N-terminal truncation of prenyltransferase enhances the biosynthesis of prenylnaringenin / 生物工程学报

8-prenylnaringenin (8-PN) is a potent estrogen with high medicinal values. It also serves as an important precursor for many prenylated flavonoids. Microbial synthesis of 8-PN is mainly hindered by the low catalytic activity of prenyltransferases (PTS) and insufficient supply of precursors. In this work, a SfN8DT-1 from Sophora flavescens was used to improve the efficiency of (2S)-naringenin prenylation. The predicted structure of SfN8DT-1 showed that its main body is comprised of 9 α-helices and 8 loops, along with a long side chain formed by nearly 120 amino acids. SfN8DT-1 mutants with different side-chain truncated were tested in Saccharomyces cerevisiae. A mutant expressing the truncated enzyme at K62 site, designated as SfND8T-1-t62, produced the highest 8-PN titer. Molecular docking of SfN8DT-1-t62 with (2S)-naringenin and dimethylallyl diphosphate (DMAPP) showed that K185 was a potentially crucial residue. Alanine scanning within a range of 0.5 nm around these two substrates showed that the mutant K185A may decrease its affinity to substrates, which also indicated K185 was a potentially critical residue. Besides, the mutant K185W enhanced the affinity to ligands implied by the simulated saturation mutation, while the saturated mutation of K185 showed a great decrease in 8-PN production, indicating K185 is vital for the activity of SfN8DT-1. Subsequently, overexpressing the key genes of Mevalonate (MVA) pathway further improved the titer of 8-PN to 31.31 mg/L, which indicated that DMAPP supply is also a limiting factor for 8-PN synthesis. Finally, 44.92 mg/L of 8-PN was produced in a 5 L bioreactor after 120 h, which is the highest 8-PN titer reported to date.

Progress in gene editing technologies for Saccharomyces cerevisiae / 生物工程学报

Saccharomyces cerevisiae is one of the most important hosts in metabolic engineering. Advanced gene editing technology has been widely used in the design and construction of S. cerevisiae cell factories. With the rapid development of gene editing technology, early gene editing technologies based on recombinase and homologous recombination have been gradually replaced by new editing systems. In this review, the principle and application of gene editing technology in S. cerevisiae are summarized. Here, we first briefly describe the classical gene editing techniques of S. cerevisiae. Then elaborate the genome editing system of MegNs, ZFNs and TALENs based on endonuclease. The latest research progress is especially introduced and discussed, including the CRISPR/Cas system, multi-copy integration of heterologous metabolic pathways, and genome-scale gene editing. Finally, we envisage the application prospects and development directions of Saccharomyces cerevisiae gene editing technology.

Clinical analysis of pedicle screw fixation in the treatment of type Ⅱ odontoid fracture with bone grafting and non-bone grafting / 中国基层医药

Objective To explore the clinical efficacy of bone grafting in the treatment of type Ⅱ odontoid fracture with pedicle screw fixation .Methods Of 14 cases with type Ⅱ odontoid fractures ,8 patients in group A received pedicle screw,lamina autologous bone grafts,6 cases of group B received simple posterior pedicle screw fixation.The operative time ,the amount of surgical bleeding and the postoperative cervical spine flexion and rotationwere observed at 3 months postoperatively ,and the patients were followed up for 5-45 months.Results All patients were followed up for 5-45 months,with an average of 26.5 months.The operation time was (1.83 ±0.5) h in the bone graft group,and (1.58 ±0.9)h in the non-bone graft group,the difference was statistically significant (t=2.842,P>0.05).The blood loss of the bone graft group was (150 ±16)mL,which of the non-bone graft group was (120 ±14) mL,the difference between the two groups was statistically significant (t =3.57,P >0.05).After 3 months,the flexion of the cervical spine of the bone graft group was (31.2 ±4.6)°,which in the non -grafted group was (32.3 ±5.7)°,the difference was statistically significant (t=0.675,P<0.05).The rotation of the bone graft group was (40.6 ±4.5)°,which in the non -graft group was (41.3 ±3.5)°,the difference was statistically significant (t=0.278,P<0.05).Both two groups had no vertebral artery and spinal cord injury ,wound healing. During the follow-up period,the two groups of patients had a good reduction of cervical spine ,no internal fixation lossening,fracture,fracture healing well,group A bone graft fusion.Conclusion For this type of fracture,simple atlantoaxial pedicle screw fixation compared with autologous iliac bone graft fusion treatment ,can save the operation time,reduce the amount of bleeding .