Corynebacterium glutamicum is an important industrial production strain that is widely used in amino acid fermentation, biopharmaceuticals, and other fields. It is particularly important to develop efficient genome editing methods for the targeted modification of C. glutamicum production strains. Currently, the gene editing system of C. glutamicum is inefficient and time-consuming. In this paper, we reported on a Francisella novicida (Fn) CRISPR-Cpf1-based system for genome editing. The system used linear DNA detached from the plasmid, and, with the assistance of the recombinase RecET, gene deletion was achieved by simultaneous electrotransformation of linear DNA with a plasmid carrying the FnCpf1 and crRNA expression cassette for double-strand breaks of the genome. Compared with previous all-in-one plasmids, this system reduced the time for one round of constructing recombinant plasmids and shortened the editing cycle by about 24 h. Finally, we successfully constructed an engineered strain (X−2) with high L-valine production by using the linear DNA-mediated gene deletion system. This method is of great importance for accelerating the process of metabolic engineering modification of C. glutamicum and its further application in high value-added products.

In this study, a UDP-glycosyltransferase gene from Forsythia suspensa was systematically identified and functionally characterized. Phylogenetic analysis showed that this gene clustered with members of the UGT74 subfamily, where it clustered closely with members of known function; accordingly, it was provisionally designated as FsUGT74F1. Prokaryotic expression experiments determined that soluble recombinant protein could be obtained under induction conditions of 0.1 mM IPTG at 16 °C for 8 h. In vitro enzyme activity assays suggested that FsUGT74F1 might catalyze the glycosylation of phillygenin using UDP-glucose under the tested conditions, yielding a product whose HPLC retention time was consistent with that of authentic phillyrin. Subcellular localization results indicated that FsUGT74F1 was predominantly associated with the plasma membrane. Transient overexpression of FsUGT74F1 in F. suspensa leaves resulted in upregulation of gene expression by approximately 247.6%, and the phillyrin content increased to 124.7% of that in the control group. Conversely, virus-induced gene silencing (VIGS) of FsUGT74F1 led to a reduction in transcript levels by approximately 35.6%, and the phillyrin content decreased to 72.5% of the control level. Molecular docking suggested that phillygenin could interact with FsUGT74F1, with Leu169 predicted to participate in substrate positioning near the C4′-OH group. Collectively, these results support the involvement of FsUGT74F1 in phillyrin biosynthesis and provide a theoretical basis for its application in metabolic engineering.

Background: Forsythia suspensa (Thunb.) Vahl is a perennial deciduous shrub of the Oleaceae family. Its dried mature fruits are used as medicine and hold an important position in traditional Chinese medicine. Tyrosine decarboxylase (TyDC) is a key enzyme involved in the synthesis of dopamine in Forsythia suspensa. At the same time, it also affects the growth and development of this species under biotic stress. Methods: This study examined the expression and function of FsTyDC1 under drought and salt stress. The TyDC gene identified in F. suspensa, termed FsTyDC1, has an open reading frame (ORF) of 1518 bp. Results: qRT-PCR and subcellular localization analyses indicated that FsTyDC1 is highly expressed in F. suspensa fruit and its protein is located in the cytoplasm. The gene was silenced using a pTRV2-FsPDS/FsTyDC1 vector with virus-induced gene silencing. Following exposure to drought and salt stress, the leaves of FsTyDC1-silenced plants exhibited increased curling and wilting. Conclusions: The results indicate that FsTyDC1 responds to both salt and drought stress, which provides a foundation for further investigation into the function of FsTyDC1.