非天然氨基酸的应用及生物合成策略

doi:10.13560/j.cnki.biotech.bull.1985.2023-0648

摘要/Abstract

摘要：

非天然氨基酸是一类不受遗传密码子约束的特殊氨基酸，它们具有独特的空间构型和化学性质，为多个领域提供了新的资源和机会。当前，非天然氨基酸可以通过化学合成和生物合成两种方式合成，其中相较于化学合成，生物合成具有更高的效率和立体选择性、更低的成本和污染，是一种有潜力的合成方式。本文综述了非天然氨基酸在蛋白质探针、酶工程、抗体药物偶联物、抗菌肽等方面的研究进展和应用案例，展示了非天然氨基酸如何突破天然氨基酸的限制，拓展蛋白质的结构和功能多样性。同时介绍了非天然氨基酸的生物合成策略，包括代谢工程中的静态调控和动态调控策略以及发酵优化策略。最后，展望了非天然氨基酸的生物合成和应用的未来发展趋势和挑战，为非天然氨基酸的合成和开发提供参考和启示。

关键词: 非天然氨基酸, 生物合成, 代谢工程, 非天然蛋白质

Abstract:

Unnatural amino acids（UAAs）are a special class of amino acids that are not constrained by genetic codons. They have unique spatial configurations and chemical properties, which provide new resources and opportunities in several fields. Currently, UAAs can be synthesized by both chemical synthesis and biosynthesis. Compared with chemical synthesis, biosynthesis is a promising synthesis with higher efficiency and stereoselectivity, lower cost and pollution. This paper reviews the research progress and application cases of UAAs in protein probes, enzyme engineering, antibody-drug conjugates, and antimicrobial peptides, etc. It shows how UAAs break through the limitations of natural amino acids and expand the structural and functional diversity of proteins. It also introduces the biosynthesis strategies of UAAs, including static and dynamic regulatory strategies in metabolic engineering and fermentation optimization strategies. Finally, it looks forward to the future development trends and challenges in the biosynthesis and application of unnatural amino acids, which will provide a reference and inspiration for the UAAs synthesis and development.

Key words: unnatural amino acids, biosynthesis, metabolic engineering, unnatural protein

李海宁, 张红兵, 耿革霞, 李冉, 贾振华. 非天然氨基酸的应用及生物合成策略[J]. 生物技术通报, 2023, 39(12): 43-55.

LI Hai-ning, ZHANG Hong-bing, GENG Ge-xia, LI Ran, JIA Zhen-hua. Application and Biosynthesis Strategies of Unnatural Amino Acids[J]. Biotechnology Bulletin, 2023, 39(12): 43-55.

