Enhanced L-theanine production through semi-rational design of γ-glutamylmethylamide synthetase from Methylovorus mays.

The thermal instability of γ-glutamylmethylamide synthetase (GMAS) from Methylovorus mays has imposed limitations on its industrial applications, affecting both stability and activity at reaction temperatures. In this study, disulfide bridges were introduced through a combination of directed evolution and rational design to enhance GMAS stability. Among the variants that we generated, M12 exhibited a 1.46-fold improvement in relative enzyme activity and a 6.23-fold increase in half-life at 40℃ compared to the wild-type GMAS. Employing variant M12 under optimal conditions, we achieved the production of 645.7 mM (112.49 g/L) L-theanine with a productivity of 29.3 mM/h, from 800 mM substrate in an ATP regeneration system. Our strategy significantly enhances the biosynthesis efficiency of L-theanine by preserving the structural stability of the enzyme during the catalysis process.

Multiplex gene knockout raises Ala-Gln production by Escherichia coli expressing amino acid ester acyltransferase.

L-Alanyl-L-Glutamine (Ala-Gln) is a common parenteral nutritional supplement. In our previous study, the recombinant whole-cell catalyst Escherichia coli BL21(DE3) overexpressing α-amino acid ester acyltransferase (BPA) to produce Ala-Gln has high activity and has been applied to large-scale production experiments. However, the degradation of Ala-Gln is detected under prolonged incubation, and endogenous broad-spectrum dipeptidase may be the primary cause. In this study, a CRISPR-Cas9 method was used to target pepA, pepB, pepD, pepN, dpp, and dtp to knock out one or more target genes. The deletion combination was optimized, and a triple knockout strain BL21(DE3)-ΔpepADN was constructed. The degradation performance of the knockout chassis was measured, and the results showed that the degradation rate of Ala-Gln was alleviated by 48% compared with the control. On this basis, BpADNPA (BPA-ΔpepADN) was built, and the production of Ala-Gln was 129% of the BPA's accumulation, proving that the ΔpepADN knockout conducive to the accumulation of dipeptide. This study will push forward the industrialization process of Ala-Gln production by whole-cell catalyst Escherichia coli expressing α-amino acid ester acyltransferase. KEY POINTS: • Endogenous dipeptidase knockout alleviates the degradation of Ala-Gln by the chassis • The balanced gene knockout combination is pepA, pepD, and pepN • The accumulation of Ala-Gln with BpADNPA was 129% of the control.

[Clinical study on real-time three-dimensional CT navigation-guided full-endoscopic lumbar interbody fusion].

Objective: To analyze the technical notes, effectiveness, and current issues of real-time three-dimensional CT navigation-guided full-endoscopic lumbar interbody fusion. Methods: Between April 2020 and October 2021, a total of 27 patients received real-time three-dimensional CT navigation-guided full-endoscopic lumbar interbody fusion. There were 18 males and 9 females with an average age of 63.2 years (range, 48-84 years). There were 6 cases of lumbar spinal stenosis, 1 case of lumbar instability, 9 cases of lumbar spinal stenosis with instability, 3 cases of degenerative spondylolisthesis, 6 cases of isthmus spondylolisthesis, and 2 cases of recurrent lumbar disc herniation. All patients showed neurological symptoms before operation (ipsilateral symptom for 15 cases and bilateral symptom for 12 cases). The symptom duration was 1-300 months (median, 24 months). The operations were performed via transforaminal approach in 8 cases, trans-facet joint approach in 18 cases, and combined approaches in 1 case. A total of 32 levels were fused, including 23 single-level cases, 3 two-level cases, and 1 three-level case. Lumbar fusion segment was L 2, 3 in 1 case, L 3, 4 in 4 cases, L 4, 5 in 20 cases, and L 5, S 1 in 7 cases. The operation time, intraoperative estimated blood loss (IEBL), and perioperative complications were recorded. The improvement of intervertebral space height at fusion level was measured, and the accuracy of percutaneous pedicle screw (PPS) and Cage placement was also evaluated based on CT images performed at 1 week postoperatively. Visual analogue scale (VAS) score for both low back pain and leg pain, Japanese Orthopaedic Association (JOA) score, and Oswestry disability index (ODI) were evaluated before operation, at 1 week postoperatively, and at last follow-up. Satisfaction to effectivenss were assessed by patients using modified MacNab criteria at last follow-up. Results: The operation time was ranged from 255 to 805 minutes (mean, 424.9 minutes). IEBL was 150-290 mL (mean, 219.3 mL). All patients received follow-up with the duration from 4 to 22 months (mean, 12.4 months). At 1 week postoperatively and last follow-up, VAS scores of low back pain and leg pain, JOA score, and ODI were significantly improved when compared with those before operation ( P<0.05). At last follow-up, the clinical indicators were similar in comparison with those at 1 week postoperatively ( P>0.05). There were 26 patients and 1 patient who respectively ranked excellent and mild in terms of effectiveness according to the modified MacNab criteria, with the excellent and good rate of 96.3%. There was 1 patient who suffered from incomplete injury of L 5 nerve root and partial neurological function recovered after 3-month conservative treatments. There were 118 implanted PPSs, and 116 of them were implanted under navigation. There were 33 Cages that were implanted under navigation. The accuracy of PPS and Cage placement was 99.1% and 97.0% respectively based on CT performed at 1 week postoperatively. The postoperative intervertebral space height was significantly increased in comparison with that before operation ( P<0.05). During follow-up, mild Cage subsidence was observed in 1 patient, whereas no fixation loosing was found. Conclusion: Real-time three-dimensional CT navigation-guided full-endoscopic lumbar interbody fusion has great safety and effectiveness with satisfactory preliminary clinical results. Design and further improvement of surgical equipment and instruments are expected to resolve the current technical difficulties.

The novel sRNA s015 improves nisin yield by increasing acid tolerance of Lactococcus lactis F44.

Nisin, a polycyclic antibacterial peptide produced by Lactococcus lactis, is stable at low pH. Improving the acid tolerance of L. lactis could thus enhance nisin yield. Small non-coding RNAs (sRNAs) play essential roles in acid tolerance by regulating their target mRNAs at the post-transcriptional level. In this study, a novel sRNA, s015, was identified in L. lactis F44 via the use of RNA sequencing, qRT-PCR analysis, and Northern blotting. s015 improved the acid tolerance of L. lactis and boosted nisin yield at low pH. In silico predictions enabled us to construct a library of possible s015 target mRNAs. Statistical analysis and validation suggested that s015 contains a highly conserved region (5'-GAAAAAAAC-3') that likely encompasses the regulatory core of the sRNA. atpG, busAB, cysD, ilvB, tcsR, ung, yudD, and ywdA were verified as direct targets of s015, and the interactions between s015 and its target genes were elucidated. This work provided new insight into the adaptation mechanism of L. lactis under acid stress.