5-氨基乙酰丙酸(5-aminolevulinic acid,5-ALA)是血红素和叶绿素等四氢吡咯化合物合成的前体,在医药和农业等领域有着广泛应用,构建带有高活性5-氨基乙酰丙酸合酶(5-aminolevulinic acid synthase,ALAS)的细胞工厂是生物合成5-ALA的重要方法,但目前针对ALAS的改造研究较少。该研究对来源于荚膜红杆菌(Rhodobacter capsulatus)的ALAS进行改造,通过定点突变ALAS活性位点周围的氨基酸残基,筛选得到酶活力提高1.5倍的突变体RC/I361V,证明通过突变ALAS活性点的氨基酸来改变其结构特征和空间构象可以实现酶活力的提高,对ALAS的改造提供了新的设计思路。进一步围绕甘氨酸代谢途径对大肠杆菌(Escherichia coli)W3110(DE3)进行代谢改造,通过突变降低甘氨酸裂解酶系H蛋白的活力,敲除编码甘氨酸C-乙酰转移酶的基因(kbL)、编码丙酮酸脱氢酶基因(poxB),降低了细胞宿主对甘氨酸的消耗,显著提高了E.coli合成5-ALA的能力。将筛选到的突变体RC/I361V转入改造后的宿主S165细胞中,经摇瓶发酵后5-ALA的产量达到2.01 g/L,为5-ALA合成中甘氨酸的有效利用提供了思路。
5-aminolevulinic acid (5-ALA) is a precursor for the synthesis of tetrahydropyrrole compounds such as heme and chlorophyll, and it has wide applications in fields such as medicine and agriculture.Constructing cell factories with highly active 5-aminolevulinic acid synthase (ALAS) is an important method for the biological synthesis of 5-ALA.But there is currently limited research on the modification of ALAS.In this study, the ALAS derived from Rhodobacter capsulatus was modified by site-directed mutagenesis of the amino acid residues around the active site of ALAS.Thus, a mutant RC/I361V with a 1.5-fold increase in enzyme activity was screened.It was demonstrated that changing the structural characteristics and spatial conformation of the ALAS enzyme by mutating the amino acids at the ALAS active site can achieve an increase in enzyme activity, provided a new design approach for ALAS modification.Furthermore, metabolic modification of Escherichia coli W3110 (DE3) was carried out around the glycine metabolism pathway, by mutating to reduce the activity of the glycine cleavage system H protein, knocking out the genes encoding glycine C-acetyltransferase (kbL) and pyruvate dehydrogenase (poxB), reducing the consumption of glycine by the host cell.Then the screened mutant RC/I361V was transferred into the modified host cell S165.The results showed that the genetically modified strain could increase the synthetic ability of 5-ALA, and the yield of 5-ALA reached 2.01 g/L after shake flask fermentation, which would provide new insights into for the effective utilization of glycine in 5-ALA synthesis.
[1] 安玉涵, 马伊丹, 杨佳, 等.5-氨基乙酰丙酸(5-ALA)的生物学作用及其在动物生产中的应用[J].饲料研究, 2023, 46(19):150-154.
AN Y H, MA Y D, YANG J, et al.Biological function of 5-aminolevulinic acid(5-ALA) and its application in animals production[J].Feed Research, 2023, 46(19):150-154.
[2] 高艳霞, 刘睿, 刘南南, 等.5-氨基乙酰丙酸及其前体药物介导的光动力疗法在恶性肿瘤治疗中的应用研究进展[J].山东医药, 2022, 62(8):90-94.
GAO Y X, LIU R, LIU N N, et al.Progress in the application of 5- aminolevulinic acid and its prodrug mediated photodynamic therapy in the treatment of malignant tumors[J].Shandong Medical Journal, 2022, 62(8):90-94.
[3] 魏敏华, 李宇虹, 张佳蓉, 等.代谢工程构建谷氨酸棒杆菌合成5-氨基乙酰丙酸[J].食品与发酵工业, 2022, 48(14):9-15.
WEI M H, LI Y H, ZHANG J R, et al.Metabolic engineering of Corynebacterium glutamicum chassis for biosynthesis of 5-aminolevulinic acid[J].Food and Fermentation Industries, 2022, 48(14):9-15.
[4] 王帅, 张乐乐, 刘恩昊, 等.5-氨基乙酰丙酸的生物合成及应用研究进展[J].天津化工, 2023, 37(S2):77-81.
WANG S, ZHANG L L, LIU E H, et al.Progress in biosynthesis and application of 5-aminolevulinic acid[J].Tianjin Chemical Industry, 2023, 37(S2):77-81.
