Synthesis of Glycolate by <i>Bacillus</i> <i>subtilis</i> through Glyoxylate Bypass Pathway

LIU Yufei; FU Cong; XIE Yukang; SHI Jiping; SUN Junsong

doi:10.13386/j.issn1002-0306.2023020003

Volume 44 Issue 20

Oct. 2023

Turn off MathJax

Article Contents

Abstract

References

Science and Technology of Food Industry > 2023 > 44(20): 143-151. > DOI: 10.13386/j.issn1002-0306.2023020003

LIU Yufei, FU Cong, XIE Yukang, et al. Synthesis of Glycolate by Bacillus subtilis through Glyoxylate Bypass Pathway[J]. Science and Technology of Food Industry, 2023, 44(20): 143−151. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023020003.

Citation:

PDF (2772 KB)

Synthesis of Glycolate by Bacillus subtilis through Glyoxylate Bypass Pathway

LIU Yufei^{1, 2,},
FU Cong^{1, 2},
XIE Yukang^{1, 2},
SHI Jiping^{1, 2, 3},
SUN Junsong^{1, 2, 3, ,}

1.
Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201210, China
2.
University of Chinese Academy of Sciences, Beijing 100049, China
3.
ShanghaiTech University, Shanghai 201210, China

More Information

Received Date: February 01, 2023
Available Online: August 10, 2023

Graphical Abstract

Abstract

Abstract

In order to construct a food-safe strain that could produce glycolate, the metabolic modification of Bacillus subtilis was carried out. In this study, the exogenous isocitrate lyase gene (aceA) was first integrated into the genome of Bacillus subtilis by homologous recombination, and the starting strain 164MCT-GA was constructed. Then the glycolate anabolism was optimized by means of metabolic engineering in the starting strain 164MCT-GA. The results showed that 164MCT-GA could synthesize glycolate with glycerol as substrate, and the yield of shaker fermentation was 0.114 g/L. To increase the supply of the key intermediate substrates, the citrate synthase gene (citA) and the glyoxylate reductase gene (yvcT) were overexpressed by replacing the native promoter with individual T7 promoter. The Bacillus strains were further engineered at multiple loci that included lactate dehydrogenase (ldh), phosphate acetyltransferase (pta) and acetyl-CoA transacetylase (mmgA, yhfs), in an attempt to modulate the carbon flux toward the formation of glycolate with a higher efficiency. The fermentation study revealed that the accumulated concentration of glycolate from the obtained B. subtilis strain GA3-52 reached 0.572 g/L, with a conversion rate of 0.175 g/g glycerol, the titer was more than five times as much as that achieved by 164MCT-GA. Thus, this study constructed a de novo synthesis pathway in B. subtilis, and laid the foundation for the fermentation production of high yield glycolic acid by food safety bacteria.
- glycolate,
- Bacillus subtilis,
- biosynthesis,
- glyoxylate bypass pathway,
- heterologous expression

FullText(HTML)

References (33)

