Simultaneously down-regulation of multiplex branch pathways using CRISPRi and fermentation optimization for enhancingβ-amyrin production in Saccharomyces cerevisiae

The production ofβ-amyrin in Saccharomyces cerevisiae is still low due to the inability of effectively regulating the endogenous metabolic pathway for competitive synthesis ofβ-amyrin precursors.In this study,we focused on two branches ofβ-amyrin synthetics pathway that consumeβ-amyrin precursors(2,3-oxidosqualene and cytosolic acetyl-CoA)and regulated related genes(ADH1,ADH4,ADH5,ADH6,CIT2,MLS2 and ERG7).We developed a CRISPRi method by constructing a multi-gRNA plasmid to down-regulate the seven genes simultaneously,which is reported for the first time in S.cerevisiae.The average transcription inhibition efficiency of the seven genes reached as high as 75.5%.Furthermore,by optimizing the fermentation condition(including pH,inoculum size,initial glucose concentration and feed of glucose or ethanol)and increasing extracellular transportation via supplying methyl-β-cyclodextrin,β-amyrin concentration of engineered strain SGibSdCg increased by 44.3%compared with the parent strain SGib,achieving 156.7 mg/L which was the highest concentration ofβ-amyrin reported in yeast.The one-step down-regulation of multiple genes using CRISPRi showed high efficiency and promising future in improving the yields of natural products.

Resistance mechanisms and reprogramming of microorganisms for efficient biorefinery under multiple environmental stresses

In the fermentation process of biorefinery,industrial strains are normally subjected to adverse environmental stresses,which leads to their slow growth,yield decline,a substantial increase in energy consumption,and other negative consequences,which ultimately seriously hamper the development of biorefinery.How to minimize the impact of stress on microorganisms is of great significance.This review not only reveals the damaging effects of different environmental stresses on microbial strains but also introduces commonly used strategies to improve microbial tolerance,including adaptive evolution,reprogramming of the industrial host based on genetic circuits,global transcription machinery engineering(gTME)and bioprocess integration.Furthermore,by integrating the advantages of these strategies and reducing the cost of system operation,the tolerance of industrial strains,combined with production efficiency and process stability,will be greatly improved,and the development prospects of biorefinery will be more widespread.

Construction of a CaHP04-PGUSl hybrid nanoflower through protein-inorganic self-assembly, and its application in glycyrrhetinic acid 3-0-mono-β-D-glucuronide preparation

Glycyrrhetinic acid 3-0-mono-β-D-glucuronide (GAMG), an important pharmaceutical intermediate and functional sweetener, has broad applications in the food and medical industries. A green and cost-effective method for its preparation is highly desired. Using sitedirected mutagenesis, we previously obtained a variant of β-glucuronidase from Aspergillus oryzae Li-3 (PGUS1), which can specifically transform glycyrrhizin (GL) into GAMG. In this study, a facile method was established to prepare a CaHP04-PGUSl hybrid nanoflower for enzyme immobilization, based on protein-inorganic hybrid selfassembly. Under optimal conditions, 1.2 mg of a CaHP04- PGUS1 hybrid nanoflower precipitate with 71.2% immobilization efficiency, 35.60 mg·g^-1 loading capacity, and 118% relative activity was obtained. Confocal laser scanning microscope and scanning electron microscope results showed that the enzyme was encapsulated in the CaHP04-PGUSl hybrid nanoflower. Moreover, the thermostability of the CaHP04-PGUSl hybrid nanoflower at 55°C was improved, and its half-life increased by 1.3 folds. Additionally, the CaHP04-PGUSl hybrid nanoflower was used for the preparation of GAMG through GL hydrolysis, with the conversion rate of 92% in 8 h, and after eight consecutive runs, it had 60% of its original activity.