Progress in Gene Editing and Metabolic Regulation of Saccharomyces cerevisiae with CRISPR/Cas9 Tools.

The CRISPR/Cas9 systems have been developed as tools for genetic engineering and metabolic engineering in various organisms. In this review, various aspects of CRISPR/Cas9 in Saccharomyces cerevisiae, from basic principles to practical applications, have been summarized. First, a comprehensive review has been conducted on the history of CRISPR/Cas9, successful cases of gene disruptions, and efficiencies of multiple DNA fragment insertions. Such advanced systems have accelerated the development of microbial engineering by reducing time and labor, and have enhanced the understanding of molecular genetics. Furthermore, the research progress of the CRISPR/Cas9-based systems in the production of high-value-added chemicals and the improvement of stress tolerance in S. cerevisiae have been summarized, which should have an important reference value for genetic and synthetic biology studies based on S. cerevisiae.

The strategy of cell extract based metal organic frameworks (CE-MOF) for improved enzyme characteristics.

A convenient cell extract based metal organic frameworks (CE-MOF) strategy was used to produce self-assembled hybrid microparticles of enzymes with improved characteristics. It was shown that many metal ions and enzymes could be used to construct catalytically active CE-MOF microparticles. As a proof-of-principle study, the ß-xylosidase BH3683 was used to prepare FeSO4-CE-MOF-BH3683 microparticles to explore the factors influencing preparation of the microparticles. As a result, DNA, RNA, polysaccharides and proteins were found to play important roles in the formation of the microparticles and affected enzyme activities through interaction with enzyme molecules. Compared with the free BH3683, the optimum temperature of FeSO4-CE-MOF-BH3683 increased 5 °C, and the relative activity at 70 °C increased two times. Moreover, FeSO4-CE-MOF-BH3683 have stronger tolerance to different concentrations of various organic solvents and high-concentration xylose than the free BH3683, and the CE-MOF microparticles prepared by BH3683 and xylanase XynII could catalyze high-concentration xylan more efficiently than their free counterparts. In addition, FeSO4-CE-MOF-BH3683 exhibited about 40 % of its initial activity after reused for 10 times, showing satisfactory reusability. To sum up, this strategy might have wide application potential in the fields of biocatalysis, biofuel production, fertilizer industry, etc.

Novel biocatalytic strategy of levan: His-ELP-intein-tagged protein purification and biomimetic mineralization.

Here a versatile fusion tag composed of His-tag, intein, and elastin-like polypeptide (ELP) tag was prepared for the first time to be fused with levansucrase SacB to construct a recombinant His-ELP-intein-SacB (HEIS) protein to realize nonchromatographic purification of SacB. The efficient biomimetic mineralization of CaHPO4 and HEIS-based hybrid-hydrangea (CaHPO4-HEIS-HH) with good reusability, excellent storage stability and 254.3% improved relative levan yield was prepared with the biomimetic mineralization method. Additionally, the CaHPO4-HEIS-HH showed outstanding operation activity when catalyzing sucrose in solution and up to 75% sucrose conversion rate in fruit juices. The mechanism of biomimetic mineralization was analyzed to show that the HEIS protein might serve as a "binder" to assemble the nanoflakes during biomimetic mineralization. The CaHPO4-HEIS-HH was applicable for efficient production of the levan-type prebiotic polysaccharides, and this approach should be highly valuable for nonchromatographic purification and convenient preparation of various encapsulated enzymes for more efficient catalysis.