References

[1]	KOIVISTOINEN O M, KUIVANEN J, BARTH D, et al. Glycolic acid production in the engineered yeasts Saccharomyces cerevisiae and Kluyveromyces lactis[J]. Microbial Cell Factories,2013,12:82. doi: 10.1186/1475-2859-12-82
[2]	LACHAUX C, FRAZAO C J R, KRAUΒER F, et al. A new synthetic pathway for the bioproduction of glycolic acid from lignocellulosic sugars aimed at maximal carbon conservation[J]. Frontiers in Bioengineering and Biotechnology,2019,7:359. doi: 10.3389/fbioe.2019.00359
[3]	ZHAO D, ZHU T, LI J, et al. Poly (lactic-co-glycolic acid)-based composite bone-substitute materials[J]. Bioactive Materials,2021,6(2):346−360. doi: 10.1016/j.bioactmat.2020.08.016
[4]	JO D J, SEOK J K, KIM S Y, et al. Human skin-depigmenting effects of resveratryl triglycolate, a hybrid compound of resveratrol and glycolic acid[J]. International Journal of Cosmetic Science,2018,40(3):256−262. doi: 10.1111/ics.12458
[5]	WU X S. Synthesis, characterization, biodegradation, and drug delivery application of biodegradable lactic/glycolic acid polymers: Part III. Drug delivery application[J]. Artificial Cells Blood Substitutes and Immobilization Biotechnology,2004,32(4):575−591. doi: 10.1081/BIO-200039635
[6]	蔡帅, 郭秋爽, 刘炎, 等. 响应面法优化弗托氏葡糖酸杆菌产羟基乙酸工艺条件[J]. 食品工业科技,2022,43(12):138−145. [CAI Shuai, GUO Qiushuang, LIU Yan, et al. Optimization of the technological conditions for glycolic acid production by Gluconobacter frateurii using response surface methodology[J]. Science and Technology of Food Industry,2022,43(12):138−145. CAI Shuai, GUO Qiushuang, LIU Yan, et al. Optimization of the technological conditions for glycolic acid production by gluconobacter frateurii using response surface methodology[J]. Science and Technology of Food Industry, 2022, 43(12): 138−145.
[7]	ZHU Z, KANG G, YU S, et al. Process intensification in carbonylation of formaldehyde with active and passive enhancement methods[J]. Journal of Flow Chemistry,2020,10(4):605−613. doi: 10.1007/s41981-020-00103-8
[8]	ZHOU X, ZHA M, CAO J, et al. Glycolic acid production from ethylene glycol via sustainable biomass energy: Integrated conceptual process design and comparative techno-economic-society-environment analysis[J]. ACS Sustainable Chemistry & Engineering,2021,9(32):10948−10962.
[9]	SHI Q, GUO H, CHEN C, et al. An efficient brønsted acidic polymer resin for the carbonylation of formaldehyde to glycolic acid[J]. Reaction Kinetics, Mechanisms and Catalysis,2020,130(2):1027−1042. doi: 10.1007/s11144-020-01819-3
[10]	YUNHAI S, HOUYONG S, HAIYONG C, et al. Synergistic extraction of glycolic acid from glycolonitrile hydrolysate[J]. Industrial & Engineering Chemistry Research,2011,50(13):8216−8224.
[11]	KATAOKA M, SASAKI M, HIDALGO A R, et al. Glycolic acid production using ethylene glycol-oxidizing microorganisms[J]. Bioscience Biotechnology and Biochemistry,2001,65(10):2265−2270. doi: 10.1271/bbb.65.2265
[12]	SALUSJäRVI L, HAVUKAINEN S, KOIVISTOINEN O, et al. Biotechnological production of glycolic acid and ethylene glycol: Current state and perspectives[J]. Applied Microbiology and Biotechnology,2019,103(6):2525−2535. doi: 10.1007/s00253-019-09640-2
[13]	ZHAN T, CHEN Q, ZHANG C, et al. Constructing a novel biosynthetic pathway for the production of glycolate from glycerol in Escherichia coli[J]. ACS Synthetic Biology,2020,9(9):2600−2609. doi: 10.1021/acssynbio.0c00404
[14]	CABULONG R B, BAñARES A B, NISOLA G M, et al. Enhanced glycolic acid yield through xylose and cellobiose utilization by metabolically engineered Escherichia coli[J]. Bioprocess and Biosystems Engineering,2021,44(6):1081−1091. doi: 10.1007/s00449-020-02502-6
[15]	PEREIRA B, LI Z J, DE MEY M, et al. Efficient utilization of pentoses for bioproduction of the renewable two-carbon compounds ethylene glycol and glycolate[J]. Metabolic Engineering,2016,34:80−87. doi: 10.1016/j.ymben.2015.12.004
[16]	ZHU T, YAO D, LI D, et al. Multiple strategies for metabolic engineering of Escherichia coli for efficient production of glycolate[J]. Biotechnology and Bioengineering,2021,118(12):4699−4707. doi: 10.1002/bit.27934