图/表 5

参考文献 62

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非天然氨基酸 UAA	合成方式 Method of synthesis	应用 Application	使用方式 Mode of use	参考文献 Reference
L-苯甘氨酸 L-phenylghycine	化学合成 Chemical synthesis	合成紫杉醇及β-内酰胺类抗生素重要中间体 Important intermediates for the synthesis of paclitaxel and β-lactam antibiotics	药物中间体 Pharmaceutical intermediates	[28]
（S）-2-环丙基甘氨酸（S）-2-cyclopropylglycine	化学合成、生物合成 Chemical synthesis and biosynthesis	设计酶抑制剂（大环饥饿激素受体拮抗剂）抗菌、抗癌、止痛等Design enzyme inhibitor（macrocyclic starvation hormone receptor antagonist）for antibacteria, anticancer, analgesic, etc	多肽类似物、药物中间体Peptide analogues，pharmaceutical intermediates	[29]
L-2-氨基丁酸 L-2-aminobutyric acid	生物合成 Biosynthesis	重磅级抗癫痫药物左乙拉西坦、布瓦西坦和抗结核乙胺丁醇等关键中间体 Key intermediate for heavyweight anti-epileptic drugs levetiracetam, bupropion and anti-tuberculosis ethambutol	药物中间体 Pharmaceutical intermediates	[30]
N-乙酰-L-苯丙氨酸N-acetyl-L-phenylalanine	化学合成 Chemical synthesis	定点偶联单克隆抗体，杀死肿瘤细胞 Targeted coupling of monoclonal antibodies to kill tumor cells	抗体药物偶联物 Antibody-drug conjugates	[31]
γ-氨基丁酸 γ-aminobutyric acid	生物合成 Biosynthesis	神经递质的一种，脑内抑制性递质、镇静神经、抗焦虑、降血压A kind of neurotransmitter, inhibitory transmitter in the brain, calming nerve, anti-anxiety, lowering blood pressure	游离氨基酸 Free amino acids	[32]
5-氨基乙酰丙酸 5-aminolevulinic acid	生物合成 Biosynthesis	血红素、卟啉、叶绿素、维生素B₁₂重要前体，具有生物可降解、无毒无残留优点 Important precursors of heme, porphyrin, chlorophyll and vitamin B₁₂, with biodegradable, non-toxic and non-residual advantages	游离氨基酸 Free amino acids	[33]
L-2,3-二氨基丙酸 L-2,3-diaminopropionic acid	化学合成 Chemical synthesis	神经毒素的直接前体，多种抗生素合成的关键物质Direct precursor of neurotoxin, and key substance for the synthesis of many antibiotics	活性药物成分 Active pharmaceutical ingredients	[30]
4-氟脯氨酸 4-fluoroproline	化学合成 Chemical synthesis	应用于蛋白质设计与工程，增强蛋白质构象稳定性 Application to protein design and engineering for enhancing protein conformational stability	插入蛋白质 Insertion of proteins	[1]
N-乙酰化氟苯丙氨酸N-acetylated fluorophenylalanine	化学合成 Chemical synthesis	抗病毒、抗癌药物结合物 Anti-viral and anti-cancer drug conjugates	抗体药物偶联物 Antibody-drug conjugates	[34]
4-羟基苯基甘氨酸 4-hydroxy-phenylglycine	化学合成 Chemical synthesis	合成治疗心肌梗死药物 Synthetic drugs for myocardial infarction	游离氨基酸 Free amino acids	[35]

非天然氨基酸 UAA	合成方式 Method of synthesis	应用 Application	使用方式 Mode of use	参考文献 Reference
L-苯甘氨酸 L-phenylghycine	化学合成 Chemical synthesis	合成紫杉醇及β-内酰胺类抗生素重要中间体 Important intermediates for the synthesis of paclitaxel and β-lactam antibiotics	药物中间体 Pharmaceutical intermediates	[28]
（S）-2-环丙基甘氨酸（S）-2-cyclopropylglycine	化学合成、生物合成 Chemical synthesis and biosynthesis	设计酶抑制剂（大环饥饿激素受体拮抗剂）抗菌、抗癌、止痛等Design enzyme inhibitor（macrocyclic starvation hormone receptor antagonist）for antibacteria, anticancer, analgesic, etc	多肽类似物、药物中间体Peptide analogues，pharmaceutical intermediates	[29]
L-2-氨基丁酸 L-2-aminobutyric acid	生物合成 Biosynthesis	重磅级抗癫痫药物左乙拉西坦、布瓦西坦和抗结核乙胺丁醇等关键中间体 Key intermediate for heavyweight anti-epileptic drugs levetiracetam, bupropion and anti-tuberculosis ethambutol	药物中间体 Pharmaceutical intermediates	[30]
N-乙酰-L-苯丙氨酸N-acetyl-L-phenylalanine	化学合成 Chemical synthesis	定点偶联单克隆抗体，杀死肿瘤细胞 Targeted coupling of monoclonal antibodies to kill tumor cells	抗体药物偶联物 Antibody-drug conjugates	[31]
γ-氨基丁酸 γ-aminobutyric acid	生物合成 Biosynthesis	神经递质的一种，脑内抑制性递质、镇静神经、抗焦虑、降血压A kind of neurotransmitter, inhibitory transmitter in the brain, calming nerve, anti-anxiety, lowering blood pressure	游离氨基酸 Free amino acids	[32]
5-氨基乙酰丙酸 5-aminolevulinic acid	生物合成 Biosynthesis	血红素、卟啉、叶绿素、维生素B₁₂重要前体，具有生物可降解、无毒无残留优点 Important precursors of heme, porphyrin, chlorophyll and vitamin B₁₂, with biodegradable, non-toxic and non-residual advantages	游离氨基酸 Free amino acids	[33]
L-2,3-二氨基丙酸 L-2,3-diaminopropionic acid	化学合成 Chemical synthesis	神经毒素的直接前体，多种抗生素合成的关键物质Direct precursor of neurotoxin, and key substance for the synthesis of many antibiotics	活性药物成分 Active pharmaceutical ingredients	[30]
4-氟脯氨酸 4-fluoroproline	化学合成 Chemical synthesis	应用于蛋白质设计与工程，增强蛋白质构象稳定性 Application to protein design and engineering for enhancing protein conformational stability	插入蛋白质 Insertion of proteins	[1]
N-乙酰化氟苯丙氨酸N-acetylated fluorophenylalanine	化学合成 Chemical synthesis	抗病毒、抗癌药物结合物 Anti-viral and anti-cancer drug conjugates	抗体药物偶联物 Antibody-drug conjugates	[34]
4-羟基苯基甘氨酸 4-hydroxy-phenylglycine	化学合成 Chemical synthesis	合成治疗心肌梗死药物 Synthetic drugs for myocardial infarction	游离氨基酸 Free amino acids	[35]

