Enhancing 5-Aminolevulinic Acid Biosynthesis through Tandem Rare Codon-mediated Attenuation of HemB Expression

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

Abstract

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

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.

Figures/Tables 7

References 35

[1]	Tang HT, Lin SM, Deng JL, et al. Engineering yeast for the de novo synthesis of jasmonates [J]. Nat Synth, 2023, 3(2): 224-235.
[2]	Yang D, Park SY, Park YS, et al. Metabolic engineering of Escherichia coli for natural product biosynthesis [J]. Trends Biotechnol, 2020, 38(7): 745-765.
[3]	Wang K, Song XT, Cui BY, et al. Metabolic engineering of Escherichia coli for efficient production of ectoine [J]. J Agric Food Chem, 2025, 73(1): 646-654.
[4]	Chen JZ, Wang Y, Guo X, et al. Efficient bioproduction of 5-aminolevulinic acid, a promising biostimulant and nutrient, from renewable bioresources by engineered Corynebacterium glutamicum [J]. Biotechnol Biofuels, 2020, 13: 41.
[5]	Das S, Sharma K, Sharmmah D, et al. Metabolic rewiring of microbial cell factories for improved production of succinic acid [J]. Biotechnol Sustain Mater, 2024, 1(1): 15.
[6]	Nevoigt E, Kohnke J, Fischer CR, et al. Engineering of promoter replacement cassettes for fine-tuning of gene expression in Saccharomyces cerevisiae [J]. Appl Environ Microbiol, 2006, 72(8): 5266-5273.
[7]	Lata H, Sharma A, Chadha S, et al. RNA interference (RNAi) mechanism and application in vegetable crops [J]. J Hortic Sci Biotechnol, 2022, 97(2): 160-170.
[8]	Tang Q, Khvorova A. RNAi-based drug design: considerations and future directions [J]. Nat Rev Drug Discov, 2024, 23(5): 341-364.
[9]	Zheng Y, Shen W, Zhang J, et al. CRISPR interference-based specific and efficient gene inactivation in the brain [J]. Nat Neurosci, 2018, 21(3): 447-454.
[10]	Izert MA, Klimecka MM, Górna MW. Applications of bacterial degrons and degraders - toward targeted protein degradation in bacteria [J]. Front Mol Biosci, 2021, 8: 669762.
[11]	Sabi R, Tuller T. Modelling the efficiency of codon-tRNA interactions based on codon usage bias [J]. DNA Res, 2014, 21(5): 511-526.
[12]	Zhang SY, Lin R, Cui LY, et al. Alter codon bias of the P. pastoris genome to overcome a bottleneck in codonoptimization strategy development and improve protein expression [J]. Microbiol Res, 2024, 282: 127629.
[13]	Fu HG, Liang YB, Zhong XQ, et al. Codonoptimization with deep learning to enhance protein expression [J]. Sci Rep, 2020, 10(1): 17617.
[14]	Xu YC, Liu KS, Han Y, et al. Codon usage bias regulates gene expression and protein conformation in yeast expression system P. pastoris [J]. Microb Cell Fact, 2021, 20(1): 91.
[15]	Zhang XY, Xi ZW, Zhao HT, et al. Efficient heterologous expression of bovine lactoferrin in Pichia pastoris and characterization of antibacterial activity [J]. Syst Microbiol Biomanuf, 2025, 5(1): 237-248.
[16]	Zhang JL, Kang Z, Chen J, et al. Optimization of the heme biosynthesis pathway for the production of 5-aminolevulinic acid in Escherichia coli [J]. Sci Rep, 2015, 5: 8584.
[17]	Jiang MR, Hong KQ, Mao YF, et al. Natural 5-aminolevulinic acid: sources, biosynthesis, detection and applications [J]. Front Bioeng Biotechnol, 2022, 10: 841443.
