Anaerobic Expression of Lactate Dehydrogenase to Improve the D-lactic Acid Optical Purity Procluced by Escherichia coli

doi:10.13560/j.cnki.biotech.bull.1985.2023-0979

Abstract

Abstract:

【Objective】This work aims to solve the problem that the light purity of the final product D-lactic acid is reduced while using cheap raw materials（containing a small amount of L-lactic acid）such as agricultural rough processing or waste in the industrial fermentation production of D-lactic acid.【Method】The expressing plasmids of L-lactate dehydrogenase gene lldD, pUC19-PLD（PpflBp6）, pUC19-NLD（PnirB）and pUC19-PNLD（PpflBp6-PnirB）with different promoters, were constructed and transformed into Escherichia coli D-lactic acid engineering strain HBUT-D, and strain HBUT-D3, HBUT-D5 and HBUT-D7 was acquired respectively. Through LldD enzymatic activity test and TOPSIS multivariate evaluation on NBS fermentative experiments（supplemented with 1 g/L L-lactic acid）, one strain was screened by its capability for fast elimination of L-lactic and high fermentative efficiency of D-lactic acid, for following fermentative experiments with cheap raw materials. 【Result】HBUT-D7 was evaluated as the optimal strain based on the LldD specific enzyme activity of 64 U/g, L-lactate consumption rate of 34 mg/(L·h), and D-lactic acid productivity of 4.09 g/(L·h). When fermented with corn syrup, the L-lactic acid consumption rates of HBUT-D and HBUT-D7 were 10.41 mg/(L·h)and 34.75 mg/(L·h), respectively. The productivities of D-lactic acid were 4.24 g/(L·h)and 3.87 g/(L·h), respectively. The optical purities of D-lactic acid were 99.07% and 99.92%, respectively. When fermented with molasses, the L-lactic acid consumption rates of HBUT-D and HBUT-D7 were 6.87 mg/(L·h)and 17.18 mg/(L·h), respectively. The productivities of D-lactic acid were 1.93 g/(L·h)and 1.88 g/(L·h), respectively. The optical purities of D-lactic acid were 99.22% and 99.99%, respectively. 【Conclusion】The expressing plasmid of the anaerobic inducible promoter may increase the L-lactate dehydrogenase enzyme activity in strain HBUT-D7, thus it can eliminate the L-lactic acid to increase the optical purity of D-lactic acid while using cheap raw materials for fermentation.

Key words: D-lactic acid, optical purity, Escherichia coli, L-lactate dehydrogenase, nirB promoter, pflB promoter, tandem promoters

WANG Zhou, YU Jie, WANG Jin-hua, WANG Yong-ze, ZHAO Xiao. Anaerobic Expression of Lactate Dehydrogenase to Improve the D-lactic Acid Optical Purity Procluced by Escherichia coli[J]. Biotechnology Bulletin, 2024, 40(5): 290-299.

