Lactobacillus casei has drawn increasing attention as a health-promoting probiotic, while effective genetic manipulation tools are often not available, e.g., the single-gene knockout in L. casei still depends on the classic homologous recombination-dependent double-crossover strategy, which is quite labor-intensive and time-consuming. In the present study, a rapid and precise genome editing plasmid, pLCNICK, was established for L. casei genome engineering based on CRISPR-Cas9 ^D10A . In addition to the P ₂₃ -Cas9 ^D10A and P _ldh -sgRNA (single guide RNA) expression cassettes, pLCNICK includes the homologous arms of the target gene as repair templates. The ability and efficiency of chromosomal engineering using pLCNICK were evaluated by in-frame deletions of four independent genes and chromosomal insertion of an enhanced green fluorescent protein (eGFP) expression cassette at the LC2W_1628 locus. The efficiencies associated with in-frame deletions and chromosomal insertion is 25 to 62%. pLCNICK has been proved to be an effective, rapid, and precise tool for genome editing in L. casei , and its potential application in other lactic acid bacteria (LAB) is also discussed in this study. IMPORTANCE The lack of efficient genetic tools has limited the investigation and biotechnological application of many LAB. The CRISPR-Cas9 ^D10A nickase-based genome editing in Lactobacillus casei , an important food industrial microorganism, was demonstrated in this study. This genetic tool allows efficient single-gene deletion and insertion to be accomplished by one-step transformation, and the cycle time is reduced to 9 days. It facilitates a rapid and precise chromosomal manipulation in L. casei and overcomes some limitations of previous methods. This editing system can serve as a basic technological platform and offers the possibility to start a comprehensive investigation on L. casei . As a broad-host-range plasmid, pLCNICK has the potential to be adapted to other Lactobacillus species for genome editing.

Background: Endophytic fungi represent underexplored resource of novel lead compounds and have a capacity to produce diverse class of plant secondary metabolites. Here we investigated endophytic fungi diversity and screening of paclitaxel-producing fungi from Taxus x media. Results: Eighty-one endophytic fungi isolated from T. media were grouped into 8 genera based on the morphological and molecular identification. Guignardia and Colletotrichum were the dominant genera, whereas the remaining genera were infrequent groups. The genera Glomerella and Gibberella were first reported in Taxus. Three representative species of the distinct genera gave positive hits by molecular marker screening and were capable of producing taxol which were validated by HPLC-MS. Among these 3 taxol-producing fungi, the highest yield of taxol was 720 ng/l by Guignardia mangiferae HAA11 compared with those of Fusarium proliferatum HBA29 (240 ng/l) and Colletotrichum gloeosporioides TA67 (120 ng/l). This is the first report of taxol producer from Guignardia. Moreover, the lower similarities of ts and bapt between microbial and plant origin suggested that fungal taxol biosynthetic cluster might be repeatedly invented during evolution, nor horizontal gene transfer from Taxus species. Conclusions: Taxol-producing endophytic fungi could be a fascinating reservoir to generate taxol-related drug lead and to elucidate the remained 5 unknown genes or the potential regulation mechanism in the taxol biosynthesis pathway.

Marine microbial natural products (MMNPs) have attracted increasing attention from microbiologists, taxonomists, ecologists, agronomists, chemists and evolutionary biologists during the last few decades. Numerous studies have indicated that diverse marine microbes appear to have the capacity to produce an impressive array of MMNPs exhibiting a wide variety of biological activities such as antimicrobial, anti-tumor, anti-inflammatory and anti-cardiovascular agents. Marine microorganisms represent an underexplored reservoir for the discovery of MMNPs with unique scaffolds and for exploitation in the pharmaceutical and agricultural industries. This review focuses on MMNPs discovery and development over the past decades, including innovative isolation and culture methods, strategies for discovering novel MMNPs via routine screenings, metagenomics, genomics, combinatorial biosynthesis, and synthetic biology. The potential problems and future directions for exploring MMNPs are also discussed.

Hypercholesterolemia is closely associated with cardiovascular disease. Supplementation with probiotics has been shown to contribute to improving lipid metabolism. The probiotic mechanisms of cholesterol reduction are complicated and remain unclear. One of the potential probiotic mechanisms for cholesterol reduction is the deconjugation of bile salts. We previously found a high bile salt hydrolase (BSH) activity of Lactobacillus casei pWQH01 (overexpression of bsh1) and Lactobacillus plantarum AR113, but found no BSH activity for Lactobacillus casei LC2W in vitro. Therefore, we decided to investigate whether the high BSH activity of L. plantarum AR113 and L. casei pWQH01 could exert a cholesterol-reducing effect in vivo. Compared to the high-cholesterol diet (HCD) group, AR113 and pWQH01 groups had a significantly lower body weight (BW), serum total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) levels and atherogenic index (AI), whereas the LC2W group had a poor capability to mitigate the blood lipid levels in the hypercholesterolemic mice. In addition, the AR113 and pWQH01 groups decreased the hepatic levels of TC and LDL-C and improved hepatic steatosis compared with the HCD group. To explore their cholesterol-lowering mechanisms of action, we determined the expression levels of these genes on the cholesterol metabolic pathways. AR113 and pWQH01 groups downregulated the expression of farnesoid X receptor (FXR) and small heterodimer partner (SHP) genes, but upregulated the expression of the cholesterol 7α-hydroxylase (CYP7A1) gene in the liver. Simultaneously, the expression of cholesterol liver X receptor (LXR) and low density lipoprotein receptor (LDLR) genes was upregulated in the liver. These results indicated that L. plantarum AR113 and L. casei pWQH01 could inhibit the cholesterol absorption and accelerate the cholesterol transportation. Taken together, these findings suggest that Lactobacillus strain overexpression of bile salt hydrolase has beneficial effects against hypercholesterolemia by reducing cholesterol absorption and increasing cholesterol catabolism.

Ligilactobacillus salivarius (basonym: Lactobacillus salivarius, L. salivarius) is a type of lactic acid bacteria (LAB) commonly found in the oropharyngeal-gastrointestinal tract (OGT). It has gained significant attention due to its probiotic and functional properties as well as its various health-promoting roles. L. salivarius strains exhibit strong resistance and adhesion in the OGT along with outstanding antioxidant and antimicrobial properties. Additionally, numerous L. salivarius strains have the ability to produce bacteriocins with antagonistic activity. These probiotic characteristics of L. salivarius indicate its remarkable potential in promoting favorable effects on human health. It has also been observed that L. salivarius has a positive effect on the composition of intestinal microbiota, thereby improving the metabolic profiling of intestinal microbiota, promoting a healthy and balanced internal environment. In recent years, multi-omics technologies such as genomics, transcriptomics, proteomics and metabolomics have been employed to gain a deeper understanding of the roles and mechanisms of L. salivarius associated with its functional properties. This review aims to provide an overview of the probiotic characteristics of L. salivarius, containing its specific interactions with the host microflora, as well as insights from omics studies.