厌氧表达乳酸脱氢酶以提高大肠杆菌产D-乳酸光学纯度

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

摘要/Abstract

摘要：

【目的】为解决在D-乳酸工业发酵生产中使用农业粗加工或废弃物等廉价原料（含有少量L-乳酸）而导致终产物D-乳酸光纯降低的问题。【方法】构建了带有不同启动子的L-乳酸脱氢酶基因lldD的表达质粒：pUC19-PLD（PpflBp6）、pUC19-NLD（PnirB）及pUC19-PNLD（PpflBp6-PnirB），并将它们分别转化入大肠杆菌D-乳酸工程菌HBUT-D中，得到菌株HBUT-D3、HBUT-D5和HBUT-D7。通过LldD的酶活检测以及对NBS培养基（添加1 g/L L-乳酸）发酵结果的TOPSIS多元评估，优选出可快速去除L-乳酸且不影响D-乳酸发酵的菌株，并使用农业廉价原料进行发酵。【结果】HBUT-D7的LldD比酶活为64 U/g，L -乳酸的消耗速率为34 mg/（L·h），D-乳酸的生产强度为4.09 g/（L·h），综合评估为最优菌株。以玉米浆为原料进行发酵时，HBUT-D及HBUT-D7的L-乳酸消耗速率分别为10.41 mg/（L·h）及34.75 mg/（L·h）；D-乳酸生产强度分别为4.24 g/（L·h）及3.87 g/（L·h）；D-乳酸光学纯度分别为99.07%及99.92%。以糖蜜为原料进行发酵时，HBUT-D及HBUT-D7的L-乳酸消耗速率为6.87 mg/（L·h）和17.18 mg/（L·h）；D-乳酸生产强度分别为1.93 g/（L·h）及1.88 g/（L·h）；D-乳酸光学纯度分别为99.22%及99.99%。【结论】厌氧诱导启动子的表达质粒可提高菌株HBUT-D7的L-乳酸脱氢酶酶活，使其在使用廉价原料发酵时能有效消除L-乳酸，提高发酵终产物D-乳酸的光学纯度。

关键词: D-乳酸, 光学纯度, 大肠杆菌, L-乳酸脱氢酶, nirB启动子, pflB启动子, 串联启动子

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

王周, 余杰, 王金华, 王永泽, 赵筱. 厌氧表达乳酸脱氢酶以提高大肠杆菌产D-乳酸光学纯度[J]. 生物技术通报, 2024, 40(5): 290-299.

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.

图/表 15

表1 本研究菌株及质粒

Table 1 Strains and plasmids in this study

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

表2 本研究所用引物

Table 2 Primers in this study

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

图1 不同质粒的验证 A：质粒pUC19-PLD及pUC19-NLD验证（M：5000 marker；1：pUC19-PLD；2-3：pUC19；4：pUC19-PNLD）；B：质粒pUC19-PNLD验证（M：5000 marker；1：pUC19-NLD；2：pUC19）

Fig. 1 Validation of different plasmids A: Validation of plasmid pUC19-PLD and plasmid pUC19-NLD（M：5000 marker；1：pUC19-PLD；2-3：pUC19；4：pUC19-PNLD）. B: Validation of plasmid pUC19-PNLD（M：5000 marker；1：pUC19-NLD；2：pUC19）

图2 不同启动子的lldD表达质粒 A：PnirB启动子重组质粒；B：PpflBP6启动子重组质粒；C：PpflBP6-PnirB串联启动子质粒

Fig. 2 Plasmids expressing lldD from different promoters A: PnirB promoter recombinant plasmid. B: PpflBP6 promoter recombinant plasmid. C: PpflBP6-PnirB tandem promoter plasmid

图3 不同菌株LldD比酶活菌株HBUT-D为空白对照；菌株HBUT-DP为HBUT-D转入pUC19质粒；菌株HBUT-D3为HBUT-D转入质粒pUC19-PLD；菌株HBUT-D5为HBUT-D转入质粒pUC19-NLD；菌株HBUT-D7为HBUT-D转入质粒pUC19-PNLD。不同小写字母代表不同菌株的LldD比酶活的差异显著性（P<0.05）

Fig. 3 LldD specific enzyme activities from different strains Strain HBUT-D is the blank control, strain HBUT-DP was transferred to the plasmid pUC19 by HBUT-D, strain HBUT-D3 to plasmid pUC19-PLD, strain HBUT-D5 to plasmid pUC19-NLD by HBUT-D, and strain HBUT-D7 to plasmid pUC19-PNLD. Different lowercase letters indicate significant differences in LldD specific enzyme activities of different strains（P<0.05）

