动态调控小热休克蛋白表达以提升大肠杆菌耐热性并高效生产L-乳酸

doi:10.13560/j.cnki.biotech.bull.1985.2026-0179

摘要/Abstract

摘要：

目的增强工业微生物的耐热性可一定程度缩短发酵时间、减少杂菌污染及促进产物的溶解，拟通过小热休克蛋白的筛选及动态表达提高大肠杆菌的耐热性。方法对不同种属的小热休克蛋白在大肠杆菌BL21(DE3)中进行表达和筛选，筛选得到的小热休克蛋白进一步使用不同强度的组成型启动子及温敏启动子P_L驱动表达，研究小热休克蛋白表达对大肠杆菌耐热性的影响。结果来自腾冲热厌氧杆菌Thermoanaerobacter tengcongensis MB4的小热休克蛋白（sHsp）能提高大肠杆菌在高温环境下的耐热性，蛋白质序列比对进一步验证了蛋白β3片层序列Pro-Gly片段的重要作用。不同强度组成型启动子驱动小热休克蛋白来提高宿主在45 ℃发酵性能，结果表明强启动子P_M193条件下L-乳酸的产量最高但生物量较低。温敏性启动子P_L被进一步选用驱动sHsp的动态表达，通过种子阶段低温培养减少sHsp的表达，减少种子阶段的生长负荷以期提高生物量。最终L-乳酸产量可达到30±0.58 g/L，最大生物量（OD₆₀₀）达到8.1±0.32，比P_M193组成型启动子表达的乳酸产量和生物量（OD₆₀₀）分别提高6%和17%。结论动态调控小热休克蛋白表达可以提高大肠杆菌在高温条件下L-乳酸发酵效果，可作为一种提高工业微生物耐热性的有效手段。

关键词: 大肠杆菌, 小热休克蛋白, 动态调控, L-乳酸, 高温发酵

Abstract:

Objective Enhanced thermotolerance of industrial microorganisms shortens fermentation time, reduces microbial contamination, and promotes product solubility. This study aimed to enhance thermotolerance of Escherichia coli (E. coli) by screening and dynamic expression of small heat shock proteins (sHsps). Method Small heat shock proteins from diverse species were expressed and screened in E. coli BL21(DE3). The selected sHsp was further expressed under the control of constitutive promoters with different strengths and the thermosensitive promoter P_L, and the effect of sHsp expression on E. coli thermotolerance was examined. Result The sHsp from Thermoanaerobacter tengcongensis MB4 enhanced thermotolerance of E. coli under high-temperature conditions. Protein sequence alignment verified that the Pro-Gly segment in the β3 sheet of this protein played an important role. Constitutive promoters with different strengths were used to drive sHsp expression to improve host fermentation performance at 45 ℃. The results showed that the strong promoter P_M193 gave the highest L-lactic acid titer but relatively low biomass. The thermosensitive promoter P_L was further used to drive dynamic sHsp expression. Low-temperature seed culture reduced sHsp expression and relieved growth burden in the seed stage, thereby increasing biomass. The final L-lactic acid titer reached 30±0.58 g/L, and the maximum biomass (OD₆₀₀) reached 8.1±0.32, which were 6% and 17% higher than those under the P_M193 constitutive promoter, respectively. Conclusion Dynamically regulated expression of heat shock proteins (sHsp) can enhance the L-lactic acid fermentation performance of E. coli under high-temperature conditions, and this approach can serve as an effective strategy to improve the thermal tolerance of industrial microorganisms.

Key words: Escherichia coli, small heat shock protein, dynamic regulation, L-lactic acid, high-temperature fermentation

梁浩, 黄恩园, 朱丽雯, 赵锦芳, 刘晓楠, 王永泽. 动态调控小热休克蛋白表达以提升大肠杆菌耐热性并高效生产L-乳酸[J]. 生物技术通报, 2026, 42(10): 305-316.

LIANG Hao, HUANG En-yuan, ZHU Li-wen, ZHAO Jin-Fang, LIU Xiao-nan, WANG Yong-ze. Enhancing E. coli Thermotolerance via Dynamic Control of Small Heat Shock Protein Expression for Efficient L-lactate Production[J]. Biotechnology Bulletin, 2026, 42(10): 305-316.

