核糖体结合位点(ribosome binding site, RBS)是一种重要的生物控制元件,是实现基因表达精细调控的重要工具,在微生物代谢工程和合成生物学中具有广泛的应用。谷氨酸棒杆菌(Corynebacterium glutamicum)是一种重要的工业微生物,被广泛应用于多种氨基酸和有机酸的工业化发酵生产。然而目前,关于谷氨酸棒杆菌RBS元件文库的报道仍然较少。该研究基于谷氨酸棒杆菌RBS序列基本特征,设计构建了一个随机RBS文库。利用流式细胞分选技术和96孔板筛选技术,建立了RBS文库高通量筛选方法。通过2步筛选,构建了一个调控范围广,覆盖范围均匀的人工RBS元件文库,调控范围达到29.04倍。利用α-淀粉酶表达系统对构建的人工RBS文库进行测试和表征,结果表明人工RBS文库具有较高的稳定性和通用性。随后该研究将人工RBS文库应用于L-高丝氨酸生物合成途径的调控优化。利用不同强度RBS元件对L-高丝氨酸合成途径关键酶LysCr和Homr进行精细表达调控,获得了具有高效L-高丝氨酸合成能力的最佳组合,L-高丝氨酸产量到达12.7 g/L,比野生型菌株高丝氨酸含量提高了3.6倍。综上所述,该研究构建了一个调控范围广、稳定性高的谷氨酸棒杆菌人工RBS文库,为谷氨酸棒杆菌代谢工程改造和基因精细表达调控提供了有效的控制元件。
Ribosome binding site (RBS) is an important biological control element for fine-regulation of genes expression, which has been widely applied in microbial metabolic engineering and synthetic biology. Corynebacterium glutamicum is an important industrial microorganism for industrial fermentation of several amino acids and organic acids. However, there were few researches on the RBS element for C. glutamicum. In this study, a random RBS library was designed and constructed based on the common RBS characteristics in C. glutamicum. Then, a high-throughput screening technology was developed by coupling flow cytometry and 96-well plate screening technology for screening of the artificial RBS library. After two-step screening, an artificial RBS element library with wide range of regulation and uniform coverage was constructed, and the regulation strength range of the library was 29.04-fold. Next, the artificial RBS library was further characterized by α-amylase expression system, and the results showed that the RBS library had high stability and versatility. Subsequently, the artificial library was applied for the regulation and optimization of the L-homoserine biosynthetic pathway. The expressions of LysCr and Homr, the key enzymes in L-homoserine synthesis pathway, were fine-regulated using RBS elements with different intensities. Finally, the best combination of LysCr and Homr for L-homoserine synthesis was obtained, and the resulted strain accumulated 12.7 g/L of L-homoserine, which was 3.6 times higher than that of the wild-type strain. Therefore, an artificial RBS library was constructed and exhibited wide regulation range and high stability in this study, which provided the effective control elements for metabolic engineering and gene expression regulation in C. glutamicum.
[1] 严伟, 信丰学, 董维亮, 等.合成生物学及其研究进展[J].生物学杂志, 2020, 37(5):1-9.
YAN W, XIN F X, DONG W L, et al.Synthetic biology and research progress[J].Journal of Biology, 2020, 37(5):1-9.
[2] CHEN Y, BANERJEE D, MUKHOPADHYAY A, et al.Systems and synthetic biology tools for advanced bioproduction hosts[J].Current Opinion in Biotechnology, 2020, 64:101-109.
[3] HANCOCK J M.Ribosome Binding Site (RBS)[M].New York:American Cancer Society, 2014:1-14.
[4] 刘莫识, 刘娇, 孙冠男, 等.谷氨酸棒杆菌人工合成启动子文库的构建及应用[J].生物工程学报, 2022, 38(2):831-842.
LIU M S, LIU J, SUN G N, et al.Construction and application of a synthetic promoter library for Corynebacterium glutamicum[J].Chinese Journal of Biotechnoligy, 2022, 38(2):831-842.