表3 不同菌株LldD酶活结果

Table 3 LldD enzyme activity results for different strains

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

图4 不同菌株无机盐培养基的发酵 A：不同菌株的L-乳酸消耗曲线；B：不同菌株的D-乳酸生产曲线；C：不同菌株葡萄糖消耗曲线；D：不同菌株生长曲线

Fig. 4 Fermentation of different strains in inorganic salt medium A: L-lactate consumption curves of different strains. B: Acid production curves of D-lactic acid of different strains. C: Glucose consumption curves of different strains. D: Growth curves of different strains

表4 不同菌株无机盐培养基发酵数据

Table 4 Data on the fermentation of different strains in inorganic salt medium

菌株 Strain	L-乳酸消耗速率Rate of L-lactate depletion/（mg·L^-1·h^-1）	L-乳酸消耗量L-lactate depletion concentration/（g·L^-1）	D-乳酸生产强度D-lactate productivity/（g·L^-1）	葡萄糖消耗速率Rate of glucose consumption/（g·L^-1·h^-1）	OD_{600 nm}	D-乳酸糖酸转化率D-lactate yield/%	初始L-乳酸浓度Initial L -lactate concentration/（g·L^-1）
HBUT-D	8±0.13^d	0.21±0.01^d	4.09±0.11^a	4.16±0.04^a	3.22±0.01^a	98.31±0.21	1.01±0.11
HBUT-D3	40±0.32^a	0.98±0.02^a	2.45±0.04^c	2.52±0.06^d	2.75±0.02^b	97.21±0.11	1.02±0.02
HBUT-D5	14±0.33^c	0.34±0.01^c	4.09±0.04^a	4.15±0.08^b	3.12±0.01^a	98.55±0.33	1.04±0.02
HBUT-D7	34±0.23^b	0.82±0.01^b	3.92±0.03^b	3.98±0.01^c	2.97±0.01^a	98.49±0.31	1.02±0.05

图5 不同重组菌株发酵指标聚类热图

Fig. 5 Clustering heat map of fermentation indexes of different recombinant strains

图6 不同指标与相对理想指标的可视化

Fig. 6 Visualization of different indicators and relatively ideal indicators

表5 各目标值与理想点之间的欧氏距离 D+和 D-以及相对距离C-

Table 5 Euclidean distance D+ and D- between each target value and the ideal point and the relative closeness C- of each target

菌株 Strain	D⁺	D^-	C^-	排序Sort
HBUT-D	0.334 3	0.139 7	0.294 8	4
HBUT-D3	0.141 2	0.334 3	0.702 9	2
HBUT-D5	0.271 6	0.154 2	0.362 2	3
HBUT-D7	0.070 0	0.296 3	0.808 8	1

图7 菌株HBUT-D7 （A）和HBUT-D （B）玉米浆培养基发酵曲线图

Fig. 7 Fermentation curves of strain HBUT-D7 (A) and HBUT-D (B) in corn syrup medium

表6 菌株HBUT-D7和HBUT-D玉米浆培养基发酵数据

Table 6 Data on fermentation of strain HBUT-D7 and HBUT-D in corn syrup medium

菌株 Strain	D-乳酸糖酸转化率D-lactate yield/%	D-乳酸光纯D-lactate optical purty/%	D-乳酸生产强度D-lactate productivity/（g·L^-1·h^-1）	L-乳酸消耗速率Rate of L-lac consumption/（mg·L^-1·h^-1）	初始L-乳酸浓度Initial L-lac concentration/（g·L^-1）
HBUT-D	97.64±0.01	99.22±0.02	4.24±0.04	10.41±0.01***	1.0±0.02
HBUT-D7	96.89±0.01	99.93±0.01	3.87±0.02	34.75±0.01***	1.0±0.02

图8 菌株 HBUT-D （A）和HBUT-D7 （B）糖蜜培养基发酵

Fig. 8 Strain HBUT-D (A) and HBUT-D7 (B) fermentation in molasses medium

表7 菌株HBUT-D和HBUT-D7糖蜜培养基发酵数据

Table 7 Data on the fermentation of strain HBUT-D and HBUT-D7 in molasses medium

菌株 Strain	D-乳酸糖酸转化率D-lactate yield/%	D-乳酸光纯 D-lactate optical purty/%	L-乳酸消耗速率Rate of L-lactate consumption/（mg·L^-1·h^-1）	D-乳酸生产强度D-lactate productivity/（g·L^-1·h^-1）
HBUT-D	61.82±0.01	99.01±0.01	6.87±0.02***	1.93
HBUT-D7	60.43±0.01	99.99±0.01	17.18±0.01***	1.88

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