Dynamically Regulating Glucose Uptake to Reduce Overflow Metabolism with a Quorum-Sensing Circuit for the Efficient Synthesis of d-Pantothenic Acid in Bacillus subtilis.

In response to a high concentration of glucose, Bacillus subtilis, a microbial chassis for producing many industrial metabolites, rapidly takes up glucose using the phosphotransferase system (PTS), leading to overflow metabolism, a common phenomenon observed in many bacteria. Although overflow metabolism affects cell growth and reduces the production of many metabolites, effective strategies that reduce overflow metabolism while maintaining normal cell growth remain to be developed. Here, we used a quorum sensing (QS)-mediated circuit to tune the glucose uptake rate and thereby relieve overflow metabolism in an engineered B. subtilis for producing d-pantothenic acid (DPA). A low-efficiency non-PTS system was used for glucose uptake at the early growth stages to avoid a rapid glycolytic flux, while an efficient PTS system, which was activated by a QS circuit, was automatically activated at the late growth stages after surpassing a threshold cell density. This strategy was successfully applied as a modular metabolic engineering process for the high production of DPA. By enhancing the translation levels of key enzymes (3-methyl-2-oxobutanoate hydroxymethytransferase, pantothenate synthetase, aspartate 1-decarboxylase proenzyme, 2-dehydropantoate 2-reductase, dihydroxy-acid dehydratase, and acetolactate synthase) with engineered 5'-untranslated regions (UTRs) of mRNAs, the metabolic flux was promoted in the direction of DPA production, elevating the yield of DPA to 5.11 g/L in shake flasks. Finally, the engineered B. subtilis produced 21.52 g/L of DPA in fed-batch fermentations. Our work not only revealed a new strategy for reducing overflow metabolism by adjusting the glucose uptake rate in combination with promoting the translation of key metabolic enzymes through engineering the 5'-UTR of mRNAs but also showed its power in promoting the bioproduction of DPA in B. subtilis, exhibiting promising application prospects.

A Type I Restriction Modification System Influences Genomic Evolution Driven by Horizontal Gene Transfer in Paenibacillus polymyxa.

Considered a "Generally Recognized As Safe" (GRAS) bacterium, the plant growth-promoting rhizobacterium Paenibacillus polymyxa has been widely applied in agriculture and animal husbandry. It also produces valuable compounds that are used in medicine and industry. Our previous work showed the presence of restriction modification (RM) system in P. polymyxa ATCC 842. Here, we further analyzed its genome and methylome by using SMRT sequencing, which revealed the presence of a larger number of genes, as well as a plasmid documented as a genomic region in a previous report. A number of mobile genetic elements (MGEs), including 78 insertion sequences, six genomic islands, and six prophages, were identified in the genome. A putative lysozyme-encoding gene from prophage P6 was shown to express lysin which caused cell lysis. Analysis of the methylome and genome uncovered a pair of reverse-complementary DNA methylation motifs which were widespread in the genome, as well as genes potentially encoding their cognate type I restriction-modification system PpoAI. Further genetic analysis confirmed the function of PpoAI as a RM system in modifying and restricting DNA. The average frequency of the DNA methylation motifs in MGEs was lower than that in the genome, implicating a role of PpoAI in restricting MGEs during genomic evolution of P. polymyxa. Finally, comparative analysis of R, M, and S subunits of PpoAI showed that homologs of the PpoAI system were widely distributed in species belonging to other classes of Firmicute, implicating a role of the ancestor of PpoAI in the genomic evolution of species beyond Paenibacillus.