Figures/Tables 15

References 25

[1]	吕汉强, 赵文花, 李含婷, 等. 聚乳酸可降解地膜对绿洲灌区玉米产量和农田水热特性的影响[J]. 甘肃农业大学学报, 2022, 57(5): 72-79, 88.
	Lyu HQ, Zhao WH, Li HT, et al. Effects of polylactic acid degradable film on maize yield and soil hydrothermal characteristics in oasis irrigation area[J]. J Gansu Agric Univ, 2022, 57(5): 72-79, 88.
[2]	王健群, 张斌. 聚乳酸-羟基乙酸共聚物微球在骨组织工程中的应用[J]. 口腔医学研究, 2020, 36(9): 817-820. doi: 10.13701/j.cnki.kqyxyj.2020.09.005
	Wang JQ, Zhang B. Application of poly(lactic-co-glycolic acid)copolymer microspheres in bone tissue engineering[J]. J Oral Sci Res, 2020, 36(9): 817-820.
[3]	吴一帆. D-乳酸嵌段共聚物的合成设计及其对左旋聚乳酸结晶行为和性能的影响[D]. 成都: 西南交通大学, 2020.
	Wu YF. Synthesis design of D-lactic acid block copolymer and investigation on crystallization behaviors and properties of block copolymer/poly(L-lactic acid)blends[D]. Chengdu: Southwest Jiaotong University, 2020.
[4]	Grabar TB, Zhou S, Shanmugam KT, et al. Methylglyoxal bypass identified as source of chiral contamination in l(+)and d(-)-lactate fermentations by recombinant Escherichia coli[J]. Biotechnol Lett, 2006, 28(19): 1527-1535. doi: 10.1007/s10529-006-9122-7 pmid: 16868860
[5]	张媛, 王小艳, 陈博, 等. 基因工程改造植物乳杆菌生产光学纯D-乳酸[J]. 当代化工, 2019, 48(4): 674-678, 682.
	Zhang Y, Wang XY, Chen B, et al. Genetically engineered Lactobacillus plantarum for production of optical pure D-lactic acid[J]. Contemp Chem Ind, 2019, 48(4): 674-678, 682.
[6]	李义, 周卫强, 刘海军, 等. 玉米浆及其制备方法和应用以及高光学纯度乳酸的生产方法: CN114181980A[P]. 2022-03-15.
	Li Y, Zhou W, Liu H, et al. Corn steep liquor, preparation method and application thereof, and production method of high-optical-purity lactic acid: CN114181980A[P]. 2022-03-15.
[7]	Alexandri M, Hübner D, Schneider R, et al. Towards efficient production of highly optically pure d-lactic acid from lignocellulosic hydrolysates using newly isolated lactic acid bacteria[J]. N Biotechnol, 2022, 72: 1-10.
[8]	Zhou SD, Iverson AG, Grayburn WS. Doubling the catabolic reducing power(NADH)output of Escherichia coli fermentation for production of reduced products[J]. Biotechnol Prog, 2010, 26(1): 45-51.
[9]	Nasr R, Akbari Eidgahi MR. Construction of a synthetically engineered nirB promoter for expression of recombinant protein in Escherichia coli[J]. Jundishapur J Microbiol, 2014, 7(7): e15942.
[10]	田甜. 高效利用蔗糖产D-乳酸工程菌的构建及其发酵研究[D]. 武汉: 湖北工业大学, 2013.
	Tian T. Construction and fermentation of engineering bacteria producing D- lactic acid from sucrose with high efficiency[D]. Wuhan: Hubei University of Technology, 2013.
[11]	Kimura H, Futai M. Effects of phospholipids on L-lactate dehydrogenase from membranes of Escherichia coli. Activation and stabilization of the enzyme with phospholipids[J]. J Biol Chem, 1978, 253(4): 1095-1110. pmid: 342518
[12]	刘红雨, 刘友存, 孟丽红, 等. 熵权法在水资源与水环境评价中的研究进展[J]. 冰川冻土, 2022, 44(1): 299-306. doi: 10.7522/j.issn.1000-0240.2021.0131
	Liu HY, Liu YC, Meng LH, et al. Research progress of entropy weight method in water resources and water environment[J]. J Glaciol Geocryol, 2022, 44(1): 299-306. doi: 10.7522/j.issn.1000-0240.2021.0131