[17]	XU S, ZHANG L, ZHOU S, et al. Biosensor-based multigene pathway optimization for enhancing the production of glycolate[J]. Applied and Environmental Microbiology,2021,87(12):e0011321. doi: 10.1128/AEM.00113-21
[18]	XIANG M, KANG Q, ZHANG D. Advances on systems metabolic engineering of Bacillus subtilis as a chassis cell[J]. Synthetic and Systems Biotechnology,2020,5(4):245−251. doi: 10.1016/j.synbio.2020.07.005
[19]	YANG S, KANG Z, CAO W, et al. Construction of a novel, stable, food-grade expression system by engineering the endogenous toxin-antitoxin system in Bacillus subtilis[J]. Journal of Biotechnology,2016,219:40−47. doi: 10.1016/j.jbiotec.2015.12.029
[20]	VAN TILBURG A Y, CAO H, VAN DER MEULEN S B, et al. Metabolic engineering and synthetic biology employing Lactococcus lactis and Bacillus subtilis cell factories[J]. Current Opinion in Biotechnology,2019,59:1−7. doi: 10.1016/j.copbio.2019.01.007
[21]	PRAMASTYA H, SONG Y, ELFAHMI E Y, et al. Positioning Bacillus subtilis as terpenoid cell factory[J]. Journal of Applied Microbiology,2021,130(6):1839−1856. doi: 10.1111/jam.14904
[22]	NIU T, LV X, LIU Z, et al. Synergetic engineering of central carbon and nitrogen metabolism for the production of N-acetylglucosamine in Bacillus subtilis[J]. Biotechnology and Applied Biochemistry,2020,67(1):123−132. doi: 10.1002/bab.1845
[23]	AMJAD ZANJANI F S, AFRASIABI S, NOROUZIAN D, et al. Hyaluronic acid production and characterization by novel Bacillus subtilis harboring truncated hyaluronan synthase[J]. AMB Express,2022,12(1):88. doi: 10.1186/s13568-022-01429-3
[24]	JI M, LIU Y, XIE S, et al. De novo synthesis of 2'-fucosyllactose in engineered Bacillus subtilis ATCC 6051a[J]. Process Biochemistry,2022,120:178−185. doi: 10.1016/j.procbio.2022.06.007
[25]	ZHANG M, ZHAO X, CHEN X, et al. Enhancement of riboflavin production in Bacillus subtilis via in vitro and in vivo metabolic engineering of pentose phosphate pathway[J]. Biotechnology Letters,2021,43(12):2209−2216. doi: 10.1007/s10529-021-03190-2
[26]	CHEN A, XIE Y, XIE S, et al. Production of citramalate in Escherichia coli by mediating colonic acid metabolism and fermentation optimization[J]. Process Biochemistry,2022,121:1−9. doi: 10.1016/j.procbio.2022.06.018
[27]	JI M, LIU Y, WU H, et al. Engineering Bacillus subtilis ATCC 6051a for the production of recombinant catalases[J]. Journal of Industrial Microbiology & Biotechnology, 2021, 48(5-6).
[28]	JI M, LI S, CHEN A, et al. A wheat bran inducible expression system for the efficient production of α-L-arabinofuranosidase in Bacillus subtilis[J]. Enzyme and Microbial Technology,2021,144:109726. doi: 10.1016/j.enzmictec.2020.109726
[29]	KABISCH J, PRATZKA I, MEYER H, et al. Metabolic engineering of Bacillus subtilis for growth on overflow metabolites[J]. Microbial Cell Factories,2013,12:72. doi: 10.1186/1475-2859-12-72
[30]	ALKIM C, TRICHEZ D, CAM Y, et al. The synthetic xylulose-1 phosphate pathway increases production of glycolic acid from xylose-rich sugar mixtures[J]. Biotechnology for Biofuels,2016,9:201. doi: 10.1186/s13068-016-0610-2
[31]	马宁, 朱康佳, 毛银, 等. 代谢工程改造大肠杆菌提高乙醇酸产率[J]. 生物工程学报,2018,34(2):224−234. [MA N, ZHU K J, MAO Y, et al. Improving glycolic acid yield by metabolic engineering in Escherichia coli[J]. Chin J Biotech,2018,34(2):224−234. Ma N, Zhu K J, Mao Y, et al. Improving glycolic acid yield by metabolic engineering in Escherichia coli[J]. Chin J Biotech, 2018, 34(2): 224–234.
[32]	ZHU K, LI G, WEI R, et al. Systematic analysis of the effects of different nitrogen source and ICDH knockout on glycolate synthesis in Escherichia coli[J]. Journal of Biological Engineering,2019,13:30. doi: 10.1186/s13036-019-0159-2
[33]	DENG Y, MA N, ZHU K, et al. Balancing the carbon flux distributions between the TCA cycle and glyoxylate shunt to produce glycolate at high yield and titer in Escherichia coli[J]. Metabolic Engineering,2018,46:28−34. doi: 10.1016/j.ymben.2018.02.008

