Improvement of l-Valine Production by Atmospheric and Room Temperature Plasma Mutagenesis and High-Throughput Screening in Corynebacterium glutamicum

Abstract

As one of the branched-chain amino acids, L-valine is an essential nutrient for most mammalian species. In this study, the L-valine producer Corynebacterium glutamicum ΔppcΔaceEΔalatΔpqo was first constructed. Additionally, an improved biosensor based on the Lrp-type transcriptional regulator and temperature-sensitive replication was built. Then, the C. glutamicum strain was mutagenized by atmospheric and room temperature plasma. A sequential three-step procedure was carried out to screen L-valineproducing strains, including the fluorescence-activated cell sorting (FACS), 96-well plate screening, and flask fermentation. The final mutant HL2-7 obtained by screening produced 3.20 g/L of L-valine, which was 21.47% higher than the titer produced by the starting strain. This study demonstrates that the L-valine-producing mutants can be successfully isolated based on the Lrp sensor system in combination with FACS screening after random mutagenesis.

Programming Adaptive Laboratory Evolution of 4-Hydroxyisoleucine Production Driven by a Lysine Biosensor in Corynebacterium Glutamicum

“…Some studies have utilized ALE to force strains to adapt to alternative growth environments and to obtain mutants with signi cantly improved performance. For example, a C. glutamicum strain with improved tolerance to high concentrations of methanol was successfully obtained by ALE, thereby enhancing methanol biotransformation, meanwhile, the cell growth was also improved (Tuyishime et al 2018;Wang et al 2020). ALE was successfully applied for improving the tolerance of E. coli to sabinene, and the sabinene production was increased to 8.43-fold (Wu et al 2020).…”

“…However, the Lys titer (11.44 − 15.77 mM) did not reduce signi cantly as compared to SZ06 (9.29 mM). Some studies have utilized ALE to force strains to adapt to alternative growth environments, thus accelerating the growth rate of cells and obtaining mutants with signi cantly improved performance (Lee et al 2010;Tuyishime et al 2018;Wang et al 2020). The shortest evolution period of most ALE was 15 days (Stella et al 2019), comparable with 8 rounds evolution here.…”

“…Because normally, the adaptive rate under speci c selection pressures is directly coupled to the growth rate, this coupled metabolic design will result in signi cantly higher production and lower by-products (Fong et al 2005). For example, ALE was successfully applied to improve cellular tolerance of a C. glutamicum strain to high concentrations of methanol, thereby enhancing methanol biotransformation, meanwhile, the cell growth was also improved (Tuyishime et al 2018; Wang et al 2020). ALE was used to develop potential evolutionary strains of Zymomonas mobilis that can co-utilize glucose and xylose (Millán et al 2020).…”

“…ALE combined with metabolic engineering was successfully used to improve the production of putrescine in C. glutamicum (Li et al 2018). Through ALE driven by an L-valine (Val) biosensor Lrp-P brnFE in combination with atmospheric and room temperature plasma mutagenesis, an mutant strain HL2-7 was successfully obtained and its Val production increased by 21.47% (Han et al 2020).…”

Programming adaptive laboratory evolution of 4-hydroxyisoleucine production driven by a lysine biosensor in Corynebacterium glutamicum

“…ALE combined with metabolic engineering was successfully used to improve the production of putrescine in C. glutamicum (Li et al 2018 ). Through ALE driven by an L-valine (Val) biosensor Lrp-P brnFE in combination with atmospheric and room temperature plasma mutagenesis, an mutant strain HL2-7 was successfully obtained and its Val production increased by 21.47% (Han et al 2020 ).…”