Characterization of an efficient CRISPR-iCas9 system in Yarrowia lipolytica for the biosynthesis of carotenoids.

The application of clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas9) technology in the genetic modification of Yarrowia lipolytica is challenged by low efficiency and low throughput. Here, a highly efficient CRISPR-iCas9 (with D147Y and P411T mutants) genetic manipulation tool was established for Y. lipolytica, which was further utilized to integrate carotene synthetic key genes and significantly improve the target product yield. First, CRISPR-iCas9 could shorten the time of genetic modification and enable the rapid knockout of nonsense suppressors. iCas9 can lead to more than 98% knockout efficiency for NHEJ-mediated repair after optimal target disruption of a single gene, 100% knockout efficiency for a single gene-guided version, and more than 80% knockout efficiency for multiple genes simultaneously in Y. lipolytica. Subsequently, this technology allowed for rapid one-step integration of large fragments (up to 9902-bp) of genes into chromosomes. Finally, YL-ABTG and YL-ABTG2Z were further constructed through CRISPR-iCas9 integration of key genes in a one-step process, resulting in a maximum ß-carotene and zeaxanthin content of 3.12 mg/g and 2.33 mg/g dry cell weight, respectively. Therefore, CRISPR-iCas9 technology provides a feasible approach to genetic modification for efficient biosynthesis of biological compounds in Y. lipolytica. KEY POINTS: • iCas9 with D147Y and P411T mutants improved the CRISPR efficiency in Y. lipolytica. • CRISPR-iCas9 achieved efficient gene knockout and integration in Y. lipolytica. • CRISPR-iCas9 rapidly modified Y. lipolytica for carotenoid bioproduction.

Developing efficient vanillin biosynthesis system by regulating feruloyl-CoA synthetase and enoyl-CoA hydratase enzymes.

Vanillin is one of the most commonly used natural-occurring flavors in the world. This study successfully constructed an efficient whole-cell catalytic system for vanillin biosynthesis from ferulic acid by regulating feruloyl-CoA synthetase (FCS) and enoyl-CoA hydratase (ECH). First, we constructed an efficient cell-free catalytic system with FCS-Str (fcs from Streptomyces sp. V-1) and ECH-Str (ech from Streptomyces sp. V-1) combination at 1:1. The efficient cell-free catalytic system provided necessary strategies for optimizing the whole-cell catalytic system. Then, we constructed the recombinant Escherichia coli by heterologously expressing the fcs-Str and ech-Str combination. Moreover, E. coli JM109 was a better recombinant Escherichia coli than E. coli BL21 with higher vanillin production. Finally, we first adjusted the ratio of FCS and ECH in E. coli JM109 to 1:1 using two copies of fcs-Str. For higher vanillin production, we further optimized the induction conditions of E. coli JM109 to increase the amount of FCS and ECH. The optimized E. coli JM109-FE-F constructed in this study has the highest vanillin synthesis rate of converting 20 mM ferulic acid to 15 mM vanillin in 6 h among all of the E. coli catalytic systems. Our study made a significant contribution to the construction of the vanillin biosynthesis system and provided a valuable strategy for increasing vanillin production. KEY POINTS: • The efficient cell-free vanillin biosynthesis system was constructed by FCS-Str and ECH-Str combination at 1:1. • Escherichia coli JM109 was determined as a better recombinant Escherichia coli than E. coli BL21 with higher vanillin production. • Escherichia coli JM109-FE-F with two copies of fcs-Str and one copy of ech-Str has the highest catalytic efficiency for vanillin production.

[Clinical analysis of 15 cases of saddle pulmonary embolism].

OBJECTIVE: To improve recognition of saddle pulmonary embolism (SPE). METHODS: A retrospectively review was performed for patients diagnosed with SPE determined by CTPA from Jan 2004 to Jan 2012. RESULTS: Fifteen SPE patients(4.44%) were found in 338 documented PE patients confirmed by CTPA. There were 7 males and 8 females, with an average age of (57 ± 13) years. The bifurcation of the main pulmonary artery was completely blocked in one case, while partial obstruction was found in the others. Hemodynamic stability was observed in 11 cases, shock in 1 case, and hypotension in 3 cases. Thromboembolectomy was performed in 1 case accompanied by patent foramen ovale straddling thrombus, and thrombolytic therapy was administered in 5 cases while anticoagulant therapy alone in 9 cases. All the cases survived. Minor bleeding was observed in 2 patients and no major bleeding occurred. CONCLUSION: The prevalence of SPE in this series was similar to that reported in the literature. But the incidence might be underestimated. Mortality rate was low. No more aggressive therapeutic interventions (thrombolytics or catheter thrombectomy) were needed in those patients with hemodynamic stability and without patent foramen ovale straddling thrombus.