谷氨酸棒状杆菌合成L-高丝氨酸的代谢改造与发酵条件探究

郭秋爽; 包倩倩; 许银彪; 蔡帅; 孙杨; 李华; 刘宇鹏

doi:10.13386/j.issn1002-0306.2022030161

谷氨酸棒状杆菌合成L-高丝氨酸的代谢改造与发酵条件探究

doi: 10.13386/j.issn1002-0306.2022030161

郭秋爽^{1, 2,},
包倩倩^{1, 2},
许银彪^{1, 2},
蔡帅^{1, 2},
孙杨^{1, 2},
李华^{1, 2, ,},
刘宇鹏^{1, 2, ,}

1.
河南大学生命科学学院微生物工程研究所，河南开封 475004
2.
河南省应用微生物工程研究中心，河南开封 475004

基金项目: 河南省自然科学基金资助项目（202300410055）；河南省重点研发与推广专项(No.192102110179）。

详细信息

作者简介:
郭秋爽（1995−），女，硕士研究生，研究方向：代谢工程改造，E-mail：1528693877@qq.com

通讯作者:
李华（1986−），男，博士，副教授，研究方向：工业微生物理性设计育种和发酵工程研究， E-mail： 10140152@vip.henu.edu.cn

刘宇鹏（1977−），男，博士，教授，研究方向：发酵工程和生物催化研究， E-mail： liuyupenglw@126.com

中图分类号: TS201.3
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- 被引次数: 0
出版历程
- 收稿日期: 2022-03-15
- 网络出版日期: 2022-12-15
- 刊出日期: 2023-01-17

Metabolic Transformation and Fermentation Condition of L-homoserine Synthesis by Corynebacterium glutamicum

GUO Qiushuang^{1, 2
,},
BAO Qianqian^{1, 2},
XU Yinbiao^{1, 2},
CAI Shuai^{1, 2},
SUN Yang^{1, 2},
LI Hua^{1, 2
, ,},
LIU Yupeng^{1, 2
, ,}

1.
Institute of Microbial Engineering, College of Life Sciences, Henan University, Kaifeng 475004, China
2.
Henan Applied Microbial Engineering Research Center, Kaifeng 475004, China

摘要

摘要: 目的：本研究以谷氨酸棒状杆菌ATCC 13032为底盘细胞，构建1株L-高丝氨酸合成菌株并分析溶氧环境对其产物合成的影响。方法：首先通过外源添加0~40 g/L的L-高丝氨酸分析谷氨酸棒状杆菌的产物耐受性；随后，通过基因thrB敲除阻断L-高丝氨酸的降解途径，获得谷氨酸棒状杆菌重组菌H1；在此基础上利用挡板摇瓶进行细胞培养以增强发酵过程中氧气供给能力。结果：与大肠杆菌相比，谷氨酸棒状杆菌对L-高丝氨酸具有更强耐受性。研究中通过敲除基因thrB构建了L-苏氨酸缺陷型谷氨酸棒状杆菌重组菌H1，发现基础培养基中加入0.5 g/L的L-苏氨酸后，该重组菌生长恢复正常水平。挡板摇瓶条件下重组菌H1的L-高丝氨酸产量增加至836.7 mg/L，较普通摇瓶产量44.6 mg/L提高了17.76倍。结论：通过阻断L-苏氨酸的合成，成功构建L-高丝氨酸合成菌株谷氨酸棒状杆菌H1，并且发现利用挡板摇瓶增强发酵过程中供氧能力是促进谷氨酸棒状杆菌高效合成L-高丝氨酸的有效手段，为后续提高L-高丝氨酸发酵产量提供了参考。
- L-高丝氨酸 /
- 谷氨酸棒状杆菌重组菌 /
- 强耐受性 /
- 溶氧
Abstract: Objective: In this study, Corynebacterium glutamicum ATCC 13032 was used as the chassis cell for synthesizing L-homoserine and analyzing the effect of dissolved oxygen on product synthesis. Methods: First, the product tolerance of C. glutamicum was analyzed by exogenously adding 0~40 g/L L-homoserine. Second, the degradation pathway of L-homoserine was blocked by gene thrB knockout, namely C. glutamicum recombinant strain H1. On this basis, the shake flask with baffles was used for cell culture to enhance oxygen supply capacity in the fermentation process. Results: Compared with Escherichia coli, C. glutamicum had a stronger tolerance to L-homoserine. In the study, C. glutamicum recombinant strain H1 was constructed by deleting the gene thrB. It was found that the growth of recombinant strain H1 returned to normal after adding 0.5 g/L L-threonine in the basal medium. The L-homoserine production of recombinant strain H1 increased to 836.7 mg/L using shake flask with baffles, which was 17.76 times higher than that using ordinary shake flask, which was 44.6 mg/L. Conclusion: C. glutamicum recombinant strain H1 was successfully constructed for producing L-homoserine via blocking the synthesis of L-threonine. It was found that the using of shake flask with baffles to enhance the oxygen supply capacity during fermentation was an effective means to promote the production of L-homoserine by C. glutamicum. This study provides a reference for improving L-homoserine production subsequently.
- L-homoserine /
- Corynebacterium glutamicum recombinant strain /
- strong tolerance /
- dissolved oxygen