非天然氨基酸 UAA	生产菌株Strain	改造策略 Modifying strategies	发酵方式 Fermentation method	产量 Titer/（g·L^-1）	参考文献 Reference
5-ALA	E. coli E. coli C. glutamicum C. glutamicum	过表达ALAS，敲除ldhA、sdhA iclR，下调hemB Overexpression of ALAS, deletion of ldhA, sdhA and iclR, downregulation of hemB 过表达KatE和SodB 强化抗氧化防御系统 Reinforcing the antioxidant defense system by expression KatE and SodB 过表达rhtA，敲除sucCD Overexpression of rhtA, and deletion of sucCD 过表达ppc Overexpression of ppc	摇瓶发酵 Shake flask 摇瓶发酵 Shake flask 分批补料 Fed-batch 分批补料 Fed-batch	6.93 11.50 14.70 16.30	[54-55] [55] [56] [57]
4-HIL	C. glutamicum C. glutamicum E. coli	过表达双加氧酶基因ido和ppc基因 Overexpression of double oxygenase gene ido and ppc 整合Lys-OFF核糖开关到dapA上游，并利用Ile激活型传感器Lrp-P_brnFEN控制ido表达，利用强启动子P_brnFE7动态控制odh I和vgb表达 Integrate the Lys-OFF riboswitch to the upstream of dapA and control ido expression using the Ile-activated sensor Lrp-P_brnFEN and dynamically control odh I and vgb expression using the strong promoter P_brnFE7 敲除α-酮戊二酸脱氢酶基因sucAB,及异柠檬酸脱氢酶激酶基因aceAK Deletion of sucAB 和aceAK	摇瓶发酵 Shake flask 摇瓶发酵 Shake flask 摇瓶发酵 Shake flask	14.10 24.40 24.10	[39] [49] [58]
L-ABA	E. coli E. coli	突变ilvA基因从而减弱ilvA受到的反馈抑制 Mutating the ilvA gene, and downregulating the feedback inhibition of ilvA 引入定点饱和突变的苏氨酸脱氨酶分子，敲除ilvH基因，对苏氨酸脱氨酶和亮氨酸脱氢酶进行协同表达调控 Introduction of a fixed-point saturated and mutated threonine deaminase molecule, deletion of the ilvH gene, and co-expression regulation of threonine deaminase and leucine dehydrogenase	摇瓶发酵 Shake flask 摇瓶发酵 Shake flask	5.39 4.86	[59] [59]
L-HS	E. coli E. coli E. coli	过表达高丝氨酸脱氢酶 I基因thrA Overexpression of thrA 过表达高丝氨酸脱氢酶II基因metL Overexpression metL 过表达高丝氨酸外排蛋白编码基因rhtA及敲除tdcC基因 Overexpression of rhtA and deletion of tdcC	分批补料 Fed-batch 分批补料 Fed-batch 分批补料 Fed-batch	1.20 1.04 39.50	[60] [60] [60]
Trans-4-hydroxy-L-proline	E. coli	引入T7 RNA聚合酶，敲除L-脯氨酸基因putA，整合L-脯氨酸-4-羟基化酶基因 Introduction of T7 RNA polymerase, deletion of putA and integration of the L-proline-4-hydroxylase gene	分批补料 Fed-batch	48.60	[53]
5-HTP	E. coli	定向进化XcP4H与辅酶MH4再生系统共表达 Directed evolution of XcP4H co-expressed with the auxin MH4 regeneration system	摇瓶发酵 Shake flask	1.11	[61]
L-DOPA	E. coli	用半乳糖渗透酶基因galP和葡萄糖激酶基因glk替代PTS功能 Replacement of PTS function with galP and glk	分批补料 Fed-batch	1.51	[62]

