Overproduction of Patchoulol in Metabolically Engineered Komagataella phaffii.

Patchoulol, a plant-derived sesquiterpene compound, is widely used in perfumes, cosmetics, and pharmaceuticals. Microbial production provides a promising alternative approach for the efficient and sustainable production of patchoulol. However, there are no systematic engineering studies on Komagataella phaffii aimed at achieving high-yield patchoulol production. Herein, by fusion overexpression of FPP synthase and patchoulol synthase (ERG20LPTS), increasing the precursor supply, adjusting the copy number of ERG20LPTS and PTS, and combined with adding auxiliary carbon source and methanol concentration optimization, we constructed a high-yield patchoulol-producing strain P6H53, which produced 149.64 mg/L patchoulol in shake-flask fermentation with methanol as the substrate. In fed-batch fermentation, strain P6H53 achieved the highest production (2.47 g/L, 21.48 mg/g DCW, and 283.25 mg/L/d) to date in a 5 L fermenter. This study will lay a good foundation for the development of K. phaffii as a promising chassis microbial cell for the synthesis of patchoulol and other sesquiterpenes with methanol as the carbon source.

The in vitro fermentation of compound oral liquid by human colonic microbiota altered the abundance of probiotics and short-chain fatty acid production.

Compound oral liquid (COL), made from functional herbal foods, has gained immense popularity in China for healthcare. However, the interaction between the nutrients in COL and gut microbiota is still unclear. In our study, the content of total flavonoids, polyphenols, and proteins was increased and the total sugar reduced by crushing raw ingredients to 10 mesh (COL-C). After 24 h incubation with supplemented COL by human gut microbiota, the results of 16S rRNA high-throughput sequencing revealed that Faecalibacterium, Collinsella, Bifidobacterium, Megamonas, Lactobacillus, Phascolarctobacterium, and Dialister were enriched by COL. In particular, the latter three genera were observed to be significantly enriched after incubation with COL-C. Meanwhile, the abundance of Dorea, Clostridium XIVa, and Escherichia/Shigella was inhibited by COL. Moreover, the increased levels of acetate, propionate, and butyrate in COL were jointly contributed by supplementary carbohydrates and the enrichment of short-chain fatty acid (SCFA)-producing bacteria. In summary, our results indicated that the optimized extraction facilitated the nutrients to be dissolved out and enhanced the potential prebiotic effects for promoting the abundance of probiotics, suggesting that the nutrients in COL-C might improve the microbial structure by strengthening the metabolism of beneficial bacteria and restricting the conditioned pathogens more efficiently.

Association of human gut microbiota composition and metabolic functions with Ficus hirta Vahl dietary supplementation.

Ficus hirta Vahl (FHV), a traditional herbal ingredient of the tonic diet, receives increasing popularity in southern China. However, it is largely unknown that how a FHV diet (FHVD) affects the human gut microbiome. In this exploratory study, a total of 43 healthy individuals were randomized into the FHVD (n = 25) and Control (n = 18) groups to receive diet intervention for 8 weeks. 16S rRNA gene sequencing, metagenomic sequencing and metabolic profile of participants were measured to assess the association between FHV diet and gut microbiome. A preservation effect of Faecalibacterium and enrichment of Dialister, Veillonella, Clostridium, and Lachnospiraceae were found during the FHVD. Accordingly, the pathway of amino acid synthesis, citrate cycle, coenzyme synthesis, and partial B vitamin synthesis were found to be more abundant in the FHVD. In addition, serine, glutamine, gamma-aminobutyric acid, tryptamine, and short-chain fatty acids (SCFAs) were higher after the FHVD. The conjoint analysis of FHV components and in-vitro fermentation confirmed that the improved SCFAs concentration was collectively contributed by the increasing abundance of key enzyme genes and available substrates. In conclusion, the muti-omics analysis showed that the FHVD optimized the structure of the gut microbial community and its metabolic profile, leading to a healthy tendency, with a small cluster of bacteria driving the variation rather than a single taxon.

Modulation of Gut Microbiota Composition and Short-Chain Fatty Acid Synthesis by Mogroside V in an In Vitro Incubation System.

Mogroside V (MV), a sweetener, is one of the major components inSiraitia grosvenorii. In our research, after in vitro incubation with MV for 24 h, the human gut microbiota diversity changed, with an enrichment of the genera Bacteroides, Lactobacillus, Prevotella, Megasphaera, and Olsenella and the inhibition of Clostridium XlVa, Dorea, and Desulfovibrio. Moreover, the synthesis of short-chain fatty acids, such as acetate, propionate, and butyrate, was increased by gut microbiota. According to ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) analysis, MV was decomposed into secondary mogrosides, such as mogroside II/I and mogrol, by gut microbiota. Enhanced antioxidant abilities of the metabolites were found in the broth. The results suggested that MV, as a potential prebiotic, could benefit human health through its interaction with gut microbiota.

Enhancing co-translational folding of heterologous protein by deleting non-essential ribosomal proteins in Pichia pastoris.

BACKGROUND: Translational regulation played an important role in the correct folding of heterologous proteins to form bioactive conformations during biogenesis. Translational pausing coordinates protein translation and co-translational folding. Decelerating translation elongation speed has been shown to improve the soluble protein yield when expressing heterologous proteins in industrial expression hosts. However, rational redesign of translational pausing via synonymous mutations may not be feasible in many cases. Our goal was to develop a general and convenient strategy to improve heterologous protein synthesis in Pichia pastoris without mutating the expressed genes. RESULTS: Here, a large-scale deletion library of ribosomal protein (RP) genes was constructed for heterologous protein expression in Pichia pastoris, and 59% (16/27) RP deletants have significantly increased heterologous protein yield. This is due to the delay of 60S subunit assembly by deleting non-essential ribosomal protein genes or 60S subunit processing factors, thus globally decreased the translation elongation speed and improved the co-translational folding, without perturbing the relative transcription level and translation initiation. CONCLUSION: Global decrease in the translation elongation speed by RP deletion enhanced co-translational folding efficiency of nascent chains and decreased protein aggregates to improve heterologous protein yield. A potential expression platform for efficient pharmaceutical proteins and industrial enzymes production was provided without synonymous mutation.