[18]	Wu Y, Liao WB, Dawuda MM, et al. 5-Aminolevulinic acid (ALA) biosynthetic and metabolic pathways and its role in higher plants: a review [J]. Plant Growth Regul, 2019, 87(2): 357-374.
[19]	Wang JW, He YM, Wang GZ, et al. Exogenous 5-aminolevulinic acid promotes carotenoid accumulation in tomato fruits by regulating ethylene biosynthesis and signaling [J]. Physiol Plant, 2024, 176(6): e14648.
[20]	Casas A. Clinical uses of 5-aminolaevulinic acid in photodynamic treatment and photodetection of cancer: a review [J]. Cancer Lett, 2020, 490: 165-173.
[21]	Yadav V, Mai Y, McCoubrey LE, et al. 5-aminolevulinic acid as a novel therapeutic for inflammatory bowel disease [J]. Biomedicines, 2021, 9(5): 578.
[22]	Zou YL, Chen T, Feng LL, et al. Enhancement of 5-aminolevulinic acid production by metabolic engineering of the Glycine biosynthesis pathway in Corynebacterium glutamicum [J]. Biotechnol Lett, 2017, 39(9): 1369-1374.
[23]	Ding WW, Weng HJ, Du GC, et al. 5-Aminolevulinic acid production from inexpensive glucose by engineering the C4 pathway in Escherichia coli [J]. J Ind Microbiol Biotechnol, 2017, 44(8): 1127-1135.
[24]	Ramzi AB, Hyeon JE, Kim SW, et al. 5-Aminolevulinic acid production in engineered Corynebacterium glutamicum via C5 biosynthesis pathway [J]. Enzyme Microb Technol, 2015, 81: 1-7.
[25]	Luo ZS, Pan F, Zhu YF, et al. Synergistic improvement of 5-aminolevulinic acid production with synthetic scaffolds and system pathway engineering [J]. ACS Synth Biol, 2022, 11(8): 2766-2778.
[26]	Erskine PT, Senior N, Maignan S, et al. Crystallization of 5-aminolaevulinic acid dehydratase from Escherichia coli and Saccharomyces cerevisiae and preliminary X-ray characterization of the crystals [J]. Protein Sci, 1997, 6(8): 1774-1776.
[27]	Miscevic D, Mao JY, Kefale T, et al. Strain engineering for high-level 5-aminolevulinic acid production in Escherichia coli [J]. Biotechnol Bioeng, 2021, 118(1): 30-42.
[28]	Replogle JM, Bonnar JL, Pogson AN, et al. Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors [J]. eLife, 2022, 11: e81856.
[29]	Pu W, Chen JZ, Zhou YY, et al. Systems metabolic engineering of Escherichia coli for hyper-production of 5-aminolevulinic acid [J]. Biotechnol Biofuels Bioprod, 2023, 16(1): 31.
[30]	Zhang J, Yang FY, Yang YP, et al. Optimizing a CRISPR-Cpf1-based genome engineering system for Corynebacterium glutamicum [J]. Microb Cell Fact, 2019, 18(1): 60.
[31]	Cardiff RAL, Carothers JM, Zalatan JG, et al. Systems-level modeling for CRISPR-based metabolic engineering [J]. ACS Synth Biol, 2024, 13(9): 2643-2652.
[32]	Cui SX, Lv XQ, Xu XH, et al. Multilayer genetic circuits for dynamic regulation of metabolic pathways [J]. ACS Synth Biol, 2021, 10(7): 1587-1597.
[33]	Ikemura T. Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system [J]. J Mol Biol, 1981, 151(3): 389-409.
[34]	Gustafsson C, Govindarajan S, Minshull J. Codon bias and heterologous protein expression [J]. Trends Biotechnol, 2004, 22(7): 346-353.
[35]	Kim S, Lee SB. Rare codon clusters at 5'-end influence heterologous expression of archaeal gene in Escherichia coli [J]. Protein Expr Purif, 2006, 50(1): 49-57.

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

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