异源表达透明颤菌血红蛋白结合调控策略提高全细胞催化合成苯乳酸产量的研究

丁心成; 孙瑞杰; 贾士儒; 侯颖

doi:10.12187/2026.01.007

异源表达透明颤菌血红蛋白结合调控策略提高全细胞催化合成苯乳酸产量的研究

天津科技大学生物工程学院, 天津 300457

作者简介: 丁心成(1997—),女,湖北省武汉市人,天津科技大学硕士研究生,主要研究方向为生物合成。E-mail:xinchengding@yeah.net;

通讯作者: 侯颖,houying@tust.edu.cn

基金项目: 工业发酵微生物教育部重点实验室暨天津市工业微生物重点实验室资助课题项目(2020KF006)
天津市研究生科研创新项目(2022SKYZ092)
中图分类号: TS201;Q789

Study on improving the yield of whole-cell catalytic production of phenyllactic acid by heterologous expression of Vitreoscilla hemoglobin combined with regulation strategy

School of Biological Engineering, Tianjin University of Science and Technology, Tianjin 300457, China

Corresponding author: HOU Ying, houying@tust.edu.cn

Received Date: 2025-03-31
Accepted Date: 2025-06-26

CLC number: TS201;Q789

摘要: 【目的】提高全细胞催化反应体系中菌株对O₂的利用率,高效催化苯丙氨酸(Phenylalanine,PHE)合成苯乳酸(Phenyllactic Acid,PLA)。【方法】基于异源表达透明颤菌血红蛋白(Vitreoscilla Hemoglobin,VHb)对宿主大肠杆菌生长情况的影响,采用基因工程技术构建包括VHb、氨基酸脱氨酶(L-amino Acid Deaminase,L-AAD)和乳酸脱氢酶(Lactate Dehydrogenase,LDH)的多酶偶联表达体系,优化关键酶的表达调控策略,并通过连续传代实验评价重组菌株的遗传稳定性。【结果】异源表达VHb能提高菌株对O₂的利用率,明显促进宿主大肠杆菌的生长速度和最大生物量。在摇瓶培养中,菌株在扩大培养阶段进入对数生长期的时间缩短了1.5 h,且在单独诱导表达VHb时最大生物量提高了46%。经VHb表达调控策略优化,获得优势重组菌株大肠杆菌BL21(pRSFDuet-aad-ldh-T7-rbs2-2vhb);进一步优化全细胞催化反应体系,确定最优反应条件为PBS缓冲液浓度0.010 mol/L、pH值7.0、温度37 ℃、转速250 r/min,在此条件下,优势重组菌株的PLA最大产量为53.7 g/L;优势重组菌株稳定性良好,连续传代10次,每一代菌株PLA产量的变化率均小于5%,具有成为工业化菌株的潜力。【结论】异源共表达VHb可促进大肠杆菌的生长,提高全细胞催化合成PLA的产量。
- 多酶偶联 /
- 全细胞催化 /
- 透明颤菌血红蛋白 /
- 苯乳酸 /
- 重组菌株
Abstract: 【Objective】 This study aims to improve the oxygen (O₂) utilization rate of strains in the whole-cell catalytic reaction system and efficiently catalyze the synthesis of phenyllactic acid (PLA) from phenylalanine (PHE). 【Methods】 Based on the effect of heterologous expression of Vitreoscilla hemoglobin (VHb) on the growth of host Escherichia coli, genetic engineering technology was used to construct a multi-enzyme coupled expression system including VHb, L-amino acid deaminase (L-AAD) and lactate dehydrogenase (LDH). Subsequently, the expression regulation strategies of key enzymes were optimized, and the genetic stability of recombinant strains was evaluated by continuous passage experiments. 【Results】 Heterologous expression of VHb could improve the oxygen utilization rate of strains and significantly promote the growth rate and maximum biomass of host E.coli. In shake flask culture, the time for the strain to enter the logarithmic growth phase during the scale-up culture stage was shortened by 1.5 h, and the maximum biomass was increased by 46% when VHb expression was induced alone. The dominant recombinant strain E.coli BL21 (pRSFDuet-aad-ldh-T7-rbs2-2vhb) was obtained by optimizing the VHb expression regulation strategy. Furthermore, the whole-cell catalytic reaction system was optimized, and the optimal reaction conditions were determined as follows: 0.010 mol/L PBS buffer, pH 7.0, temperature 37 ℃, and rotation speed 250 r/min. Under these conditions, the maximum PLA yield of the dominant recombinant strain was 53.7 g/L; The dominant recombinant strain had good stability; after 10 consecutive passages, the change rate of PLA yield in each generation was less than 5%, showing potential to become an industrial strain. 【Conclusion】 Heterologous co-expression of VHb can promote the growth of E.coli and improve the yield of PLA via whole-cell catalysis.
- multi-enzyme conjugation /
- whole-cell catalysis /
- Vitreoscilla hemoglobin /
- phenyllactic acid /
- recombinant strain
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沈阳化工大学材料科学与工程学院沈阳 110142

异源表达透明颤菌血红蛋白结合调控策略提高全细胞催化合成苯乳酸产量的研究

作者简介: 丁心成(1997—),女,湖北省武汉市人,天津科技大学硕士研究生,主要研究方向为生物合成。E-mail:xinchengding@yeah.net;

通讯作者: 侯颖,houying@tust.edu.cn

Study on improving the yield of whole-cell catalytic production of phenyllactic acid by heterologous expression of Vitreoscilla hemoglobin combined with regulation strategy

Corresponding author: HOU Ying, houying@tust.edu.cn

计量

通讯作者: 陈斌, bchen63@163.com

异源表达透明颤菌血红蛋白结合调控策略提高全细胞催化合成苯乳酸产量的研究

作者简介:丁心成(1997—),女,湖北省武汉市人,天津科技大学硕士研究生,主要研究方向为生物合成。E-mail:xinchengding@yeah.net

通讯作者: 侯颖, houying@tust.edu.cn

English Abstract

Study on improving the yield of whole-cell catalytic production of phenyllactic acid by heterologous expression of Vitreoscilla hemoglobin combined with regulation strategy

Corresponding author: HOU Ying, houying@tust.edu.cn

目录

异源表达透明颤菌血红蛋白结合调控策略提高全细胞催化合成苯乳酸产量的研究

作者简介: 丁心成(1997—),女,湖北省武汉市人,天津科技大学硕士研究生,主要研究方向为生物合成。E-mail:xinchengding@yeah.net;

通讯作者: 侯颖,houying@tust.edu.cn

Study on improving the yield of whole-cell catalytic production of phenyllactic acid by heterologous expression of Vitreoscilla hemoglobin combined with regulation strategy

Corresponding author: HOU Ying, houying@tust.edu.cn

计量

文章相关

通讯作者: 陈斌, bchen63@163.com

异源表达透明颤菌血红蛋白结合调控策略提高全细胞催化合成苯乳酸产量的研究

作者简介:丁心成(1997—),女,湖北省武汉市人,天津科技大学硕士研究生,主要研究方向为生物合成。E-mail:xinchengding@yeah.net

通讯作者: 侯颖, houying@tust.edu.cn

English Abstract

Study on improving the yield of whole-cell catalytic production of phenyllactic acid by heterologous expression of Vitreoscilla hemoglobin combined with regulation strategy

Corresponding author: HOU Ying, houying@tust.edu.cn

目录