稀有密码子串联介导的HemB表达弱化提升5-氨基乙酰丙酸的含量

doi:10.13560/j.cnki.biotech.bull.1985.2025-0095

摘要/Abstract

摘要：

目的应用稀有密码子串联策略弱化5-氨基乙酰丙酸分解代谢途径中关键酶（5-氨基乙酰丙酸脱水酶）HemB表达，降低5-氨基乙酰丙酸分解代谢，进而提高5-氨基乙酰丙酸含量。方法以谷氨酸棒杆菌为底盘细胞合成5-氨基乙酰丙酸，依据谷氨酸棒杆菌密码子偏好性，在HemB编码基因的5′引入含有稀有密码子串联的DNA序列来弱化HemB表达。结果引入稀有密码子的谷氨酸棒杆菌工程细胞生长速度降低，发酵液中5-氨基乙酰丙酸分解代谢产物含量下降，5-氨基乙酰丙酸含量得以明显提升。结论稀有密码子串联策略有效弱化了HemB表达，提高了5-氨基乙酰丙酸含量，此策略亦可用于其他蛋白质表达弱化和代谢途径的重塑。

关键词: 谷氨酸棒杆菌, 5-氨基乙酰丙酸脱水酶, 5-氨基乙酰丙酸, 弱化表达, 稀有密码子

Abstract:

Objective To reduce 5-aminolevulinic acid degradation and enhance its accumulation, the expression of 5-aminolevulinic acid dehydratase (HemB), which is a key enzyme in the catabolic pathway of 5-aminolevulinic acid (5-ALA), was attenuated via a tandem rare codon engineering strategy. Method Corynebacterium glutamicum was used as the chassis cell for the synthesis of 5-ALA. To reduce the catabolism of 5-ALA, tandem rare codons according to codon usage bias of C. glutamicum were introduced at the 5' end of the gene encoding HemB to weaken its expression. Result The metabolically engineered C. glutamicum strain showed a reduced growth rate, simultaneously the content of 5-ALA degradation intermediates decreased and 5-ALA accumulation was significantly enhanced in the fermentation broth. Conclusion The rare codon engineering strategy effectively attenuates HemB expression to enhance 5-ALA accumulation, and this strategy can be applied to fine-tuning expression of other proteins and remodeling other metabolic pathways.

Key words: Corynebacterium glutamicum, 5-aminolevulinic acid dehydratase, 5-aminolevulinic acid, attenuating expression, rare codon

李加仪, 李尽益, 白雪, 柏映国, 刘波, 张志伟. 稀有密码子串联介导的HemB表达弱化提升5-氨基乙酰丙酸的含量[J]. 生物技术通报, 2025, 41(8): 74-81.

LI Jia-yi, LI Jin-yi, BAI Xue, BAI Ying-guo, LIU Bo, ZHANG Zhi-wei. Enhancing 5-Aminolevulinic Acid Biosynthesis through Tandem Rare Codon-mediated Attenuation of HemB Expression[J]. Biotechnology Bulletin, 2025, 41(8): 74-81.

