途径工程改造谷氨酸棒杆菌产莽草酸

doi:10.13560/j.cnki.biotech.bull.1985.2021-1058

摘要/Abstract

摘要：

莽草酸是一种芳香族中间代谢产物,也是合成抗禽流感药物磷酸奥司他韦的前体。目前,国内外莽草酸的生产主要依靠成本较高,周期较长的植物提取法。微生物发酵法合成莽草酸具有生产成本低、周期短等优势成为研究的热点。为了构建产莽草酸的重组谷氨酸棒杆菌,此次研究从基因组水平上对谷氨酸棒杆菌体内的莽草酸代谢途径进行代谢工程改造。通过阻断莽草酸分解代谢途径、解除反馈抑制以及阻断竞争性代谢途径的策略,实现了莽草酸产量的大幅提升。结果显示,所构建的重组谷氨酸棒杆菌SKA06经72 h摇瓶发酵,莽草酸产量达到7.61 g/L,相较出发菌种提升了68倍。并且,基于染色体工程的遗传改造策略克服了引入质粒带来传代不稳定、需要添加抗生素等问题,可以为莽草酸工程菌种的选育提供重要参考。

关键词: 谷氨酸棒杆菌, 莽草酸, 启动子工程, 途径工程

Abstract:

Shikimic acid,as an intermediate metabolite of aromatic family,is the precursor of oseltamivir phosphate for the synthesis of an anti-avian influenza drug. Currently,the production of shikimic acid mainly depends on the plant extraction method with shortcoming in high cost and long period. Due to the advantages in low production cost and short period,microbial fermentation for the synthesis of shikimic acid has turned into the hot spot of research. In order to construct a recombinant C. glutamicum capable of producing shikimic acid,the metabolic pathways of shikimic acid in C. glutamicum was modified via metabolic engineering at genetic level. The yield of shikimic acid significantly increased by blocking the catabolic pathway,relieving the feedback inhibition,and blocking the competitive metabolic pathway. The final strain C. glutamicum SKA06 produced 7.61 g/L of shikimic acid during 72 h shake flask cultivation,which was 68 times higher than that of the original strain. Moreover,genetic modification based on chromosome engineering overcame the problems of unstable passage and antibiotics addition caused by the introduction of plasmid,which may provide important reference for the development of shikimic acid producing strains.

Key words: Corynebacterium glutamicum, shikimic acid, promoter engineering, pathway engineering

聂立斌, 易铃欣, 邓妍, 盛琦, 吴晓玉, 张斌. 途径工程改造谷氨酸棒杆菌产莽草酸[J]. 生物技术通报, 2022, 38(6): 93-102.

NIE Li-bin, YI Ling-xin, DENG Yan, SHENG Qi, WU Xiao-yu, ZHANG Bin. Pathway Engineering Modification of Corynebacterium glutamicum for Shikimic Acid Production[J]. Biotechnology Bulletin, 2022, 38(6): 93-102.

图/表 8

表1 本实验所用的菌种

Table 1 Corynebacterium glutamicum and Escherichia coli strains used in this study

Strain	Relevant genotype	Reference
Escherichia coli DH5α	Cloning host	Lab stock
Corynebacterium. glutamicum CICC 20189	Wild type,L-phenylalanine producing strain	Purchased from CICC
SKA01	CICC 20189 with deletion of aroK	This study
SKA02	CICC 20189 with deletion of aroK and a strong PNCgl0824 promoter insertion in the upstream region of aroG	This study
SKA03	CICC 20189 with deletion of aroK and a strong Psod promoter insertion in the upstream region of aroG	This study
SKA04	CICC 20189 with deletion of aroK,a strong PNCgl0824 promoter insertion in the upstream region of aroG,and deletion of qsuB	This study
SKA05	CICC 20189 with deletion of aroK,a strong PNCgl0824 promoter insertion in the upstream region of aroG,deletion of qsuB,and insertion of SaroG^E.coli at the △qsuB site.	This study
SKA06	CICC 20189 with deletion of aroK,a strong PNCgl0824 promoter insertion in the upstream region of aroG,deletion of qsuB,and insertion of ParoG^E.coli at the △qsuB site.	This study

表2 本实验所用的质粒

Table 2 Plasmids used in this study

Plasmid	Characteristics	Reference
pK18mobsacB	Mobilizable vector,allows for selection of double crossover in C. glutamicum,Km^R,sacB	Lab stock
pK18-△aroK	A derivative of pK18mobsacB,harboring △aroK fragment	This study
pK18-ParoG	A derivative of pK18mobsacB,harboring ParoG fragment	This study
pK18-SaroG	A derivative of pK18mobsacB,harboring SaroG fragment	This study
pK18-△qsuB	A derivative of pK18mobsacB,harboring △qsuB fragment	This study
pK18-△qsuB-SaroG^E.coli	A derivative of pK18mobsacB,harboring △qsuB-SaroG^E.coli fragment	This study
pK18-△qsuB-Paro^GE.coli	A derivative of pK18mobsacB,harboring △qsuB-ParoG^E.coli fragment	This study

