Identification and functional characterization of a potential l-Homoserine exporter in Corynebacterium glutamicum

Abstract

Exporter protein systems play a crucial role in the efficient production of valuable chemicals. However, the lack of active exporters significantly limits the application of industrial bio-based production, making the identification and utilization of novel exporters highly important. In this study, we discovered a novel l-Homoserine exporter, Cg0701, in Corynebacterium glutamicum through homology analysis. First, tolerance assays revealed that the cg0701 overexpression strain (CgH-2) exhibited a 10.45% increase in cell growth compared to the control when cultivated with 30 g/L-Homoserine. Additionally, export assays demonstrated that the l-Homoserine export capacity of CgH-2 increased by approximately 30%. Furthermore, genomic overexpression of cg0701 in an l-Homoserine-producing chassis also enhanced both tolerance and export activity. As a result, the recombinant strain CgH-11 produced 10.79 g/L-Homoserine in shake flask cultures and 48.72 g/L in a 5 L fermenter, representing improvements of 19.89% and 24.44%, respectively. In summary, our results indicate that Cg0701 is a novel l-Homoserine exporter in C. glutamicum, enriching our understanding of amino acid export systems and providing a valuable target for the construction of l-Homoserine microbial cell factories.

Keywords

Amino acid exporter / Cg0701 / Corynebacterium glutamicum / L-Homoserine production

Data Availability Statement

All data generated during this study are included in this published article and its supplementary information files. The original data and resources are available from the corresponding author on reasonable request.

Declaration of Competing Interest

There are no conflicts of interest to declare.

CRediT authorship contribution statement

Xiaodi Liu: Writing - original draft, Conceptualization. Xiangyu Zhu: Methodology, Investigation, Data curation. Wenxin Jiang: Validation, Methodology. Huanmin Du: Writing - review & editing, Supervision, Conceptualization.

Acknowledgements

The authors thank Dr. Jun Liu and Dr. Chia-Hung CHEN for constructive discussions.

References

[1] J.O. Krömer, C. Wittmann, H. Schröder, E. Heinzle, Metabolic pathway analysis for rational design of l -methionine production by Escherichia coli and Corynebacterium glutamicum, Metab. Eng. 8 (2006) 353-369, doi:10.1016/j.ymben.2006.02.001.

[2] K.H. Lee, J.H. Park, T.Y. Kim, H.U. Kim, S.Y. Lee, Systems metabolic engineer- ing of Escherichia coli for L-threonine production, Mol. Syst. Biol. 3 (2007) 149, doi:10.1038/msb4100196.

[3] Y. Zhang, M.H. Wei, G.H. Zhao, W.J. Zhang, Y.Z. Li, B.B. Lin, Y.J. Li, Q.Y. Xu, N. Chen, C.L. Zhang, High-level production of l -Homoserine using a non-induced, non-auxotrophic Escherichia coli chassis through metabolic engineering, Bioresour. Technol. 327 (2021) 124814, doi:10.1016/j.biortech.2021.124814.

[4] G.H. Zhao, Y.Q. Tang, Z.H. Li, G. Liu, D.Z. Zhang, X.Q. Hu, X.Y. Wang, Engineer- ing Corynebacterium glutamicum for efficient l -Homoserine production, Bioresour. Technol. 431 (2025) 132617, doi:10.1016/j.biortech.2025.132617.

[5] Y.Y. Chen, L.G. Huang, T. Yu, Y. Yao, M.M. Zhao, A.P. Pang, J.P. Zhou, B. Zhang, Z.Q. Liu, Y.G. Zheng, Balancing the AspC and AspA pathways of Escherichia coli by systematic metabolic engineering strategy for high-efficient l -Homoserine produc- tion, ACS Synth. Biol. 13 (2024) 2457-2469, doi:10.1021/acssynbio.4c00208.

