代谢工程改造毕赤酵母生产赤藓糖醇

doi:10.13560/j.cnki.biotech.bull.1985.2023-0138

摘要/Abstract

摘要：

本研究旨在以毕赤酵母为底盘细胞构建赤藓糖醇生产菌株。通过调控糖酵解途径中磷酸果糖激酶基因pfk的表达，敲除副产物阿拉伯糖醇和核糖醇生产相关基因，过表达不同来源的4-磷酸赤藓糖磷酸化酶、赤藓糖还原酶和糖醇磷酸酶，构建毕赤酵母赤藓糖醇生产菌株，对过表达戊糖磷酸途径关键酶转酮酶（TKL）、磷酸核酮糖差向异构酶（RPE）及赤藓糖还原酶对赤藓糖醇产量的影响也进行了探究。结果表明，过表达酿酒酵母来源的糖醇磷酸酶基因pyp1及大肠杆菌来源的4-磷酸赤藓糖磷酸化酶基因yidA的工程菌株C8具有赤藓糖醇生产能力，摇瓶发酵产量为30 mg/L；进一步过表达tkl和rpe后，菌株C10摇瓶发酵产量提高约40倍，达到1.2 g/L，高密度发酵产量为10.6 g/L；赤藓糖还原酶的过量表达并没有提升赤藓糖醇的产量，反而提高了副产物的产量。本研究首次在毕赤酵母中成功构建了赤藓糖醇合成通路，为改造毕赤酵母高效生产赤藓糖醇及其他高价值化合物奠定基础。

关键词: 赤藓糖醇, 毕赤酵母, 代谢工程, 发酵

Abstract:

The aim of this study is to construct an erythritol-producing strain using Pichia pastoris as the chassis cell. An erythritol-producing P. pastoris strain was developed by regulating the expression of phosphofructokinase gene（pfk）in glycolysis pathway, knocking out the genes associated with the production of by-products arabitol and ribitol, and overexpressing 4-phosphate erythrose phosphorylase, erythrose reductase and sugar alcohol phosphatase genes derived from different organisms. Next, we investigated the effects of overexpressions of erythrose reductase and two key enzymes transketolase（TKL）and ribulose-phosphate epimerase（RPE）involved in pentose phosphate pathway on the erythritol production. The results showed that strain C8 harboring pyp1 gene derived from Saccharomyces cerevisiae and yidA gene derived from Escherichia coli had the ability to produce erythritol. The erythritol production of the C8 strain in shake-flask fermentation was 30 mg/L. Furthermore, the overexpression of tkl and rpe genes enhanced the erythritol production of C10 strain by about 40-fold. The erythritol production of C10 reached 1.2 and 10.6 g/L in the shake-flask and high-cell-density fermentations, respectively. Further the overexpression of erythrose reductase did not cause the erythritol production increased, while caused the production of by-products increased. In this study, the erythritol synthetic pathway was successfully constructed in P. pastoris for the first time, which laid a foundation for engineering P. pastoris for efficient production of erythritol and other high-value compounds.

Key words: erythritol, Pichia pastoris, metabolic engineering, fermentation

赵思佳, 王晓璐, 孙纪录, 田健, 张杰. 代谢工程改造毕赤酵母生产赤藓糖醇[J]. 生物技术通报, 2023, 39(8): 137-147.

ZHAO Si-jia, WANG Xiao-lu, SUN Ji-lu, TIAN Jian, ZHANG Jie. Modification of Pichia pastoris for Erythritol Production by Metabolic Engineering[J]. Biotechnology Bulletin, 2023, 39(8): 137-147.

图/表 11

图1 毕赤酵母中赤藓糖醇合成通路的构建

Fig. 1 Construction of erythritol biosynthetic pathway in P. pastoris

图2 毕赤酵母菌株C0和C2的表型验证 A：菌株C0和C2的生长曲线；B：阿拉伯糖醇和核糖醇的生产

Fig. 2 Phenotype verification of P. pastoris C0 and C2 strains A: Growth curves of C0 and C2 strains; B: productions of arabitol and ribitol