表3 本实验所用的引物

Table 3 Primers used in this study

Primer name	Sequence（5'-3'）	Size/bp
aroK-up-F	aacgacggccagtgccaagctCAATCGATGATTCCCCAGTTC	42
aroK -up-R	GAAAAGCTTGGGAAACGTCTAGAGCGTCGGAGTCGACGAGTTCAGTG	47
aroK -down-F	GCTCTAGACGTTTCCCAAGCTTTTCAAGCACTGAAATCCTCCGGAGT	47
aroK -down-R	cggtacccggggatcctctagCGGTGTCAATGAATACTGCGTC	43
aroK -check-F	AAAAGCCTGTGGCGCCGTGTT	21
aroG-up-F	aacgacggccagtgccaagctCTTGTGAGCGCTTCTTTGATC	42
aroG-up-R（sod）	CAAGCCCGGAATAATTGGCAATGGGATGGGGTGAATTTAGG	41
aroG-up-R（P）	CTATCCGTATTAGTGGCACAGTTATGGGATGGGGTGAATTTAGG	44
sod-F	TGCCAATTATTCCGGGCTTG	20
sod-R	TCCGCACCGAGCATATACATCTT	23
PNCgl0824-F	AACTGTGCCACTAATACGGATAG	23
PNCgl0824-R	CAATTTCGCCTGCTTCCGATT	21
aroG-down-F（sod）	AAGATGTATATGCTCGGTGCGGATGCATAGCCCTGAAAGGCAAG	44
aroG-down-F（P）	AATCGGAAGCAGGCGAAATTGTGCATAGCCCTGAAAGGCAAG	42
aroG-down-R	cggtacccggggatcctctagTCGTCGGAGGTTCCGAAGAAG	42
qsuB-up-F	aacgacggccagtgccaagctGCTCGCGTTGCTATTGCTG	40
qsuB-up-R	GAAAAGCTTGGGAAACGTCTAGAGCGAAACCACCAAGTCCTGCTCG	46
qsuB-down-F	GCTCTAGACGTTTCCCAAGCTTTTCATCTGTTCCAGCCGTTTCGAG	46
qsuB-down-R	cggtacccggggatcctctagCACCCAAGCGGAAACCCAATT	42
qsuB-check-F	GGCTCAGGATTTGGGATTAAC	21
Sod-F（qsuB）	ACTTGGTGGTTTCGCTCTAGATGCCAATTATTCCGGGCTTG	41
Sod-R（aroG^{E. coli}）	AAATCGTCGTTCTGATAATTCATTCCGCACCGAGCATATACATCTT	46
P-F（qsuB）	ACTTGGTGGTTTCGCTCTAGAAACTGTGCCACTAATACGGATAG	44
P-R（aroG^{E. coli}）	AAATCGTCGTTCTGATAATTCATCAATTTCGCCTGCTTCCGATT	44
aroG^{E. coli}-F1	ATGAATTATCAGAACGACGATTT	23
aroG^{E. coli}-R1	CGACCGGACAAAAAAGCCCTGATGCCAGTTCG	32
aroG^{E. coli}-F2	GCATCAGGGCTTTTTTGTCCGGTCGGCTTCAAAAATG	37
aroG^{E. coli}-R2	ACGGCTGGAACAGATGAAAAGCTTTTACCCGCGACGCGCTTTTACT	46

图1 谷氨酸棒杆菌的莽草酸代谢途径

Fig.1 Pathway of shikimic acid in C. glutamicum PEP：phosphoenolpyruvate;E4P：erythrose-4-phosphate;DAHP：3-deoxy-D-arabinoheptulosonate-7-phosphate;DHQ：3-dehydro-quinate;DHS：3-dehydroshikimate;PCA：protocatechuate;S3P：shikimate-3-phosphate SA,shikimic acid;aroG：encoding DAHP synthetase;aroGE. coli：encoding DAHP synthetase from E. coli;aroB：encoding DHQ synthase;aroD：encoding DHQ dehydratase;aroE：encoding shikimate dehydrogenase;aroK：encoding shikimate kinase;qsuB：encoding DHS dehydratase;qsuD：encoding QA/ shikimate dehydrogenase

图2 aroK基因缺失株的构建及发酵性能评价 A：PCR鉴定aroK阳性基因敲除株;M：DNA marker;1：利用引物aroK-check-F和aroK-down-R扩增谷氨酸棒杆菌CICC 20189;4：阴性对照;2,3,5,6：利用引物aroK-check-F和aroK-down-R扩增谷氨酸棒杆菌SKA01。B：谷氨酸棒杆菌CICC 20189和SKA01发酵48和72 h的莽草酸产量。C：不同菌种产莽草酸的HPLC色谱图及标准品对照