Supplements (0)

Cited By

Cited by

Periodical cited type(12)

1.	赵智慧，董建方，刘爱龙，马艳，党文宏，朱银龙，罗文华，王伟仁. 枸杞酒混菌酿造工艺的研究. 酿酒科技. 2025(01): 55-59 .
2.	李银凤，刘昕，付伟，刘晓柱. 一株野生刺梨来源葡萄汁有孢汉逊酵母的鉴定及酿造学性能分析. 食品科技. 2024(07): 18-23 .
3.	陆进舟，蒋贵兰，李银凤，黄名正，唐维媛，刘晓柱. 异常威克汉姆酵母发酵米酒品质特性分析. 贵州农机化. 2024(04): 25-27+31 .
4.	李银凤，刘晓柱. 贵州刺梨生态食品开发现状调查研究. 食品工业. 2024(11): 261-265 .
5.	马文瑞，孙志伟，石俊，张小娜，于佳俊，裴疆森，王德良，贾士儒，薛洁，钟成. 非酿酒酵母Nakazawaea ishiwadae GDMCC 60786产乙酸乙酯的诱变菌株筛选及其安全性评价. 食品科学. 2023(10): 165-172 .
6.	李银凤，黎华，唐小玉，朱文丽，刘晓柱. 空心李酿酒酵母的酿造学性能分析. 食品研究与开发. 2023(18): 193-197 .
7.	莫皓然，黄名正，张群，唐维媛，李婷婷，许存宾，刘晓柱，于志海，李鑫. 顶空固相微萃取结合溶剂辅助风味蒸发分析无籽刺梨挥发性成分及其呈香贡献. 食品工业科技. 2023(20): 289-297 . 本站查看
8.	马冬，吕树萍，米兰芳，许赛冰，许佳琦，钟八莲. 赣脐4号脐橙果实挥发性组分分析. 赣南师范大学学报. 2023(03): 76-82 .
9.	李银凤，刘晓柱. 海拔高度对刺梨根际土壤真菌与细菌多样性的影响. 河南农业科学. 2023(10): 82-91 .
10.	郭志君，杨磊，骆红霞，周模美，房玉林. 苹果酸—乳酸发酵对刺梨酒香气的影响. 食品与机械. 2022(03): 197-204+233 .
11.	荆丰雪，钟斌，万娅琼，樊洪泓，程江华，徐雅芫. 微生物源β-葡萄糖苷酶在发酵食品中的应用. 食品安全质量检测学报. 2022(24): 8041-8049 .
12.	黄贺敏，吴丽香，邓梅忠，王雨晴，张雯，陈善义，张恩仁，詹仁锋，方璟，倪莉，李菁菁. 不同品质烟叶微生物群落与其挥发性成分的关联研究. 食品与生物技术学报. 2022(12): 85-95 .