[13]	Aguilera L, Campos E, Giménez R, et al. Dual role of LldR in regulation of the lldPRD operon, involved in L-lactate metabolism in Escherichia coli[J]. J Bacteriol, 2008, 190(8): 2997-3005. doi: 10.1128/JB.02013-07 pmid: 18263722
[14]	Futai M, Kimura H. Inducible membrane-bound L-lactate dehydrogenase from Escherichia coli. Purification and properties[J]. J Biol Chem, 1977, 252(16): 5820-5827. pmid: 18473
[15]	Bekker M, Alexeeva S, Laan W, et al. The ArcBA two-component system of Escherichia coli is regulated by the redox state of both the ubiquinone and the menaquinone pool[J]. J Bacteriol, 2010, 192(3): 746-754.
[16]	许琼丹. 基于糖转运及代谢相关基因敲除的产D-乳酸大肠杆菌工程菌的构建[D]. 武汉: 湖北工业大学, 2020.
	Xu QD. Construction of D-lactic acid-producing Escherichia coli strains by knockout of sugar transport and metabolism genes[D]. Wuhan: Hubei University of Technology, 2020.
[17]	鲁春阳, 文枫, 杨庆媛, 等. 基于改进TOPSIS法的城市土地利用绩效评价及障碍因子诊断——以重庆市为例[J]. 资源科学, 2011, 33(3): 535-541.
	Lu CY, Wen F, Yang QY, et al. An evaluation of urban land use performance based on the improved TOPSIS method and diagnosis of its obstacle indicators: a case study of Chongqing[J]. Resour Sci, 2011, 33(3): 535-541.
[18]	张帆, 陈梦茹, 邢英英, 等. 基于熵权法和TOPSIS对马铃薯施肥和滴灌量组合的优化[J]. 植物营养与肥料学报, 2023, 29(4): 732-744.
	Zhang F, Chen MR, Xing YY, et al. Optimization of fertilizer and drip irrigation levels for efficient potato production based on entropy weight method and TOPSIS[J]. J Plant Nutr Fertil, 2023, 29(4): 732-744.
[19]	Fantino JR, Py B, Fontecave M, et al. A genetic analysis of the response of Escherichia coli to cobalt stress[J]. Environ Microbiol, 2010, 12(10): 2846-2857.
[20]	Zaslaver A, Mayo AE, Rosenberg R, et al. Just-in-time transcription program in metabolic pathways[J]. Nat Genet, 2004, 36(5): 486-491. pmid: 15107854
[21]	张玲, 林荣, 宋祖坤, 等. 启动子串联及改造提高FAD为辅基的葡萄糖脱氢酶在Bacillus subtilis中的表达[J]. 食品与发酵工业, 2019, 45(8): 15-21. doi: 10.13995/j.cnki.11-1802/ts.018685
	Zhang L, Lin R, Song ZK, et al. Promoter tandem and transformation for FAD-conjugated glucose dehydrogenase expression in Bacillus subtilis[J]. Food Ferment Ind, 2019, 45(8): 15-21.
[22]	葛春蕾, 刘中美, 崔文璟, 等. 通过串联启动子实现纳豆激酶在枯草芽孢杆菌中的高效表达[J]. 现代食品科技, 2016, 32(11): 72-77, 15.
	Ge CL, Liu ZM, Cui WJ, et al. Efficient overexpression of recombinant nattokinase in Bacillus subtilis by tandem promoters[J]. Mod Food Sci Technol, 2016, 32(11): 72-77, 15.
[23]	Chauhan V, Bahrudeen MNM, Palma CSD, et al. Analytical kinetic model of native tandem promoters in E. coli[J]. PLoS Comput Biol, 2022, 18(1): e1009824.
[24]	Kasho K, Ozaki S, Katayama T. IHF and fis as Escherichia coli cell cycle regulators: activation of the replication origin oriC and the regulatory cycle of the DnaA initiator[J]. Int J Mol Sci, 2023, 24(14): 11572.
[25]	Kim D, Seo SW, Gao Y, et al. Systems assessment of transcriptional regulation on central carbon metabolism by Cra and CRP[J]. Nucleic Acids Res, 2018, 46(6): 2901-2917. doi: 10.1093/nar/gky069 pmid: 29394395