图3 毕赤酵母菌株C2、C3、C4阿拉伯糖醇和核糖醇的生产

Fig. 3 Production of arabitol and ribitol by P. pastoris C2, C3 and C4 strains

表1 本研究中涉及的外源基因

Table 1 Exogenous genes involved in this study

基因Gene name	功能Function	来源Source	蛋白分子量Protein molecular weight/kD
pyp1	糖醇磷酸酶基因Sugar alcohol phosphatase gene	S. cerevisiae	30.7
yidA	去磷酸化酶基因Dphosphorylase gene	E. coli MG1655	32.9
err1	赤藓糖还原酶基因Erythrose reductase gene	T. reesei	39.5
er（NADH）	赤藓糖还原酶基因Erythrose reductase gene	Y. lipolytica	39.4
er（NADPH）	赤藓糖还原酶基因Erythrose reductase gene	C. magnoliae JH110	34.6
er1	赤藓糖还原酶基因Erythrose reductase gene	M. megachiliensis	39.6
er3	赤藓糖还原酶基因Erythrose reductase gene	M. megachiliensis	39.8
ER10	赤藓糖还原酶基因Erythrose reductase gene	Y. lipolytica CLIB122	39.1
ER25	赤藓糖还原酶基因Erythrose reductase gene	Y. lipolytica CLIB122	38.2
JX885	赤藓糖还原酶基因Erythrose reductase gene	Y. lipolytica	31.4
rpe1	差向异构酶基因Epimerase gene	S. cerevisiae	29.2

图4 外源基因在毕赤酵母中表达 A：T-ARS-PGAP质粒图谱；B：SDS-PAGE检测外源基因表达

Fig. 4 Expressions of exogenous genes in P. pastoris A: T-ARS-PGAP plasmid map; B: SDS-PAGE detection of the expression of exogenous genes. 1-11: err1, er（NADH）, er（NADPH）, er, er3, pyp1, ER10, ER25, JX885, rpe1, yidA

表2 突变株C5高密度发酵产物

Table 2 Products of high-density fermentation of mutant C5

化合物名称Compound name	最高浓度Maximum concentration/(g·L^-1)
赤藓糖醇 Erythritol	0
阿拉伯糖醇 Arabitol	1.4
核糖醇 Ribitol	5.6

图5 毕赤酵母菌株C0和C8的表型验证 A：生长曲线；B：赤藓糖醇产量；C：阿拉伯糖醇和核糖醇产量

Fig. 5 Fermentation profiles of P. pastoris C0 and C8 strains A: Growth curve. B: Production of erythritol. C: Production of arabitol and ribitol

图6 毕赤酵母菌株C0和C10的表型验证 A：生长曲线；B：赤藓糖醇产量；C：核糖醇产量；D：阿拉伯糖醇产量

Fig. 6 Phenotype verification of P. pastoris C0 and C10 strains A: Growth curve. B: Production of erythritol. C: Production of ribitol. D: Production of arabitol

图7 毕赤酵母菌株C10高密度发酵

Fig. 7 High-cell-density fermentation with strain P. pastoris C10

表3 突变株C11和C12摇瓶发酵产物

Table 3 Products of shake-flask fermentation of mutant C11 and C12

化合物名称 Compound name	最高浓度Maximum concentration/（g·L^-1）
化合物名称 Compound name	C11	C12
赤藓糖醇 Erythritol	1.2	1.2
核糖醇 Ribitol	1.6	4.0
阿拉伯糖醇 Arabitol	4.9	5.1

图8 毕赤酵母菌株C11高密度发酵 A：湿重和葡萄糖含量；B：赤藓糖醇、核糖醇和阿拉伯糖醇产量

Fig. 8 High-cell-density fermentation with strain P. pastoris C11 A: Wet weight and glucose concentration. B: Production of erythritol, ribitol and arabitol