图/表 6

表1 本文所用引物及序列

Table 1 Primers and sequences used in this article

Primers

Sequences （5′-3′）

TTsHsp-F

TTsHsp-R

P_M137-F

P_M137-R

P_M146-F

P_M146-R

P_M193-F

P_M193-R

P_L-F

P_L-R

pET-F

pET-R

GFP-F

GFP-R

ccdB-F

ccdB-R

ATGGCAAATGAATTAATGGA

TTATTGGATTTCAATATGAT

CTCCTGAATCTATCTATCCTTTATCTCTGGCGGTGTTGAC

TCCATTAATTCATTTGCCATAGCTGTTTCCTGGTTTAAAC

AGAATTACATGTGAGAAATTTTATCTCTGGCGGTGTTGAC

TCCATTAATTCATTTGCCATAGCTGTTTCCTGGTTTAAACGT

GTAACGAAAGAGGATAAACCTTATCTCTGGCGGTGTTGAC

TCCATTAATTCATTTGCCATCTTGTCTCTCCTGGTTTAAACGT

GGAAGGAGCTGACTGGGTTGCTCAATTGTTATCAGCTATGCGC

TCCATTAATTCATTTGCCATGGTGGTCAGTGCGTCCTGCT

TGCCACCGCTGAGCAATAAC

GGTTTATCCTCTTTCGTTAC

CCTCTAGAAATAATTTTGTTTAACT

CGAGTTTGTATAGTTCATCC

AGCAGGACGCACTGACCACCATGCAGTTTAAGGTTTACACCTATA

CTAGATCCTTTACGCGTTCGTTATATTCCCCAGAACATCAGGTTA

图1 小热休克蛋白的筛选A：不同小热休克蛋白在E. coli BL21（DE3）中的表达，1，8：180 kD蛋白Marker；2、4、6、9、11和13：细胞裂解后的上清；3、5、7、10、12和14：细胞裂解后的沉淀。B：小热休克蛋白表达对宿主在52 ℃热激下存活率的影响；C：小热休克蛋白的表达对宿主在45 ℃培养条件下生物量的影响。TT、OO、PF、OP：分别为Thermoanaerobacter tengcongensis MB4、Oenococcus oeni、Pyrococcus furiosus、Oceanithermus profundus的小热休克蛋白

Fig. 1 Screening of small heat shock proteinsA: Expression of different small heat shock proteins in E. coli BL21(DE3). Lanes 1 and 8: 180 kD protein marker; 2, 4, 6, 9, 11, 13: supernatant after cell lysis; 3, 5, 7, 10, 12, 14: pellet after cell lysis. B: Effect of small heat shock protein expression on host cell survival under heat shock at 52 ℃. C: Effect of small heat shock protein expression on host biomass under cultivation at 45 ℃. TT, OO, PF and OP refer to the small heat shock proteins derived from Thermoanaerobacter tengcongensis MB4, Oenococcus oeni, Pyrococcus furiosus and Oceanithermus profundus, respectively

图2 小热休克蛋白氨基酸多序列比对

Fig. 2 Multiple sequence alignment of amino acid for small heat shock proteins

图3 不同启动子表达小热休克蛋白对宿主耐热性影响A：不同强度的启动子表达小热休克蛋白sHsp的示意图；B：在52 ℃热应激条件下不同强度启动子驱动小热休克蛋白表达对宿主存活率影响；C：不同组成型启动子驱动小热休克蛋白表达对乳酸45 ℃发酵的影响；D：不同强度启动子驱动小热休克蛋白表达对宿主最高温度耐受度的影响

Fig. 3 Effects of heat shock protein expression driven by different promoters on host thermotoleranceA: Schematic diagram of small heat shock protein (sHsp) expression driven by promoters of different strengths. B: Effects of the expression of small heat shock proteins driven by promoters of different strengths on host cell survival rate under 52 ℃ heat stress. C: Effects of the expression of small heat shock proteins driven by different constitutive promoters on lactic acid fermentation at 45 ℃. D: Effect of different strength promoters driving the expression of small heat shock proteins on the host maximum temperature tolerance

图4 温敏性启动子PL对小热休克蛋白表达耐受发酵产酸情况A：温敏性启动子PL动态调控示意图［20］；B-C：致死基因ccdB（B）和绿色荧光蛋白GFP（C）分别作为报告基因测试PL启动子的温控性能；D：PL启动子相对于组成型启动子在种子阶段30 ℃培养对宿主生物量的影响；E：PL启动子相对于组成型启动子在发酵阶段45 ℃下培养对宿主生物量的影响；F：PL启动子相对于组成型启动子对L-乳酸产量的影响

Fig. 4 Effects of thermosensitive promoter PL mediated heat shock protein expression on acid production during stress-tolerant fermentationA: Schematic diagram of dynamic regulation of thermosensitive promoter PL[20]. B-C: Evaluation of the temperature-controlled performance of the PL promoter using the lethal gene ccdB (B) and green fluorescent protein GFP (C) as reporter genes. D: Effect of the PL promoter on host biomass in comparison with a constitutive promoter during seed culture at 30 ℃. E: Effect of the PL promoter on host biomass in comparison with a constitutive promoter during fermentation at 45 ℃; F: Effect of the PL promoter on L‑lactic acid production in comparison with a constitutive promoter

表 2 常见的小热休克蛋白启动子

Table 2 Common promoters for small heat shock proteins

启动子 Promoter	热休克蛋白 Heat shock protein	来源 Source	特点 Characteristics	参考文献 References
T7	Hsp20	Tepidimonas sediminis（沉积物暖单胞菌）	强表达；泄漏表达、IPTG依赖、代谢负担重、菌株限制	［8］
P_Lac	IbpA/IbpB	Escherichia coli	适应菌株范围广；IPTG依赖	［25］
P_IbpA	IbpA/IbpB	Escherichia coli	σ32调控，在受到胁迫时才表达，生长负荷小；诱导温度严格、表达时间窗口窄	［26］
P_gapA	Hsp20	Thermoanaerobacter tengcongensis MB4	适用菌株范围广，组成型强表达，σ32/σ70双调控	［12］
P_L	Hsp20	Thermoanaerobacter tengcongensis MB4	无需诱导剂，通过温度控制，使小热休克蛋白在生长阶段和发酵阶段处于不同开关状态，减少生长期间负荷；生长阶段必须控制在较低温度30 ℃，会一定程度影响生长	本研究

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