianjing:Hebei University of Technology, 2017.