Fig.2 Construction and fermentation evaluation of aroK-deleted strain A：PCR identification of positive strain with aroK deleted. M：DNA marker;1：amplifying C. glutamicum CICC 20189 using aroK-check-F and aroK-down-R as primers. 4：Negative control. 2,3,5,6：amplifying C. glutamicum SKA01 using aroK-check-F and aroK-down-R as primers. B：Shikimic acid production at fermented 48 h and 72 h of strain C. glutamicum CICC 20189 and SKA01.C：HPLC analysis of shikimic acid produced by different strains in contrast with standard sample

图3 重组谷氨酸棒杆菌SKA02和SKA03的PCR鉴定及摇瓶发酵性能评价 A：PCR鉴定重组菌SKA02;M：DNA marker;1,2：利用引物PNCgl0824-F和aroG-down-R扩增谷氨酸棒杆菌SKA02;3：利用引物PNCgl0824-F和aroG-down-R扩增谷氨酸棒杆菌SKA01;B：PCR鉴定重组菌SKA03;M：DNA marker;1-5：利用引物 sod-F和aroG-down-R扩增谷氨酸棒杆菌SKA03;6：利用引物sod-F和aroG-down-R扩增谷氨酸棒杆菌SKA01;C：谷氨酸棒杆菌SKA02和SKA03的莽草酸发酵曲线;D：谷氨酸棒杆菌SKA02和SKA03的生长曲线

Fig.3 PCR identification and shake flask fermentation evaluation of recombinant strain C. glutamicum SKA02 and SKA03 A：PCR identification of recombinant strain SKA02. M：DNA marker;1,2：amplifying C. glutamicum SKA02 using PNCgl0824-F and aroG-down-R as primers;3：amplifying C. glutamicum SKA01 using PNCgl0824-F and aroG-down-R as primers. B：PCR identification of recombinant strain SKA03. M：DNA marker;1-5：amplifying C. glutamicum SKA03 using sod-F and aroG-down-R;6：amplifying C. glutamicum SKA01 using sod-F and aroG-down-R. C：Shikimic acid production curves of C. glutamicum strain SKA02 and SKA03. D：Cell growth curves of C. glutamicum strain SKA02 and SKA03

图4 重组谷氨酸棒杆菌SKA04的构建及发酵性能评价 A：PCR鉴定重组菌SKA04;M：DNA marker;1-3：利用引物qsuB-check-F和qsuB-down-R扩增谷氨酸棒杆菌SKA04;4：利用引物qsuB-check-F和qsuB-down-R扩增谷氨酸棒杆菌SKA02;B：谷氨酸棒杆菌SKA02和SKA04的莽草酸发酵曲线;C：谷氨酸棒杆菌SKA02和SKA04的生长曲线

Fig.4 Construction and fermentation evaluation of recom-binant C. glutamicum strain SKA04 A：PCR identification of recombinant SKA04. M：DNA marker;1-3：amplifying C. glutamicum SKA04 using qsuB-check-F and qsuB-down-R as primers;4：amplifying C. glutamicum SKA02 using qsuB-check-F and qsuB-down-R as primers. B：Shikimic acid production curves of C. glutamicum strain SKA02 and SKA04. C：Cell growth curves of C. glutamicum strain SKA02 and SKA04

图5 重组谷氨酸棒杆菌SKA04、SKA05和SKA06的构建及摇瓶发酵性能评价 A：PCR鉴定重组菌SKA05;M：DNA marker;1：利用引物PNCgl0824-F和aroGE. coli-R1扩增谷氨酸棒杆菌SKA04;2-8：利用引物PNCgl0824-F和aroGE. coli-R1扩增谷氨酸棒杆菌SKA05。B：PCR鉴定重组菌SKA06;M：DNA marker;1：利用引物 sod-F和aroGE. coli-R1扩增谷氨酸棒杆菌SKA06;2-8：利用引物sod-F和aroGE. coli-R1扩增谷氨酸棒杆菌SKA01。C：谷氨酸棒杆菌SKA04、SKA05和SKA06的莽草酸发酵曲线。D：谷氨酸棒杆菌SKA04、SKA05和SKA06的生长曲线

Fig. 5 Construction and shake flask fermentation evaluation of recombinant C. glutamicum strain SKA04,SKA05,and SKA06 A：PCR identification of SKA05. M：DNA marker;1：amplifying C. glutamicum SKA04 using PNCgl0824-F and aroGE. coli-R1 as primers;2-8：amplifying C. glutamicum SKA05 using PNCgl0824-F and aroGE. coli-R1 as primers. B：PCR identification of SKA06. M：DNA marker;1,2：amplifying C. glutamicum SKA06 using sod-F and aroGE. coli-R1 as primers;3：amplifying C. glutamicum SKA04 using sod-F and aroGE. coli-R1 as primers. C：Shikimic acid production curves of C. glutamicum strain SKA04,SKA05,and SKA06. D：Cell growth curves of C. glutamicum strain SKA04,SKA05,and SKA06

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