非天然氨基酸 UAA	生产菌株Strain	改造策略 Modifying strategies	发酵方式 Fermentation method	产量 Titer/（g·L^-1）	参考文献 Reference
5-ALA	E. coli E. coli C. glutamicum C. glutamicum	过表达ALAS，敲除ldhA、sdhA iclR，下调hemB Overexpression of ALAS, deletion of ldhA, sdhA and iclR, downregulation of hemB 过表达KatE和SodB 强化抗氧化防御系统 Reinforcing the antioxidant defense system by expression KatE and SodB 过表达rhtA，敲除sucCD Overexpression of rhtA, and deletion of sucCD 过表达ppc Overexpression of ppc	摇瓶发酵 Shake flask 摇瓶发酵 Shake flask 分批补料 Fed-batch 分批补料 Fed-batch	6.93 11.50 14.70 16.30	[54-55] [55] [56] [57]
4-HIL	C. glutamicum C. glutamicum E. coli	过表达双加氧酶基因ido和ppc基因 Overexpression of double oxygenase gene ido and ppc 整合Lys-OFF核糖开关到dapA上游，并利用Ile激活型传感器Lrp-P_brnFEN控制ido表达，利用强启动子P_brnFE7动态控制odh I和vgb表达 Integrate the Lys-OFF riboswitch to the upstream of dapA and control ido expression using the Ile-activated sensor Lrp-P_brnFEN and dynamically control odh I and vgb expression using the strong promoter P_brnFE7 敲除α-酮戊二酸脱氢酶基因sucAB,及异柠檬酸脱氢酶激酶基因aceAK Deletion of sucAB 和aceAK	摇瓶发酵 Shake flask 摇瓶发酵 Shake flask 摇瓶发酵 Shake flask	14.10 24.40 24.10	[39] [49] [58]
L-ABA	E. coli E. coli	突变ilvA基因从而减弱ilvA受到的反馈抑制 Mutating the ilvA gene, and downregulating the feedback inhibition of ilvA 引入定点饱和突变的苏氨酸脱氨酶分子，敲除ilvH基因，对苏氨酸脱氨酶和亮氨酸脱氢酶进行协同表达调控 Introduction of a fixed-point saturated and mutated threonine deaminase molecule, deletion of the ilvH gene, and co-expression regulation of threonine deaminase and leucine dehydrogenase	摇瓶发酵 Shake flask 摇瓶发酵 Shake flask	5.39 4.86	[59] [59]
L-HS	E. coli E. coli E. coli	过表达高丝氨酸脱氢酶 I基因thrA Overexpression of thrA 过表达高丝氨酸脱氢酶II基因metL Overexpression metL 过表达高丝氨酸外排蛋白编码基因rhtA及敲除tdcC基因 Overexpression of rhtA and deletion of tdcC	分批补料 Fed-batch 分批补料 Fed-batch 分批补料 Fed-batch	1.20 1.04 39.50	[60] [60] [60]
Trans-4-hydroxy-L-proline	E. coli	引入T7 RNA聚合酶，敲除L-脯氨酸基因putA，整合L-脯氨酸-4-羟基化酶基因 Introduction of T7 RNA polymerase, deletion of putA and integration of the L-proline-4-hydroxylase gene	分批补料 Fed-batch	48.60	[53]
5-HTP	E. coli	定向进化XcP4H与辅酶MH4再生系统共表达 Directed evolution of XcP4H co-expressed with the auxin MH4 regeneration system	摇瓶发酵 Shake flask	1.11	[61]
L-DOPA	E. coli	用半乳糖渗透酶基因galP和葡萄糖激酶基因glk替代PTS功能 Replacement of PTS function with galP and glk	分批补料 Fed-batch	1.51	[62]