[5] KINOSHITA S, NAKAYAMA K, AKITA S.Taxonomical study of glutamic acid accumulating bacteria, Micrococcus glutamicus nov.sp[J].Bulletin of the Agricultural Chemical Society of Japan, 1958, 22(3):176-185.
[6] MCKINLAY J B, VIEILLE C, ZEIKUS J G.Prospects for a bio-based succinate industry[J].Applied Microbiology and Biotechnology, 2007, 76(4):727-740.
[7] NIIMI S, SUZUKI N, INUI M, et al.Metabolic engineering of 1, 2-propanediol pathways in Corynebacterium glutamicum[J].Applied Microbiology and Biotechnology, 2011, 90(5):1721-1729.
[8] SONG Y Y, MATSUMOTO K, YAMADA M, et al.Engineered Corynebacterium glutamicum as an endotoxin-free platform strain for lactate-based polyester production[J].Applied Microbiology and Biotechnology, 2012, 93(5):1917-1925.
[9] EGGELING L, BOTT M.A giant market and a powerful metabolism:L-lysine provided by Corynebacterium glutamicum[J].Applied Microbiology and Biotechnology, 2015, 99(8):3387-3394.
[10] HENKE N A, WIEBE D, PÉREZ-GARCÍA F, et al.Coproduction of cell-bound and secreted value-added compounds:Simultaneous production of carotenoids and amino acids by Corynebacterium glutamicum[J].Bioresource Technology, 2018, 247:744-752.
[11] WEI L, XU N, WANG Y R, et al.Promoter library-based module combination (PLMC) technology for optimization of threonine biosynthesis in Corynebacterium glutamicum[J].Applied Microbiology and Biotechnology, 2018, 102(9):4117-4130.
[12] WEI L, ZHAO J H, WANG Y R, et al.Engineering of Corynebacterium glutamicum for high-level γ-aminobutyric acid production from glycerol by dynamic metabolic control[J].Metabolic Engineering, 2022, 69:134-146.
[13] WANG Y, CHENG H J, LIU Y, et al.In-situ generation of large numbers of genetic combinations for metabolic reprogramming via CRISPR-guided base editing[J].Nature Communications, 2021, 12(1):678.
[14] ZHANG B, ZHOU N, LIU Y M, et al.Ribosome binding site libraries and pathway modules for shikimic acid synthesis with Corynebacterium glutamicum[J].Microbial Cell Factories, 2015, 14(1):1-14.
[15] 朱海霞, 石瑛, 张庆娜, 等.3, 5-二硝基水杨酸(DNS)比色法测定马铃薯还原糖含量的研究[J].中国马铃薯, 2005, 19(5):266-269.
ZHU H X, SHI Y, ZHANG Q N, et al.Applying 3, 5-dinitrosalicylic acid(DNS) method to analyzing the content of potato reducing sugar[J].Chinese Potato, 2005, 19(5):266-269.
[16] 赵金花, 魏亮, 徐宁, 等.双酶偶联法测定发酵液中L-高丝氨酸的含量[J].食品与发酵工业, 2023, 49(2):239-245.
ZHAO J H, WEI L, XU N, et al.Determination of L-homoserine in fermentation broth by double enzyme coupling method[J].Food and Fermentation Industries, 2023, 49(2):239-245.
[17] PFEIFER-SANCAR K, MENTZ A, RÜCKERT C, et al.Comprehensive analysis of the Corynebacterium glutamicum transcriptome using an improved RNAseq technique[J].BMC Genomics, 2013, 14:888.
[18] JEON J, LEE J Y, LEE C, et al.L-Glufosinate intermediate and L-glufosinate preparation method:US, US2022024955A1[P].2022-01-27.
[19] HONG K K, KIM J H, YOON J H, et al.O-succinyl-L-homoserine-based C4-chemical production:Succinic acid, homoserine lactone, γ-butyrolactone, γ-butyrolactone derivatives, and 1, 4-butanediol[J].Journal of Industrial Microbiology & Biotechnology, 2014, 41(10):1517-1524.
