Transfer of disulfide bond formation modules via yeast artificial chromosomes promotes the expression of heterologous proteins in Kluyveromyces marxianus.

Kluyveromyces marxianus is a food-safe yeast with great potential for producing heterologous proteins. Improving the yield in K. marxianus remains a challenge and incorporating large-scale functional modules poses a technical obstacle in engineering. To address these issues, linear and circular yeast artificial chromosomes of K. marxianus (KmYACs) were constructed and loaded with disulfide bond formation modules from Pichia pastoris or K. marxianus. These modules contained up to seven genes with a maximum size of 15 kb. KmYACs carried telomeres either from K. marxianus or Tetrahymena. KmYACs were transferred successfully into K. marxianus and stably propagated without affecting the normal growth of the host, regardless of the type of telomeres and configurations of KmYACs. KmYACs increased the overall expression levels of disulfide bond formation genes and significantly enhanced the yield of various heterologous proteins. In high-density fermentation, the use of KmYACs resulted in a glucoamylase yield of 16.8 g/l, the highest reported level to date in K. marxianus. Transcriptomic and metabolomic analysis of cells containing KmYACs suggested increased flavin adenine dinucleotide biosynthesis, enhanced flux entering the tricarboxylic acid cycle, and a preferred demand for lysine and arginine as features of cells overexpressing heterologous proteins. Consistently, supplementing lysine or arginine further improved the yield. Therefore, KmYAC provides a powerful platform for manipulating large modules with enormous potential for industrial applications and fundamental research. Transferring the disulfide bond formation module via YACs proves to be an efficient strategy for improving the yield of heterologous proteins, and this strategy may be applied to optimize other microbial cell factories.

Characterization and optimization of 5´ untranslated region containing poly-adenine tracts in Kluyveromyces marxianus using machine-learning model.

BACKGROUND: The 5´ untranslated region (5´ UTR) plays a key role in regulating translation efficiency and mRNA stability, making it a favored target in genetic engineering and synthetic biology. A common feature found in the 5´ UTR is the poly-adenine (poly(A)) tract. However, the effect of 5´ UTR poly(A) on protein production remains controversial. Machine-learning models are powerful tools for explaining the complex contributions of features, but models incorporating features of 5´ UTR poly(A) are currently lacking. Thus, our goal is to construct such a model, using natural 5´ UTRs from Kluyveromyces marxianus, a promising cell factory for producing heterologous proteins. RESULTS: We constructed a mini-library consisting of 207 5´ UTRs harboring poly(A) and 34 5´ UTRs without poly(A) from K. marxianus. The effects of each 5´ UTR on the production of a GFP reporter were evaluated individually in vivo, and the resulting protein abundance spanned an approximately 450-fold range throughout. The data were used to train a multi-layer perceptron neural network (MLP-NN) model that incorporated the length and position of poly(A) as features. The model exhibited good performance in predicting protein abundance (average R2 = 0.7290). The model suggests that the length of poly(A) is negatively correlated with protein production, whereas poly(A) located between 10 and 30 nt upstream of the start codon (AUG) exhibits a weak positive effect on protein abundance. Using the model as guidance, the deletion or reduction of poly(A) upstream of 30 nt preceding AUG tended to improve the production of GFP and a feruloyl esterase. Deletions of poly(A) showed inconsistent effects on mRNA levels, suggesting that poly(A) represses protein production either with or without reducing mRNA levels. CONCLUSION: The effects of poly(A) on protein production depend on its length and position. Integrating poly(A) features into machine-learning models improves simulation accuracy. Deleting or reducing poly(A) upstream of 30 nt preceding AUG tends to enhance protein production. This optimization strategy can be applied to enhance the yield of K. marxianus and other microbial cell factories.

[Application of unilateral biportal endoscopy technique in single-segment thoracic ossification of ligamentum flavum].

