Recruiting a Phosphite Dehydrogenase/Formamidase-Driven Antimicrobial Contamination System in Bacillus subtilis for Nonsterilized Fermentation of Acetoin

Abstract

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.

Nonsterile l-Lysine Fermentation Using Engineered Phosphite-Grown Corynebacterium glutamicum

“…A previous study had similarly found that ptxD from P. stutzeri was better than other genes that were tested in B. subtilis . 23 …”

“…A previous study had similarly found that ptxD from P. stutzeri was better than other genes that were tested in B. subtilis. 23 Because the original Cg-0206 strain could not grow on the Pt medium, a conventional Pi fermentation medium (Pi medium) but without CSL was used to determine its growth. As shown in Figure 1B, the growth of Cg-0206 was reduced by 35.0% (from 5.03 to 3.27 cfu/mL) and L-lysine production was reduced by 35.6% (from 38.33 to 24.67 g/L after 72 h, Figure 1C) by omitting CSL.…”

“… 21 In 2018, ptxD was introduced into cyanobacteria , 22 and in 2020, formamidase ( fmdA ) and ptxD genes were introduced into Bacillus subtilis for fermentation in nonsterile systems. 23 However, there have been no reports of C. glutamicum , one of the most important amino acid production strains in industry, being modified in this fashion. Whether Pt assimilation is effective in C. glutamicum fermentation processes to combat microbial contamination remains to be investigated.…”

“…In 2014, the Pt dehydrogenase gene ptxD was introduced into Schizosaccharomyces pombe and Saccharomyces cerevisiae to combat microbial contamination during the fermentation process . In 2018, ptxD was introduced into cyanobacteria, and in 2020, formamidase ( fmdA ) and ptxD genes were introduced into Bacillus subtilis for fermentation in nonsterile systems . However, there have been no reports of C.…”

Phosphite Reduces the Predation Impact of Poterioochromonas malhamensis on Cyanobacterial Culture

“…This concept has been an effective selection method for prokaryotic and eukaryotic microbial hosts that do not use antibiotics [ 17 , 18 ]. In particular, the cheap availability of Pt offers distinct practical benefits: an industrial fermentation process without the requirement of sterilization of nutrient broth [ 19 , 20 , 21 ] and economically viable selective microbial cultivation at large scales [ 21 , 22 ]. Previously, we and others reported the implementation of a Pt-based selective cultivation method for cyanobacteria and several other microalgae [ 23 , 24 , 25 ].…”