Microbial
contamination, especially in large-scale processes, is
partly a life-or-death issue for industrial fermentation. Therefore,
the aim of this research was to create an antimicrobial contamination
system in Bacillus subtilis 168 (an ideal acetoin
producer for its safety and acetoin synthesis potential). First, introduction
of the formamidase (FmdA) from Helicobacter pylori and the phosphite dehydrogenase (PtxD) from Pseudomonas
stutzeri enabled the engineered Bacillus subtilis to simultaneously assimilate formamide and phosphite as nitrogen
(N) and phosphorus (P) sources. Thus, the engineered B. subtilis became the dominant population in a potentially contaminated system,
while contaminated microbes were starved of key nutrients. Second,
stepwise metabolic engineering via chromosome-based
overexpression of the relevant glycolysis and acetoin biosynthesis
genes led to a 1.12-fold increment in acetoin titer compared with
the starting host. Finally, with our best acetoin producer, 25.56
g/L acetoin was synthesized in the fed-batch fermentation, with a
productivity of 0.33 g/L/h and a yield of 0.37 g/g under a nonsterilized
and antibiotic-free system. More importantly, our work fulfills many
key criteria of sustainable chemistry since sterilization is abolished,
contributing to the simplified fermentation operation with lower energy
consumption and cost.
Considerable attention has been given to the development of robust fermentation processes, but microbial contamination and phage infection remain deadly threats that need to be addressed. In this study, a robust Escherichia coli BL21(DE3) strain was successfully constructed by simultaneously introducing a nitrogen and phosphorus (N&P) system in combination with a CRISPR/Cas9 system. The N&P metabolic pathways were able to express formamidase and phosphite dehydrogenase in the host cell, thus enabled cell growth in auxotrophic 3-(N-morpholino) propanesulfonic acid medium with formamide and phosphite as nitrogen and phosphorus sources, respectively. N&P metabolic pathways also allowed efficient expression of heterologous proteins, such as green fluorescent protein (GFP) and chitinase, while contaminating bacteria or yeast species could hardly survive in this medium. The host strain was further engineered by exploiting the CRISPR/Cas9 system to enhance the resistance against phage attack. The resultant strain was able to grow in the presence of T7 phage at a concentration of up to 2 × 10 7 plaqueforming units/ml and produce GFP with a yield of up to 30 μg/10 9 colony-forming units, exhibiting significant advantages over conventional engineered E. coli. This newly engineered, robust E. coli BL21(DE3) strain therefore shows great potential for future applications in industrial fermentation.
K E Y W O R D Santimicrobial, anti-phage, CRISPR/Cas9 system, Escherichia coli, N&P pathway
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.