Breeding of Methanol-Tolerant Methylobacterium extorquens AM1 by Atmospheric and Room Temperature Plasma Mutagenesis Combined With Adaptive Laboratory Evolution

Abstract

Methylobacterium extorquens AM1, which can be used as a methylotrophic cell factory (MeCF) for the production of fine chemicals from methanol, is the most extensively studied model methylotrophic strain. However, its low tolerance for methanol limits the development of bioprocesses and there have been no reports of improved methanol tolerance of M. extorquens AM1. In this study, atmospheric and room temperature plasma (ARTP) mutagenesis, in combination with adaptive laboratory evolution (ALE), is used to generate a mutant with high methanol tolerance (referred to as CLY-2533). The final cell density of CLY-2533 is 7.10 times higher than that of the wild-type strain in medium containing 5% (v/v) methanol. Through comparative genomics analysis and overexpression of the exploited putative genes, seven mutated genes are identified as being closely related to the higher methanol tolerance of CLY-2533. Additionally, the mvt operon, which contains genes related to the biosynthesis of mevalonate acid (MEV), is introduced into CLY-2533. This recombinant strain shows significant improvements in both MEV production and cell growth in 5% methanol medium. These findings will be helpful in rational design of methanol-utilizing strain for an improved host platform for methanol based biomanufacturing.

Engineering of membrane phospholipid component enhances salt stress tolerance in Saccharomyces cerevisiae

“…In this study, redistributed membrane phospholipid driven by the module assembly of cds1 and cho1 contributed to the increase in membrane potential and membrane integrity, which significantly enhanced salt stress tolerance. Compared with long mutagenesis breeding (Cui et al, ), adaptive laboratory evolution (Walker et al, ), transporter engineering (Kell et al, ), this strategy is a more convenient and efficient way to achieve microbial tolerance. However, the rearranged phospholipid bilayer structure in strain Y03 is unknown, which may be important for exploring increased membrane potential and membrane integrity.…”

“…To increase the tolerance of industrial strains to environmental stress (Chakraborty, Winardhi, Morgan, Yan, & Kenney, ; Olin‐Sandoval et al, ), a series of strategies have been developed. These include global transcription machinery engineering (Alper, Moxley, Nevoigt, Fink, & Stephanopoulos, ), adaptive laboratory evolution (Walker, Ryu, & Trinh, ), mutagenesis breeding (Cui et al, ), transporter engineering (Kell, Swainston, Pir, & Oliver, ), and membrane engineering (Tan et al, ).…”

Potentials, Utilization, and Bioengineering of Plant Growth-Promoting Methylobacterium for Sustainable Agriculture

“…Another choice to enhance the ability of the microbiome to promote plant growth is to improve the ability of resident Methylobacteria through random mutations achieved by treating the bacteria with UV light, chemicals, or atmosphere, and using room temperature plasma (ARTP) combined with adaptive laboratory evolution [83].…”

Dynamic regulation of membrane integrity to enhance l‐malate stress tolerance in Candida glabrata

“…Several methods have been explored to improve the robustness of industrial strains, including adaptive laboratory evolution (Zhu et al, 2018), reverse engineering (Pereira et al, 2019), transporter engineering (Kusumawardhani et al, 2018), mutagenesis breeding (Cui et al, 2018), and membrane engineering (Qi et al, 2019). Membrane engineering is a feasible and efficient method to increase the robustness and production performance of industrial strains (Sandoval & Papoutsakis, 2016), because the cell membrane can separate and insulate the cytoplasm from environmental stress.…”