[6] P. Liu, B. Zhang, Z.H. Yao, Z.Q. Liu, Y.G. Zheng, Multiplex design of metabolic net- work for production of l -Homoserine in Escherichia coli, Appl. Environ. Microbiol. 86 (2020) e01477-20, doi:10.1128/AEM.01477-20.

[7] Q.X. Mu, S.S. Zhang, X.J. Mao, Y. Tao, B. Yu, Highly efficient production of l- Homoserine in Escherichia coli by engineering a redox balance route, Metab. Eng. 67 (2021) 321-329, doi:10.1016/j.ymben.2021.07.011.

[8] L. Wei, Q. Wang, N. Xu, J. Cheng, W. Zhou, G.Q. Han, H.F. Jiang, J. Liu, Y.H. Ma, Combining protein and metabolic engineering strategies for high-level production of O-acetylhomoserine in Escherichia coli, ACS Synth. Biol. 8 (2019) 1153-1167, doi:10.1021/acssynbio.9b00042.

[9] M. Liu, J.L. Lou, J.L. Gu, X.M. Lyu, F.Q. Wang, D.Z. Wei, Increasing l -Homoserine production in Escherichia coli by engineering the central metabolic pathways, J. Biotechnol. 314-315 (2020) 1-7, doi:10.1016/j.jbiotec.2020.03.010.

[10] H. Li, B.S. Wang, L.H. Zhu, S. Cheng, Y.R. Li, L. Zhang, Z.Y. Ding, Z.H. Gu, G.Y. Shi, Metabolic engineering of Escherichia coli W3110 for l -Homoserine production, Pro- cess Biochem 51 (2016) 1973-1983, doi:10.1016/j.procbio.2016.09.024.

[11] C. Ding, J.W. Zhang, J.F. Qiao, Z.P. Ma, P. Liu, J. Liu, Q.D. Liu, N. Xu, Identifi- cation and engineering efflux transporters for improved l -Homoserine production in Escherichia coli, J. Appl. Microbiol. 134 (2023) lxad075, doi:10.1093/jam- bio/lxad075.

[12] M. D’Este, M. Alvarado-Morales, I. Angelidaki, Amino acids production focus- ing on fermentation technologies-a review, Biotechnol. Adv. 36 (2018) 14-25, doi:10.1016/j.biotechadv.2017.09.001.

[13] X. Jin, S.M. Wang, Y.B. Wang, Q.S. Qi, Q.F. Liang, Metabolic engineering strategies for l -Homoserine production in Escherichia coli, Microb. Cell Fact. 23 (2024) 338, doi:10.1186/s12934-024-02623-7.

[14] K. Niu, R. Zheng, M. Zhang, M.Q. Chen, Y.M. Kong, Z.Q. Liu, Y.G. Zheng, Ad- justment of the main biosynthesis modules to enhance the production of l- Homoserine in Escherichia coli W3110, Biotechnol. Bioeng. 122 (2025) 223-232, doi:10.1002/bit.28861.

[15] M.M. Cai, Z.Q. Zhao, X.F. Li, Y.Y. Xu, Z.M. Rao, Development of a nonauxotrophic l -Homoserine hyperproducer in Escherichia coli by systems metabolic engineering, Metab. Eng. 73 (2022) 270-279, doi:10.1016/j.ymben.2022.08.003.

[16] B.Y. Sun, F.Q. Wang, J. Zhao, X.Y. Tao, M. Liu, D.Z. Wei, Engineering Es- cherichia coli for l -Homoserine production, J. Basic Microbiol. 63 (2022) 168-178, doi:10.1002/jobm.202200488.

[17] N. Li, L.H. Li, S.Q. Yu, J.W. Zhou, Dual-channel glycolysis balances cofactor supply for l -homoserine biosynthesis in Corynebacterium glutamicum, Bioresour. Technol. 369 (2023) 128473, doi:10.1016/j.biortech.2022.128473.

[18] T.M. Vo, S. Park, Metabolic engineering of Escherichia coli W3110 for effi- cient production of Homoserine from glucose, Metab. Eng. 73 (2022) 104-113, doi:10.1016/j.ymben.2022.07.001.

