Enhancing E. coli Thermotolerance via Dynamic Control of Small Heat Shock Protein Expression for Efficient L-lactate Production

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

Abstract

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

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

Figures/Tables 6

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

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

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

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

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

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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