[5] DING W W, WENG H J, DU G C, et al.5-Aminolevulinic acid production from inexpensive glucose by engineering the C4 pathway in Escherichia coli[J].Journal of Industrial Microbiology & Biotechnology, 2017, 44(8):1127-1135.
[6] CHRISTEN P, MEHTA P K, SANDMEIER E.Molecular evolution of pyridoxal-5′-phosphate-dependent enzymes[C].Biochemistry of Vitamin B6 and PQQ.Basel:Springer, 1994:9-13.
[7] MEHTA P K, CHRISTEN P.Homology of 1-aminocyclopropane-1-carboxylate synthase, 8-amino-7-oxononanoate synthase, 2-amino-6-caprolactam racemase, 2, 2-dialkylglycine decarboxylase, glutamate-1-semialdehyde 2, 1-aminomutase and isopenicillin-N-epimerase with aminotransferases[J].Biochemical and Biophysical Research Communications, 1994, 198(1):138-143.
[8] FERREIRA G C, NEAME P J, DAILEY H A.Heme biosynthesis in mammalian systems:Evidence of a Schiff base linkage between the pyridoxal 5′-phosphate cofactor and a lysine residue in 5-aminolevulinate synthase[J].Protein Science, 1993, 2(11):1959-1965.
[9] KAUFHOLZ A L, HUNTER G A, FERREIRA G C, et al.Aminolaevulinic acid synthase of Rhodobacter capsulatus:High-resolution kinetic investigation of the structural basis for substrate binding and catalysis[J].The Biochemical Journal, 2013, 451(2):205-216.
[10] HE G M, JIANG M R, CUI Z Z, et al.Construction of 5-aminolevulinic acid synthase variants by cysteine-targeted mutation to release heme inhibition[J].Journal of Bioscience and Bioengineering, 2022, 134(5):416-423.
[11] DU S, ZHENG N, ZHANG Z H, et al.Rational design engineering of 5-aminolevulinate synthase with activity and stability enhancement[J].Journal of Agricultural and Food Chemistry, 2025, 73(3):1892-1901.
[12] 陈久洲, 王钰, 蒲伟, 等.5-氨基乙酰丙酸生物合成技术的发展及展望[J].合成生物学, 2021, 2(6):1000-1016.
CHEN J Z, WANG Y, PU W, et al.Advances and perspective on bioproduction of 5-aminolevulinic acid[J].Synthetic Biology Journal, 2021, 2(6):1000-1016.
[13] WANG Q, JIA M J, LI H J, et al.Design of a genetically encoded biosensor for high-throughput screening and engineering 5-aminolevulinic acid hyper-producing Escherichia coli[J].ACS Sustainable Chemistry & Engineering, 2024, 12(12):4846-4857.
[14] ZHOU H M, ZHANG C Y, LI Z L, et al.Systematic development of a highly efficient cell factory for 5-aminolevulinic acid production[J].Trends in Biotechnology, 2024, 42(11):1479-1502.
[15] MAUZERALL D, GRANICK S.The occurrence and determination of delta-amino-levulinic acid and porphobilinogen in urine[J].The Journal of Biological Chemistry, 1956, 219(1):435-446.
[16] ZHANG H, LI Y C, NIE J L, et al.Structure-based dynamic analysis of the glycine cleavage system suggests key residues for control of a key reaction step[J].Communications Biology, 2020, 3:756.
[17] ASTNER I, SCHULZE J O, VAN DEN HEUVEL J, et al.Crystal structure of 5-aminolevulinate synthase, the first enzyme of heme biosynthesis, and its link to XLSA in humans[J].The EMBO Journal, 2005, 24(18):3166-3177.
[18] 蒲伟, 陈久洲, 孙村民, 等.琥珀酸脱氢酶或琥珀酰辅酶A合成酶缺失促进大肠杆菌积累5-氨基乙酰丙酸[J].生物工程学报, 2013, 29(10):1494-1503.
PU W, CHEN J Z, SUN C M, et al.Deficiency of succinic dehydrogenase or succinyl-CoA synthetase enhances the production of 5-aminolevulinic acid in recombinant Escherichia coli[J].Chinese Journal of Biotechnology, 2013, 29(10):1494-1503.
[19] 李凤辰, 段纪甫, 宫衡.氨基酸对大肠杆菌BL21生长影响的研究[J].工业微生物, 2013, 43(3):29-31.
LI F C, DUAN J F, GONG H.Effects of amino acid on growth of Escherichia coli BL21[J].Industrial Microbiology, 2013, 43(3):29-31.