[19] L. Eggeling, H. Sahm, New ubiquitous translocators: amino acid export by Corynebac- terium glutamicum and Escherichia coli, Arch. Microbiol. 180 (2003) 155-160, doi:10.1007/s00203-003-0581-0.

[20] C.M. Jones, N.J. Hernandez Lozada, B.F. Pfleger, Efflux systems in bacteria and their metabolic engineering applications, Appl. Microbiol. Biotechnol. 99 (2015) 9381-9393, doi:10.1007/s00253-015-6963-9.

[21] Q. Liu, Y. Liang, Y. Zhang, X.L. Shang, S.W. Liu, J.F. Wen, T.Y. Wen, YjeH is a novel exporter of l -methionine and branched-chain amino acids in Escherichia coli, Appl. Environ. Microbiol. 81 (2015) 7753-7766, doi:10.1128/AEM.02242-15.

[22] X.M. Zhang, Y.J. Gao, Z.W. Chen, G.Q. Xu, X.J. Zhang, H. Li, J.S. Shi, M.A.G. Kof- fas, Z.H. Xu, High-yield production of l -serine through a novel identified exporter combined with synthetic pathway in Corynebacterium glutamicum, Microb. Cell Fact. 19 (2020) 115, doi:10.1186/s12934-020-01374-5.

[23] V.A. Livshits, N.P. Zakataeva, V.V. Aleshin, M.V. Vitushkina, Identification and characterization of the new gene rhtA involved in Threonine and ho- moserine efflux in Escherichia coli, Res. Microbiol. 154 (2003) 123-135, doi:10.1016/S0923-2508(03)00036-6.

[24] R. Diesveld, N. Tietze, O. Fürst, A. Reth, B. Bathe, H. Sahm, L. Eggeling, Activity of exporters of Escherichia coli in Corynebacterium glutamicum and their use to in- crease l -threonine production, J. Mol. Microbiol. Biotechnol. 16 (2009) 198-207, doi:10.1159/000142530.

[25] N. Li, S. Xu, G.C. Du, J. Chen, J.W. Zhou, Efficient production of l -Homoserine in Corynebacterium glutamicum ATCC 13032 by redistribution of metabolic flux, Biochem. Eng. J. 161 (2020) 107665, doi:10.1016/j.bej.2020.107665.

[26] J. Becker, C. Wittmann, Systems and synthetic metabolic engineering for amino acid production - the heartbeat of industrial strain development, Curr. Opin. Biotechnol. 23 (2012) 718-726, doi:10.1016/j.copbio.2011.12.025.

[27] W. Leuchtenberger, K. Huthmacher, K. Drauz, Biotechnological production of amino acids and derivatives: current status and prospects, Appl. Microbiol. Biotechnol. 69 (2005) 1-8, doi:10.1007/s00253-005-0155-y.

[28] X.L. Shang, Y. Zhang, G.Q. Zhang, X. Chai, A.H. Deng, Y. Liang, T.Y. Wen, Charac- terization and molecular mechanism of AroP as an aromatic amino acid and histi- dine transporter in Corynebacterium glutamicum, J. Bacteriol. 195 (2013) 5334-5342, doi:10.1128/JB.00971-13.

[29] L. Li, Y.W. Zhao, L.J. Ruan, S. Yang, M. Ge, W.H. Jiang, Y.H. Lu, A step- wise increase in pristinamycin II biosynthesis by Streptomyces pristinaespiralis through combinatorial metabolic engineering, Metab. Eng. 29 (2015) 12-25, doi:10.1016/j.ymben.2015.02.001.

[30] N. Okibe, N. Suzuki, M. Inui, H. Yukawa, Efficient markerless gene replacement in Corynebacterium glutamicum using a new temperature-sensitive plasmid, J. Microbiol. Methods. 85 (2011) 155-163, doi:10.1016/j.mimet.2011.02.012.

