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Tunicosaponins are natural products extracted from Psammosilene tunicoides, which is an important ingredient of Yunnan Baiyao Powder, an ancient and famous Asian herbal medicine. The representative aglycones of tunicosaponins are the oleanane-type triterpenoids of gypsogenin and quillaic acid, which were found to manipulate a broad range of virus-host fusion via wrapping the heptad repeat-2 (HR2) domain prevalent in viral envelopes. However, the unknown biosynthetic pathway and difficulty in chemical synthesis hinder the therapeutic use of tunicosaponins. Here, two novel cytochrome P450-dependent monooxygenases that take part in the biosynthesis of tunicosaponins, CYP716A262 (CYP091) and CYP72A567 (CYP099), were identified from P. tunicoides. In addition, the whole biosynthesis pathway of the tunicosaponin aglycones was reconstituted in yeast by transforming the platform strain BY-bAS with the CYP716A262 and CYP716A567 genes, the resulting strain could produce 146.84 and 314.01 mg/L of gypsogenin and quillaic acid, respectively. This synthetic biology platform for complicated metabolic pathways elucidation and microbial cell factories construction can provide alternative sources of important natural products, helping conserve natural plant resources.

Large hydrophobic molecules, such as carotenoids, cannot be effectively excreted from cells by natural transportation systems. These products accumulate inside the cells and affect normal cellular physiological functions, which hinders further improvement of carotenoid production by microbial cell factories. In this study, we proposed to construct a novel artificial transport system utilizing membrane lipids to carry and transport hydrophobic molecules. Membrane lipids allow the physiological mechanism of membrane dispersion to be reconstructed and amplified to establish a novel artificial membrane vesicle transport system (AMVTS). Specifically, a few proteins in E. coli were reported or proposed to be related to the formation mechanism of outer membrane vesicles, and were individually knocked out or overexpressed to test their physiological functions. The effects on tolR and nlpI were the most significant. Knocking out both tolR and nlpI resulted in a 13.7% increase of secreted β-carotene with a 35.6% increase of specific production. To supplement the loss of membrane components of the cells due to the increased membrane vesicle dispersion, the synthesis pathway of phosphatidylethanolamine was engineered. While overexpression of AccABCD and PlsBC in TW-013 led to 15% and 17% increases of secreted β-carotene, respectively, the overexpression of both had a synergistic effect and caused a 53-fold increase of secreted β-carotene, from 0.2 to 10.7 mg/g dry cell weight (DCW). At the same time, the specific production of β-carotene increased from 6.9 to 21.9 mg/g DCW, a 3.2-fold increase. The AMVTS was also applied to a β-carotene hyperproducing strain, CAR025, which led to a 24-fold increase of secreted β-carotene, from 0.5 to 12.7 mg/g DCW, and a 61% increase of the specific production, from 27.7 to 44.8 mg/g DCW in shake flask fermentation. The AMVTS built in this study establishes a novel artificial transport mechanism different from natural protein-based cellular transport systems, which has great potential to be applied to various cell factories for the excretion of a wide range of hydrophobic compounds.

With the development of CRISPR/Cas9 technology, a new generation of editing methods that convert specific bases has enabled precise single-base mutations. To date, conversion of cytosine to thymidine and adenine to guanine has been achieved using the cytidine deaminase APOBEC1 and adenosine deaminase (TadA), respectively. However, the base editing efficiency can be unacceptably low in some cell types or at certain target loci. One reason might be the lack of a selective pressure against the survival of nonedited cells. Few studies on ABE in prokaryotes have been reported, probably due to the relatively low editing efficiency of TadA. Improving the editing efficiency is the key for establishing base editing techniques and especially the ABE technologies. In this work, a selective pressure against nonedited cells was implemented to increase the base editing efficiency. First, we fused nCas9 or dCas9 with TadA to compare the editing efficiency of nCas9-TadA and dCas9-TadA fusion complexes in the model prokar...