Streptococcus mutans has been reported as a primary cariogenic pathogen associated with dental caries. The bacteria can produce glucosyltransferases (Gtfs) to synthesize extracellular polysaccharides (EPSs) that are known as virulence factors for adherence and formation of biofilms. Therefore, an ideal inhibitor for dental caries is one that can inhibit planktonic bacteria growth and prevent biofilm formation. Bergenia crassifolia (L.), widely used as a folk medicine and tea beverage, has been reported to have a variety of bioactivities. The present study aimed to explore the effect of B. crassifolia (L.) leaf extracts on the biofilm of Streptococcus mutans. The B. crassifolia (L.) leaf extracts showed inhibitory effects by decreasing viability of bacteria within the biofilm, as evidenced by the XTT assay, live/dead staining assay and LDH activity assay, and could decrease the adherence property of S. mutans through inhibiting Gtfs to synthesize EPSs. In addition, the reduced quantity of EPSs and the inhibition of Gtfs were positively correlated with concentrations of test samples. Finally, the MTT assay showed that the extracts had no cytotoxicity against normal oral cells. In conclusion, the extracts and sub-extracts of B. crassifolia leaves were found to be antimicrobial and could reduce EPS synthesis by inhibiting activities of Gtfs to prevent bacterial adhesion and biofilm formation. Therefore, B. crassifolia leaves have potential to be developed as a drug to prevent and cure dental caries.

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Background Trichoderma reesei is widely used for cellulase production and accepted as an example for cellulase research. Cre1-mediated carbon catabolite repression (CCR) can significantly inhibit the transcription of cellulase genes during cellulase fermentation in T. reesei. Early efforts have been undertaken to modify Cre1 for the release of CCR; however, this approach leads to arrested hyphal growth and decreased biomass accumulation, which negatively affects cellulase production. Results In this study, novel fusion transcription factors (fTFs) were designed to release or attenuate CCR inhibition in cellulase transcription, while Cre1 was left intact to maintain normal hyphal growth. Four designed fTFs were introduced into the T. reesei genome, which generated several transformants, named Kuace3, Kuclr2, Kuace2, and Kuxyr1. No obvious differences in growth were observed between the parent and transformant strains. However, the transcription levels of cel7a, a major cellulase gene, were significantly elevated in all the transformants, particularly in Kuace2 and Kuxyr1, when grown on lactose as a carbon source. This suggested that CCR inhibition was released or attenuated in the transformant strains. The growth of Kuace2 and Kuxyr1 was approximately equivalent to that of the parent strain in fed-batch fermentation process. However, we observed a 3.2- and 2.1-fold increase in the pNPCase titers of the Kuace2 and Kuxyr1 strains, respectively, compared with that of the parent strain. Moreover, we observed a 6.1- and 3.9-fold increase in the pNPCase titers of the Kuace2 and Kuxyr1 strains, respectively, compared with that of Δcre1 strain. Conclusions A new strategy based on fTFs was successfully established in T. reesei to improve cellulase titers without impairing fungal growth. This study will be valuable for lignocellulosic biorefining and for guiding the development of engineering strategies for producing other important biochemical compounds in fungal species.

In Trichoderma reesei, carbon catabolite repression (CCR) significantly downregulates the transcription of cellulolytic enzymes, which is usually mediated by the zinc finger protein Cre1. It was found that there is a conserved region at the C-terminus of Cre1/CreA in several cellulase-producing fungi that contains up to three continuous S/T phosphorylation sites. Here, S387, S388, T389, and T390 at the C-terminus of Cre1 in T. reesei were mutated to valine for mimicking an unphosphorylated state, thereby generating the transformants Tr_Cre1 S387V , Tr_Cre1 S388V , Tr_Cre1 T389V , and Tr_Cre1 T390V , respectively. Transcription of cel7a in Tr_ Cre1 S388V was markedly higher than that of the parent strain when grown in glucose-containing media. Under these conditions, both filter paperase (FPase) and p-nitrophenyl-β-D-cellobioside (pNPCase) activities, as well as soluble proteins from Tr_Cre1 S388V were significantly increased by up to 2-to 3-fold compared with that of other transformants and the parent strain. The results suggested that S388 is critical site of phosphorylation for triggering CCR at the terminus of Cre1. To our knowledge, this is the first report demonstrating an improvement of cellulase production in T. reesei under CCR by mimicking dephosphorylation at the C-terminus of Cre1. Taken together, we developed a precision engineering strategy based on the modification of phosphorylation sites of Cre1 transcription factor to enhance the production of cellulase in T. reesei under CCR.