[31] C. Alkim, D. Farias, J. Fredonnet, H. Serrano-Bataille, P. Herviou, M. Picot, N. Slama, S. Dejean, N. Morin, B. Enjalbert, J.M. François, Toxic effect and inability of l- Homoserine to be a nitrogen source for growth of Escherichia coli resolved by a com- bination of in vivo evolution engineering and omics analyses, Front. Microbiol. 13 (2022) 1051425, doi:10.3389/fmicb.2022.1051425.

[32] G.H. Liu, C. Ding, Y. Ju, Z.P. Ma, L. Wei, J. Liu, Q.D. Liu, N. Xu, Directed evolution of an EamB transporter for improved l -cysteine tolerance and production in Escherichia coli, FEMS Microbiol. Lett. 368 (2022) fnac008, doi:10.1093/femsle/fnac008.

[33] H.M. Du, Y.T. Qi, J.F. Qiao, L.C. Li, N. Xu, L. Shao, L. Wei, J. Liu, Balancing re- dox homeostasis to improve l -cysteine production in Corynebacterium glutamicum, J. Agric. Food Chem. 71 (2023) 13848-13856, doi:10.1021/acs.jafc.3c03828.

[34] H.M. Du, J.F. Qiao, Y.T. Qi, L.C. Li, N. Xu, L. Shao, L. Wei, J. Liu, Reprogramming the sulfur recycling network to improve l -cysteine production in Corynebacterium glutamicum, Green Chem 25 (2023) 3152-3165, doi:10.1039/D3GC00027C.

[35] L. Wei, J.H. Zhao, Y.R. Wang, J.S. Gao, M.H. Du, Y. Zhang, N. Xu, H.M. Du, J.S. Ju, Q. D. Liu, J. Liu, Engineering of Corynebacterium glutamicum for high-level gamma- aminobutyric acid production from glycerol by dynamic metabolic control, Metab. Eng. 69 (2022) 134-146, doi:10.1016/j.ymben.2021.11.010.

[36] C. Dellomonaco, J.M. Clomburg, E.N. Miller, R. Gonzalez, Engineered reversal of the beta-oxidation cycle for the synthesis of fuels and chemicals, Nature 476 (2011) 355-359, doi:10.1038/nature10333.

[37] A.M. Kunjapur, Y. Tarasova, K.L.J. Prather, Synthesis and accumulation of aromatic aldehydes in an engineered strain of Escherichia coli, J. Am. Chem. Soc. 136 (2014) 11644-11654, doi:10.1021/ja506664a.

[38] P. Xu, L.Y. Li, F.M. Zhang, G. , Stephanopoulos, M. Koffas, Improving fatty acids pro- duction by engineering dynamic pathway regulation and metabolic control, Proc. Natl. Acad. Sci. U.S.A. 111 (2014) 11299-11304, doi:10.1073/pnas.1406401111.

[39] S.A. van der Hoek, I. Borodina, Transporter engineering in microbial cell factories: the ins, the outs, and the in-betweens, Curr. Opin. Biotechnol. 66 (2020) 186-194, doi:10.1016/j.copbio.2020.08.002.

[40] Y. Zhu, C. Zhou, Y. Wang, C. Li, Transporter engineering for microbial manufactur- ing, Biotechnol. J. 15 (2020) e1900494, doi:10.1002/biot.201900494.

[41] M.S. Ahmed, K.J. Lauersen, S. Ikram, C. Li, Efflux transporters’ engineering and their application in microbial production of heterologous metabolites, ACS Synth. Biol. 10 (2021) 646-669, doi:10.1021/acssynbio.0c00507.

[42] S. Malla, E. van der Helm, B. Darbani, S. Wieschalka, J. Forster, I. Borodina, M.O.AA. Sommer. Novel efficient l -lysine exporter identified by functional metage- nomics, Front. Microbiol. 13 (2022) 855736, doi:10.3389/fmicb.2022.855736.