参考文献 34

[1]	Hijosa-Valsero M, Garita-Cambronero J, Paniagua-García AI, et al. By-products of sugar factories and wineries as feedstocks for erythritol generation[J]. Food Bioprod Process, 2021, 126: 345-355. doi: 10.1016/j.fbp.2021.02.001 URL
[2]	中国食品科学技术学会. 赤藓糖醇的科学共识[J]. 中国食品学报, 2022, 22(12): 405-412.
	Chinese Institute of Food Science and Technology. Scientific consensus on erythritol[J]. J Chin Inst Food Sci Technol, 2022, 22(12): 405-412.
[3]	Martău GA, Coman V, Vodnar DC. Recent advances in the biotechnological production of erythritol and mannitol[J]. Crit Rev Biotechnol, 2020, 40(5): 608-622. doi: 10.1080/07388551.2020.1751057 pmid: 32299245
[4]	邱学良. 产赤藓糖醇亚罗解脂酵母的耐热机制分析及组合策略改造[D]. 无锡: 江南大学, 2020.
	Qiu XL. Thermotolerance mechanism analysis and combination strategy transformation of erythritol producing Yarrowia lipolytica[D]. Wuxi: Jiangnan University, 2020.
[5]	den Hartog GJM, Boots AW, Adam-Perrot A, et al. Erythritol is a sweet antioxidant[J]. Nutrition, 2010, 26(4): 449-458. doi: 10.1016/j.nut.2009.05.004 pmid: 19632091
[6]	张妍, 张丽, 李宝磊, 等. 赤藓糖醇的国内外研究进展[J]. 饮料工业, 2022, 25(5): 76-79.
	Zhang Y, Zhang L, Li BL, et al. Research progress of erythritol at home and abroad[J]. Beverage Ind, 2022, 25(5): 76-79.
[7]	中华人民共和国工业和信息化部. 口腔清洁护理用品牙膏中赤藓糖醇含量的测定高效液相色谱法: QB/T 5408—2019[S]. 北京: 中国轻工业出版社, 2019.
	Ministry of Industry and Information of the People's Republic of China. Oral hygiene care products determination of erythritol content in toothpaste high performance liquid chromatography: QB/T 5408—2019[S]. Beijing: China Light Industry Press, 2019.
[8]	Liang PX, Cao MF, Li J, et al. Expanding sugar alcohol industry: Microbial production of sugar alcohols and associated chemocatalytic derivatives[J]. Biotechnol Adv, 2023, 64: 108105. doi: 10.1016/j.biotechadv.2023.108105 URL
[9]	隋松森, 王松江, 郭传庄, 等. 微生物发酵法产赤藓糖醇的研究进展[J]. 中国食品添加剂, 2021, 32(6): 125-131.
	Sui SS, Wang SJ, Guo CZ, et al. Research progress in erythritol production by microbial fermentation[J]. China Food Addit, 2021, 32(6): 125-131.
[10]	Stapley JA, Genders DJ, Atherton DM, et al. Methods for the electrolytic production of erythrose or erythritol: United States: US7955489[P]. 2011-06-07.
[11]	Richter H, Vlad D, Unden G. Significance of pantothenate for glucose fermentation by Oenococcus oeni and for suppression of the erythritol and acetate production[J]. Arch Microbiol, 2001, 175(1): 26-31. pmid: 11271417
[12]	Gänzle MG, Vermeulen N, Vogel RF. Carbohydrate, peptide and lipid metabolism of lactic acid bacteria in sourdough[J]. Food Microbiol, 2007, 24(2): 128-138. pmid: 17008155
[13]	Veiga-da-Cunha M, Santos H, Van Schaftingen E. Pathway and regulation of erythritol formation in Leuconostoc oenos[J]. J Bacteriol, 1993, 175(13): 3941-3948. pmid: 8391532
[14]	Khatape AB, Dastager SG, Rangaswamy V. An overview of erythritol production by yeast strains[J]. FEMS Microbiol Lett, 2022, 369(1): fnac107. doi: 10.1093/femsle/fnac107 URL
[15]	Rakicka-Pustułka M, Mirończuk AM, Celińska E, et al. Scale-up of the erythritol production technology - Process simulation and techno-economic analysis[J]. J Clean Prod, 2020, 257: 120533. doi: 10.1016/j.jclepro.2020.120533 URL
[16]	Deshpande MS, Kulkarni PP, Kumbhar PS, et al. Erythritol production from sugar based feedstocks by Moniliella pollinis using lysate of recycled cells as nutrients source[J]. Process Biochem, 2022, 112: 45-52. doi: 10.1016/j.procbio.2021.11.020 URL
[17]	Yang SL, Pan XW, Wang Q, et al. Enhancing erythritol production from crude glycerol in a wild-type Yarrowia lipolytica by metabolic engineering[J]. Front Microbiol, 2022, 13: 1054243. doi: 10.3389/fmicb.2022.1054243 URL