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图 1 不同宿主菌对L-高丝氨酸的耐受性分析

Figure 1. Tolerance analysis of different host bacteria to L-homoserine

注：A：大肠杆菌；B：谷氨酸棒状杆菌。

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图 2 基因thrB敲除及其对C. glutamicum ATCC 13032生长的影响

Figure 2. The deletion of gene thrB and its effect on the growth of C. glutamicum ATCC 13032

注：A：基因thrB敲除质粒构建流程图；B：基因thrB敲除质粒琼脂糖凝胶电泳图，M：marker；1：上游同源臂（L）；2：下游同源臂（D）；3：L-D重叠 PCR产物；4：敲除质粒单酶切验证；5：敲除质粒双酶切验证；C：基因thrB敲除PCR验证，M：marker；1：野生菌C. glutamicum ATCC 13032；2：基因thrB敲除菌C. glutamicum H1；D：基因thrB敲除对C. glutamicum ATCC 13032生长的影响。

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图 3 L-高丝氨酸激酶编码基因thrB敲除对L-高丝氨酸合成的影响

Figure 3. The effect of gene thrB deletion on the synthesis of L-homoserine

注：A：野生菌C. glutamicum ATCC 13032与基因thrB敲除菌C. glutamicum H1 72 h发酵液高效液相色谱分析；B：野生菌C. glutamicum ATCC 13032与基因thrB敲除菌C. glutamicum H1的细胞生长以及L-高丝氨酸产量分析。

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图 4 不同培养环境下菌株的生长情况及溶氧变化

Figure 4. Growth and dissolved oxygen of strains in different culture environments

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表 1 实验菌株与质粒

Table 1. Experimental strains and plasmids

菌株/质粒	特征	来源
E. coli DH5α	用于质粒的扩增与保存	本实验室保存
E. coli W3110	用于L-高丝氨酸生长抑制测定	本实验室保存
C. glutamicum ATCC 13032	野生型，生物素缺陷型	本实验室保存
C. glutamicum H1	13032 衍生菌，∆thrB	本研究
pK18mobsacB	自杀质粒，Km^r	本实验室保存
pK18-thrB	包含thrB同源臂的pK18mobsacB	本研究

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表 2 实验引物

Table 2. Primers for the experiments

引物名称	引物序列
thrB-L-F-BamH I	CATGGATCCCCCGACTCTATTACCTGTGT
thrB-L-R	CTTGTTGGGCGTCAGTAAAATTAGTCCCTTTCGAGGCG
thrB-R-F	TTTTACTGACGCCCAACAAGGAAGGCCCCCTTC
thrB-R-R-Hind III	CCCAAGCTTCTTCCAAACACGCGTCCCCGACAAC
thrB-YZ-F	GCTATTTCTGCTCGCGTGCA
thrB-YZ-R	GATTCGAAGGGGGCCTTCCTTGTTG

下载: 导出CSV

表 3 L-高丝氨酸梯度洗脱程序

Table 3. Gradient elution procedure of L-homoserine

时间（min）	流动相A（%）	流动相B（%）	流速（mL/min）
0	90.0	10.0	1.00
5.00	90.0	10.0	1.00
11.00	33.0	67.0	1.00
14.50	0.0	100.0	1.20
15.00	0.0	100.0	1.20
17.00	90.0	10.0	1.00
25.00	90.0	10.0	1.00

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表 4 不同培养基下L-高丝氨酸的产量（mg/L）

Table 4. The production of L-homoserine in different media (mg/L)

培养基	C. glutamicum ATCC 13032	C. glutamicum H1
发酵培养基Ⅰ	0	22.4±0.35
发酵培养基Ⅱ	0	44.6±0.72

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表 5 不同摇瓶方式对L-高丝氨酸合成的影响

Table 5. The effect of different shaking flasks on the synthesis of L-homoserine

摇瓶种类	菌株	胞外L-高丝氨酸（mg/L）	胞内L-高丝氨酸（mg/g-DCW）	OD₆₀₀	残糖（g/L）
普通摇瓶	C. glutamicum H1	44.6±0.72	0.68±0.015	16.03±0.52	16.61±0.63
挡板摇瓶	C. glutamicum H1	836.7±20.54	1.78±0.143	40.2±2.12	4.24±0.25

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