Directed evolution, which is also known as Adaptive laboratory evolution (ALE) is a laboratory simulation of the natural Darwinian evolutionary process. It is a molecular level evolutionary process that involves artificially creating a large number of mutations by some scientific means, and then screening the samples by continuously increasing the survival pressure of the strains according to the production needs or other purposes[1]. Directed evolution is often used to create industrial strains with excellent properties, is a widely used and efficient tool in metabolic engineering, and is currently receiving a lot of attention in the field of synthetic biology.

C. tyrobutyricum is an anaerobic bacterium producing butyrate as a primary product which is widely used in chemical, food and biofuel applications.C. tyrobutyricum has a strong acid-producing metabolism and is able to tolerate high concentrations of acidic products, making it an ideal host for the production of butyrate and other carboxylic acids.[2] C. tyrobutyricum can be used to generate robust host chassis with established acid tolerance and fermentation optimization strategies for the production of a wide range of carboxylates and their derivatives

C. tyrobutyricum metabolizes glucose via the Embden-Meyerhof-Parnassian (EMP) pathway; therefore, the engineering strategy for C. tyrobutyricum may involve the modification of the EMP pathway. And the EMP pathway is closely related to the growth process of C. tyrobutyricum[3]. If genome-directed evolution experiments are performed on C. tyrobutyricum, strains with improved yield through evolution will negatively affect growth and cause carbon loss due to the poor targeting of the evolutionary tools, which is not conducive to industrial-scale fermentation, once they have negatively evolved in the EMP pathway.

The NOG pathway is a cyclic pathway that begins and ends with F6P. In the NOG pathway, F6P converted from glucose and two other F6P molecules are converted to three AcCoA molecules and two F6P molecules.[4]The NOG pathway can be combined with the EMP pathway. The introduction of the NOG pathway in C. tyrobutyricum adds an alternative glycolytic pathway and reduces the negative effects of EMP modification. In addition, the NOG pathway produces one more AcCoA molecule compared to the EMP pathway.

Integration of NOG and EMP pathway has brought increased carbon fixation in organic products in many microorganisms, such as Saccharomyces cerevisiae and E. coli[5-7]. Thus, this integration has the potential not only to reduce the growth side effects of various chassis cells in directed evolution, but also to produce more intermediates of valuable compounds, such as AcCoA. Taken together, we believe that the development of such C. tyrobutyricum chassis cells may be more beneficial for subsequent studies of genome-directed evolution.

[1]WILLIAMS, Thomas C.; PRETORIUS, Isak S.; PAULSEN, Ian T. Synthetic evolution of metabolic productivity using biosensors. Trends in biotechnology, 2016, 34.5: 371-381..

[2]HUANG, Jin, et al. Biosynthesis of butyric acid by Clostridium tyrobutyricum. Preparative Biochemistry and Biotechnology, 2018, 48.5: 427-434..

[3]G. Linger Jeffrey, R. Ford Leah, R. Kavita. Development of C. tyrobutyricum as a microbial cell factory for the production of fuel and chemical intermediates from lignocellulosic feedstocks. Front Energy Res 8, 183, (2020). DOI:10.3389/fenrg.2020.00183.

[4]I. Bogorad, T.S. Lin, J. Liao. Synthetic non-oxidative glycolysis enables complete carbon conservation. Nature 502, 693–697, (2013). DOI: https://doi.org/10.1038/nature12575.

[5]Y. Zheng, Q. Yuan, H. Luo, X. Yang, H. Ma. Engineering NOG-pathway in Escherichia coli for poly-(3-hydroxybutyrate) production from low cost carbon sources. Bioengineered 9, 209-213, (2018). DOI: 10.1080/21655979.2018.1467652.

[6]T. Yu, Q. Liu, X. Wang, X. Liu, Y. Chen, J. Nielsen. Metabolic reconfiguration enables synthetic reductive metabolism in yeast. Nat Metab 4,1551–1559, (2022). DOI: 10.1038/s42255-022-00654-1.

[7]K. Miyoshi, R. Kawai, T. Niide, Y. Toya, H. Shimizu. Functional evaluation of non-oxidative glycolysis in Escherichia coli in the stationary phase under microaerobic conditions. J Biosci Bioeng 135, 291-297, (2023). DOI: 10.1016/j.jbiosc.2023.01.002.