图/表 7

表1 本研究所用引物

Table 1 Primers used in this study

引物名称 Primer name	引物序列 Primer sequence （5′-3′）	引物用途 Primer usage
hemB-LF1	TGATGGGTCTTGTTGTTGGCGTACCAGAACTGATCGACGCTCT	扩增hemB左侧同源臂
hemB-LR24	TAGCCTTAGGGATAGGGACCTTAGCATTGCTTGTAACCTTTGCTCTCT	扩增hemB左侧同源臂
hemB-RF24	CTAAGGTCCCTATCCCTAAGGCTAAGCACTTCTTCTGATTACTCCCAC	扩增在hemB起始密码子ATG后添加24 bp DNA序列的hemB左侧同源臂
hemB-RR1	GTCCTCTGTATGTTGATAAACGCCCGGCATGGAG	扩增在hemB起始密码子ATG后添加24 bp DNA序列的hemB左侧同源臂
hemB-LF2	GCGTTTATCAACATACAGAGGACTCGCTGCTGCGTGCCGCCCA	扩增hemB右侧同源臂
hemB-RR2	AGTATCTTCCTGGCATCTTCCAGTCGCAGGATCTTGCTGGTAG	扩增hemB右侧同源臂
hemB-LR18	AGTGCTTAGGGATAGGGACCTTAGCATTGCTTGTAACCTTTGCTCTCT	扩增在hemB起始密码子ATG后添加18 bp DNA序列的hemB左侧同源臂
hemB-RF18	CTAAGGTCCCTATCCCTAAGCACTTCTTCTGATTACTCCCAC	扩增在hemB起始密码子ATG后添加18 bp DNA序列的hemB左侧同源臂
hemB-LR9	ATCAGAAGAAGTGCTGGACCTTAGCATTGCTTGTAACCTTTGCTCTCT	扩增在hemB起始密码子ATG后添加9 bp DNA序列的hemB左侧同源臂
hemB-RF9	CTAAGGTCCAGCACTTCTTCTGATTACTCCCAC	扩增在hemB起始密码子ATG后添加9 bp DNA序列的hemB左侧同源臂
pA15-F	CTGGAAGATGCCAGGAAGATACT	扩增大肠杆菌复制元件和gDNA
hemB/gDNA-R	CTCTCATCCGCCAAAACAGCCAGCAGCGAATCTTCAGTGTGCTGAATCTACAACAGTAGAAATTCGGATCC	扩增大肠杆菌复制元件和gDNA

图1 用于HemB表达弱化的DNA序列

Fig. 1 DNA sequence for attenuating expression of HemB

图2 稀有密码子嵌入hemB起始密码子后策略示意图A：hemB及其相邻区域的DNA序列；B：构建的基因编辑质粒上hemB及其相邻区域的DNA序列。图中黄色背景ATG为hemB起始密码子，蓝色背景TTTA为Cas12a识别的PAM基序，A中灰色背景部分为gDNA序列，B中灰色背景部分是依据同义密码子原则对gDNA进行了突变（避免Cas12a切割）

Fig. 2 Schematic diagram of the strategy after inserting rare codons into starting codon hemBA: DNA sequence of hemB and its adjacent DNA sequence. B: DNA sequence of hemB and its adjacent DNA sequence in the gene-editing plasmid. In figure A, the ATG highlighted with a yellow background indicates the start codon of hemB, while the TTTA marked with a blue background indicates the PAM motif recognized by Cas12a. The gray-highlighted region in panel A corresponds to the gDNA sequence. In figure B, the gray-highlighted region shows mutations introduced into the gDNA based on the synonymous codon principle to avoid cleavage by Cas12a

图3 HemB编辑和表达弱化的质粒hemB-L和hemB-R为基因hemB的左右同源序列，同源序列之间为用于HemB表达弱化的DNA序列w9、w18和w24序列。w9：5′-CTAAGGTCC-3′，w18：5′- CTAAGGTCCCTATCCCTA-3′，w24：5′-CTAAGGTCCCTATCCCTAAGGCTA-3′，gDNA：5′-TCAGCACACTGAAGATTCGCTGCTG-3′

Fig. 3 Plasmid for gene editing and attenuating expression of HemBhemB-L and hemB-R as the left and right homologous sequence of hemB, respectively. The DNA sequences between hemB-L and hemB-R used for attenuating expressions of HemB as follows, w9: 5′-CTAAGGTCC-3′, w18: 5′- CTAAGGTCCCTATCCCTA-3′, w24: 5′-CTAAGGTCCCTATCCCTAAGGCTA-3′, and gDNA: 5′-TCAGCACACTGAAGATTCGCTGCTG-3′

图4 不同菌株在固体LB上生长表型1：C. glutamicum hemB-w24；2：C. glutamicum hemB-w18；3：C. glutamicum hemB-w9；4：C. glutamicum hemBm

Fig. 4 Growth profiles of different strains cultured on solid medium LB

图5 不同菌株发酵液上清1：C. glutamicum-pXJM19；2：C. glutamicum hemB-w24-hemA；3：C. glutamicum hemBm-hemA

Fig. 5 Fermentated supernatants of different strains

图6 不同菌株发酵液中5-ALA含量1：C. glutamicum-pXJM19；2：C. glutamicum hemB-w24-hemA；3：C. glutamicum hemBm-hemA

Fig. 6 5-ALA content in fermentated supernatants of different strains

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