Adaptive Laboratory Evolution of Bacillus subtilis 168 for Efficient Production of Surfactin Using NH4Cl as a Nitrogen Source

Abstract

Bacillus subtilis strain 168 is commonly used as a host to produce recombinant proteins and as a chassis for bio-based chemicals production. However, its preferred nitrogen source is organic nitrogen, which greatly increases production costs. In this study, adaptive laboratory evolution (ALE) was used to improve B. subtilis 168 growth using NH4Cl as the sole nitrogen source. The cell density (OD600) of a mutant strain LJ-3 was 208.7% higher than that of the original strain. We also optimized the metal ions in the medium and this resulted in a further increase in growth rate by 151.3%. Reintroduction of the sfp+ gene into strain LJ-3 led to the LJ-31 clone, which restored LJ-3’s ability to synthesize surfactin. The fermentation system was optimized (C/N, aeration, pH) in a 5 L bioreactor. Dry cell weight of 7.4 g/L and surfactin concentration of 4.1 g/L were achieved using the optimized mineral salt medium after 22 h of batch fermentation with a YP/S value of 0.082 g/g and a YP/X of 0.55 g/g. HPLC analysis identified the surfactin isoforms produced by strain LJ-31 in the synthetic medium as C13-surfactin 13.3%, C14-surfactin 44.02%, and C15-surfactin 32.79%. Hence, the variant LJ-3 isolated by ALE is a promising engineering chassis for efficient and cost-effective production of a variety of metabolites.

Pathway and enzyme engineering for the bioconversion of lignin derivatives into homoeriodictyol in Saccharomyces cerevisiae

“…For example, utilizing single-component inorganic nitrogen sources, such as ammonium sulfate, ammonium nitrate, etc ., would demonstrate a significant advantage in terms of lowering costs of the fermentation and facilitating the process evaluation. 58 Second, it is reported that most auxotrophic strains showed decreased cell growth and production capacity during the biosynthesis process. 59 To promote the overproduction of homoeriodictyol, future efforts should be made in complementing the auxotrophic markers of the engineered strain.…”

Engineering Bacillus subtilis J46 for efficient utilization of galactose through adaptive laboratory evolution

“…In our pursuit to harness this adaptability for biotechnological applications, we employed ALE to improve the galactose utilization capability of B. subtilis . There were several trials to engineer B. subtilis with ALE (Driessen et al 2023 ; Li et al 2023 ; Zeigler and Nicholson 2017 ). Zhang et al have reported that B. subtilis was adapted under xylose as a selection pressure and validated several key mutations (Zhang et al 2015 ).…”

“…Zhang et al have reported that B. subtilis was adapted under xylose as a selection pressure and validated several key mutations (Zhang et al 2015 ). ALE was utilized not only to address carbon stress but also to promote rapid development, efficient use of nitrogen sources, and tolerance to lignocellulosic hydrolysate (Driessen et al 2023 ; Li et al 2023 ; Liu et al 2020 ).…”

“…Notable studies have explored growth under glucose as the primary carbon source (Liu et al 2020 ; Yuan et al 2019 ), while others have delved into growth on xylose as the sole carbon substrate (Averesch and Rothschild 2019 ; Zhang et al 2015 ). Additionally, investigations have been carried out with NH 4 Cl serving as the exclusive nitrogen source (Li et al 2023 ), and within the milieu of lignocellulosic hydrolysate (Driessen et al 2023 ). Through these varied explorations, ALE has emerged as a powerful tool in fine-tuning the metabolic and growth attributes of B. subtilis to adapt to distinct nutrient and environmental regimes.…”