4. Conclusions
Present biochemical production by microbial biotransformation has improved with strain development techniques and process engineering. In tandem, amino acid-based bioconversion has been used to make biotransformation more efficient and economical because amino acids such as l-lysine and l-glutamate can be produced affordably on a large scale (Georgi, Rittmann, & Wendisch, 2005; Y.-G. Hong et al., 2018; H. J. Kim et al., 2015; H. T. Kim et al., 2019; J.-H. Kim et al., 2017; Kimura, 2003; Shin et al., 2018; Stansen et al., 2005; Yang et al., 2019; Yang et al., 2020). Starting from these compounds, we can avoid the long, complex pathway from glucose to reach the final product. In addition, we can avoid individual strain construction, and we can overcome the low productivity of a precursor or intermediate from a general strain, without identifying a new pathway. In practice, high-producing strains are not widely accessible, and there is a large difference between the high-producing strains and a newly developed strain. Typically, new pathways or strains will have a lower titer than expected, and it does not necessarily solve the problems needed to develop a high-producing strain. As a result, biochemical production starting from highly accessible chemicals with bioconversion is in high demand. In addition, we can focus our efforts on essential processes to improve the pathway and final product. There are several successful examples of bioconversion including cadaverine production from l-lysine (Huang et al., 2020; H. J. Kim et al., 2015; J.-H. Kim et al., 2016; J.-H. Kim et al., 2017; Rui et al., 2020), glutaric acid production from l-lysine (Y.-G. Hong et al., 2018; Yang et al., 2019; Yang et al., 2020), and γ-aminobutyric acid production from l-glutamate (Ke et al., 2016; Park et al., 2013; Plokhov et al., 2000; Yuan et al., 2019).
For these reasons, we have continued to develop different bioconversion systems from readily available chemicals and optimized whole-cell reactions resulting in the highest titer and increase of productivity. In addition to changing the starting material, we used easily accessible tools by comparing accessible plasmids and reinforced whole-cell systems by incorporating tandem genes. The simplicity of techniques in this study demonstrates that productivity could be improved in a straightforward manner. Furthermore, this study details many parameters to adjust for large production and raises several questions for studies in the near future.
In our approach, we constructed the l-PA production system with the reinforced gene and optimized culture conditions including medium composition, temperature, IPTG concentration, agitation speed, metal ion and buffer concentration. As a result, we produced 93.5 g/L l-PA and improved production more than 5-fold over that in previous studies. Furthermore, productivity from 1 M l-lysine (monohydrochloride) was 0.78 g/L/h by modifying gene copies and systematically optimizing reaction parameters. l-lysine monohydrochloride is much cheaper and more soluble than l-lysine. Further efforts to stabilize the system for longer times or to shorten the reaction and studies on l-PA recovery from reaction solution will continue to improve l-PA production.
Acknowledgments This study was supported by the National Research Foundation of Korea (NRF) (NRF-2019R1F1A1058805 and NRF-2019M3E6A1103979), Research Program to solve social issues of the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2017M3A9E4077234). This work was also supported by R&D Program of MOTIE/KEIT (10067772) and by R&D Program for Forest Science Technology (Project No. 2020261C10-2022-AC02) provided by Korea Forest Service (Korea Forestry Promotion Institute). Consulting service provided by the Microbial Carbohydrate Resource Bank (MCRB, Seoul, Korea) is highly appreciated.