菌株及质粒 Strain and plasmid	特征 Characteristic	来源 Source
HBUT-D	W ∆frdBC ∆pta ∆adhE ∆pflB ∆aldA	本实验室保藏
HBUT-DP	HBUT-D carrying pUC19	本研究
HBUT-D3	HBUT-D carrying pUC19-PLD	本研究
HBUT-D5	HBUT-D carrying pUC19-PNLD	本研究
HBUT-D7	HBUT-D carrying pUC19-NLD	本研究
pUC19-PLD	pUC19::PpflBp6-lldD, A^pr	本研究
pUC19-NLD	pUC19::PnirB-lldD, A^pr	本研究
pUC19-PNLD	pUC19::PpflBp6-PnirB-lldD, A^pr	本研究

菌株及质粒 Strain and plasmid	特征 Characteristic	来源 Source
HBUT-D	W ∆frdBC ∆pta ∆adhE ∆pflB ∆aldA	本实验室保藏
HBUT-DP	HBUT-D carrying pUC19	本研究
HBUT-D3	HBUT-D carrying pUC19-PLD	本研究
HBUT-D5	HBUT-D carrying pUC19-PNLD	本研究
HBUT-D7	HBUT-D carrying pUC19-NLD	本研究
pUC19-PLD	pUC19::PpflBp6-lldD, A^pr	本研究
pUC19-NLD	pUC19::PnirB-lldD, A^pr	本研究
pUC19-PNLD	pUC19::PpflBp6-PnirB-lldD, A^pr	本研究

引物名称 Primer name	序列 Sequence（5'-3'）
P1	CCGTGACTTAAGAAAATTTATAC
P2	CTGGCTGCGGAAATAATCATTTTTGCCTCGATTTCTTTTC
P3	GAAAAGAAATCGAGGCAAAAATGTCTTCCATGACAACAACTG
P4	ACATACAGCGCCGAACGGTC
P5	CAGCAATATACCCATTAAGGAGTAT
P6	AACGACTATGCCGCATTC
P7	TTCGCCTGATGCTCCC
P8	CTGGCTGCGGAAATAATCATACTAACTCTCTCTTTATTAAGTCGGC
P9	GCCGACTTAATAAAGAGAGAGTTAGTATGATTATTTCCGCAGCCAG
P10	ATATGCGGTGTGAAATACC
P11	GTTTTACAACGTCGTGACTTTGGATAATCAAATATTTACTCCGT
P12	TAAGGAGAAAATACCGCATCTATGCCGCATTCCCTTT
P13	AGTCACGACGTTGTAAAAC
P14	ATGCGGTATTTTCTCCTTA
P15	ATAGTTTAGCGGCCGCATTCTTATACAGATGCGTAAGGAGAAAA
P16	ATAAGAATGCGGCCGCTAAACTATTTTTCTCCTTACGCATCTGT
P17	GCTCATACCTGAATGCGCA
P18	CGGAAGAGCGCCCAAT
P19	CTTATCGCCACTGGCAGCC
P20	TGTAGGTCGTTTCGCTCCAA

引物名称 Primer name	序列 Sequence（5'-3'）
P1	CCGTGACTTAAGAAAATTTATAC
P2	CTGGCTGCGGAAATAATCATTTTTGCCTCGATTTCTTTTC
P3	GAAAAGAAATCGAGGCAAAAATGTCTTCCATGACAACAACTG
P4	ACATACAGCGCCGAACGGTC
P5	CAGCAATATACCCATTAAGGAGTAT
P6	AACGACTATGCCGCATTC
P7	TTCGCCTGATGCTCCC
P8	CTGGCTGCGGAAATAATCATACTAACTCTCTCTTTATTAAGTCGGC
P9	GCCGACTTAATAAAGAGAGAGTTAGTATGATTATTTCCGCAGCCAG
P10	ATATGCGGTGTGAAATACC
P11	GTTTTACAACGTCGTGACTTTGGATAATCAAATATTTACTCCGT
P12	TAAGGAGAAAATACCGCATCTATGCCGCATTCCCTTT
P13	AGTCACGACGTTGTAAAAC
P14	ATGCGGTATTTTCTCCTTA
P15	ATAGTTTAGCGGCCGCATTCTTATACAGATGCGTAAGGAGAAAA
P16	ATAAGAATGCGGCCGCTAAACTATTTTTCTCCTTACGCATCTGT
P17	GCTCATACCTGAATGCGCA
P18	CGGAAGAGCGCCCAAT
P19	CTTATCGCCACTGGCAGCC
P20	TGTAGGTCGTTTCGCTCCAA

菌株 Strain	酶活Enzyme activity/U	蛋白量Protein content/mg	比酶活Specific enzyme activity/（U·g^-1）
HBUT-DP	20±6	2 489	8.5±1.1
HBUT-D	20±4	2 596	8.1±1.4
HBUT-D3	632±12	7 893	80.7±1.5
HBUT-D5	275 ±10	7 274	37.8±2.8
HBUT-D7	483±11	7 863	61.4±1.4