[18]	Li LZ, Yang TY, Guo WQ, et al. Construction of an efficient mutant strain of Trichosporonoides oedocephalis with HOG1 gene deletion for production of erythritol[J]. J Microbiol Biotechnol, 2016, 26(4): 700-709. doi: 10.4014/jmb.1510.10049 URL
[19]	Gómez-Ramírez IV, Corrales-García LL, Possani LD, et al. Expression in Pichia pastoris of human antibody fragments that neutralize venoms of Mexican scorpions[J]. Toxicon, 2023, 223: 107012. doi: 10.1016/j.toxicon.2022.107012 URL
[20]	Guo F, Dai ZX, Peng WF, et al. Metabolic engineering of Pichia pastoris for malic acid production from methanol[J]. Biotechnol Bioeng, 2021, 118(1): 357-371. doi: 10.1002/bit.v118.1 URL
[21]	Cai P, Wu XY, Deng J, et al. Methanol biotransformation toward high-level production of fatty acid derivatives by engineering the industrial yeast Pichia pastoris[J]. Proc Natl Acad Sci USA, 2022, 119(29): e2201711119.
[22]	Zhang QQ, Wang XL, Luo HY, et al. Metabolic engineering of Pichia pastoris for myo-inositol production by dynamic regulation of central metabolism[J]. Microb Cell Fact, 2022, 21(1): 112. doi: 10.1186/s12934-022-01837-x
[23]	Abdel-Mawgoud AM, Markham KA, Palmer CM, et al. Metabolic engineering in the host Yarrowia lipolytica[J]. Metab Eng, 2018, 50: 192-208. doi: S1096-7176(18)30273-8 pmid: 30056205
[24]	Kobayashi Y, Yoshida J, Iwata H, et al. Gene expression and function involved in polyol biosynthesis of Trichosporonoides megachiliensis under hyper-osmotic stress[J]. J Biosci Bioeng, 2013, 115(6): 645-650. doi: 10.1016/j.jbiosc.2012.12.004 pmid: 23294575
[25]	Lee DH, Lee YJ, Ryu YW, et al. Molecular cloning and biochemical characterization of a novel erythrose reductase from Candida magnoliae JH110[J]. Microb Cell Fact, 2010, 9: 43. doi: 10.1186/1475-2859-9-43
[26]	Carly F, Vandermies M, Telek S, et al. Enhancing erythritol productivity in Yarrowia lipolytica using metabolic engineering[J]. Metab Eng, 2017, 42: 19-24. doi: 10.1016/j.ymben.2017.05.002 URL
[27]	Jovanović B, Mach RL, Mach-Aigner AR. Characterization of erythrose reductases from filamentous fungi[J]. AMB Express, 2013, 3(1): 43. doi: 10.1186/2191-0855-3-43 pmid: 23924507
[28]	Janek T, Dobrowolski A, Biegalska A, et al. Characterization of erythrose reductase from Yarrowia lipolytica and its influence on erythritol synthesis[J]. Microb Cell Fact, 2017, 16(1): 118. doi: 10.1186/s12934-017-0733-6 URL
[29]	Shenoy A, Yalamanchili S, Davis AR, et al. Expression and display of glycoengineered antibodies and antibody fragments with an engineered yeast strain[J]. Antibodies, 2021, 10(4): 38. doi: 10.3390/antib10040038 URL
[30]	De Brabander P, Uitterhaegen E, Delmulle T, et al. Challenges and progress towards industrial recombinant protein production in yeasts: a review[J]. Biotechnol Adv, 2023, 64: 108121. doi: 10.1016/j.biotechadv.2023.108121 URL
[31]	Ávila-Fernández Á, Montiel S, Rodríguez-Alegría ME, et al. Simultaneous enzyme production, Levan-type FOS synthesis and sugar by-products elimination using a recombinant Pichia pastoris strain expressing a levansucrase-endolevanase fusion enzyme[J]. Microb Cell Fact, 2023, 22(1): 18. doi: 10.1186/s12934-022-02009-7 pmid: 36703199
[32]	Inokuma K, Miyamoto S, Morinaga K, et al. Direct production of 4-hydroxybenzoic acid from cellulose using cellulase-displaying Pichia pastoris[J]. Biotechnol Bioeng, 2023, 120(4): 1097-1107. doi: 10.1002/bit.v120.4 URL
[33]	Chen SL, Liu TS, Zhang WG, et al. Cofactor engineering for efficient production of α-farnesene by rational modification of NADPH and ATP regeneration pathway in Pichia pastoris[J]. Int J Mol Sci, 2023, 24(2): 1767. doi: 10.3390/ijms24021767 URL
[34]	Hijosa-Valsero M, Paniagua-García AI, Díez-Antolínez R. Cell immobilization for erythritol production[J]. J Fungi, 2022, 8(12): 1286. doi: 10.3390/jof8121286 URL