Objective: To investigate the safety and effectiveness of unilateral biportal endoscopy (UBE) technique in the treatment of single-segment thoracic ossification of ligamentum flavum (TOLF). Methods: Between August 2020 and December 2021, 11 patients with single-segment TOLF were treated with UBE technique. There were 6 males and 5 females, with an average of 58.2 years (range, 49-72 years). The responsible segment was T 6, 7 in 1 case, T 7, 8 in 1 case, T 8, 9 in 2 cases, T 9, 10 in 2 cases, T 10, 11 in 2 cases, and T 11, 12 in 3 cases. Imaging examination showed that the ossification were located on the left side in 4 cases, on the right side in 3 cases, and on bilateral sides in 4 cases. The main clinical symptoms were chest and back pain or lower limb pain, all accompanied by lower limb numbness and fatigue. The disease duration ranged from 2 to 28 months (median, 17 months). The operation time, postoperative hospital stay, and complications were recorded. Visual analogue scale (VAS) score was used to evaluate the chest and back pain and low limb pain, and Oswestry disability index (ODI) and Japanese Orthopedic Association (JOA) score were used to evaluate functional recovery before operation and at 3 days, 1 month, 3 months after operation, and last follow-up. The anteroposterior diameter of the coronal spinal canal was measured by CT before and after operation to evaluate the effect of surgical decompression. Results: All operations were successfully completed. The operation time was 50-105 minutes, with an average of 80.0 minutes. No postoperative complication such as dural sac tear, cerebrospinal fluid leakage, spinal nerve injury, or infection occurred. The postoperative hospital stay was 2-5 days, with an average of 3.1 days. All incisions healed by first intention. All patients were followed up 6-22 months, with an average of 14.8 months. CT measurement at 3 days after operation showed that the anteroposterior diameter of the spinal canal was (8.63±1.61) mm, which was significantly larger than that before operation [(3.67±1.37) mm] ( t=-12.181, P<0.001). The VAS score of chest and back pain and lower limb pain and ODI at each time point after operation were significantly lower than those before operation ( P<0.05). The above indexes were further improved after operation, except that there was no significant difference between at 3 months after operation and at last follow-up ( P>0.05), the differences between other time points were significant ( P<0.05). There was no recurrence during the follow-up period. Conclusion: UBE technique is a safe and effective method to treat single-segment TOLF, but its long-term effectiveness needs to be further studied.

Retrospective Study of Minimal Three-Year Follow-Up of Transforaminal Endoscopic Discectomy for Lumbar Disc Herniation: 5000 Multicenter Cases.

Objective The objective of this study was to investigate the long-term effects of percutaneous transforaminal endoscopic lumbar discectomy (PTELD) and clarify the differences between outside-in and inside-out techniques. Methodology This was a multicenter retrospective study with a chart review of questionnaires about patients' quality of life. Patients were recruited from three hospitals in China. Based on certain inclusion and exclusion criteria, we enrolled in the study 5000 patients aged ≥18 years diagnosed with lumbar disc herniation who received PTELD from September 2015 to September 2019. The outside-in technique (n=2039) was compared with the inside-out technique for PTELD (n=1890) on the Visual Analogue Scale (VAS), Oswestry Disability Index (ODI), and the Short Form 36 (SF-36) of the Health Survey Questionnaire (physical component) both pre-operatively and post-operatively. Results VAS, ODI, and SF-36 significantly improved just after surgery for both techniques compared with pre-operative status. Nevertheless, significant differences existed between the two techniques concerning VAS for leg pain, VAS for back pain, ODI, and SF-36 at 0.5 months post-operatively. The above indices steadily improved within six months after both techniques, after which they did not significantly improve. In detail, outside-in patients suffered more back pain and worse ODI and SF-36 (physical) but had more relief from leg pain 0.5 months after surgery in terms of VAS. As for recovery rate from symptoms, there were only significant differences in recovery rate for leg pain and back pain at the first 1.5 months post-operatively. As for satisfaction rates, the outside-in technique had better results than the inside-out technique at both 0.5 months and 12 months. Conclusion Both techniques could relieve the symptoms of lumbar disc herniation. However, patients in the outside-in group suffered more back pain and a bigger risk of nerve injury than those in the inside-out group.

Identification of a Novel NtLRR-RLK and Biological Pathways That Contribute to Tolerance of TMV in Nicotiana tabacum.

Tobacco mosaic virus (TMV) infection can causes serious damage to tobacco crops. To explore the approach of preventing TMV infection of plants, two tobacco cultivars with different resistances to TMV were used to analyze transcription profiling before and after TMV infection. The involvement of biological pathways differed between the tolerant variety (Yuyan8) and the susceptible variety (NC89). In particular, the plant-virus interaction pathway was rapidly activated in Yuyan8, and specific resistance genes were enriched. Liquid chromatography tandem mass spectrometry analysis detected large quantities of antiviral substances in the tolerant Yuyan8. A novel Nicotiana tabacum leucine-rich repeat receptor kinase (NtLRR-RLK) gene was identified as being methylated and this was verified using bisulfite sequencing. Transient expression of TMV-green fluorescent protein in pRNAi-NtLRR-RLK transgenic plants confirmed that NtLRR-RLK was important for susceptibility to TMV. The specific protein interaction map generated from our study revealed that levels of BIP1, E3 ubiquitin ligase, and LRR-RLK were significantly elevated, and all were represented at node positions in the protein interaction map. The same expression tendency of these proteins was also found in pRNAi-NtLRR-RLK transgenic plants at 24 h after TMV inoculation. These data suggested that specific genes in the infection process can activate the immune signal cascade through different resistance genes, and the integration of signal pathways could produce resistance to the virus. These results contribute to the overall understanding of the molecular basis of plant resistance to TMV and in the long term could identify new strategies for prevention and control virus infection.