Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening.

Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, glycoside hydrolase family 7 (GH7) CBHs are critically important for the bioeconomy and typically difficult to engineer. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity. Using experimentally assayed activities of genome mined CBHs, we applied sequence and structural alignments to top performers to identify key point mutations linked to improved activity. From ∼1500 known GH7 sequences, an evolutionarily diverse subset of 57 GH7 CBH genes was expressed in Trichoderma reesei and screened using a multiplexed activity screening assay. Ten catalytically enhanced natural variants were identified, produced, purified, and tested for efficacy using industrially relevant conditions and substrates. Three key amino acids in CBHs with performance comparable or superior to Penicillium funiculosum Cel7A were identified and combinatorially engineered into P. funiculosum cel7a, expressed in T. reesei, and assayed on lignocellulosic biomass. The top performer generated using this combined approach of natural diversity genome mining, experimental assays, and computational modeling produced a 41% increase in conversion extent over native P. funiculosum Cel7A, a 55% increase over the current industrial standard T. reesei Cel7A, and 10% improvement over Aspergillus oryzae Cel7C, the best natural GH7 CBH previously identified in our laboratory.

A multi-plex protein expression system for production of complex enzyme formulations in Trichoderma reesei.

Heterologous protein production has been challenging in the hyper-cellulolytic fungus, Trichoderma reesei as the species is known for poor transformation efficiency, low homologous recombination frequency, and marginal screening systems for the identification of successful transformants. We have applied the 2A-peptide multi-gene expression system to co-express four proteins, which include three cellulases: a cellobiohydrolase (CBH1), an endoglucanase (EG1), and a ß-D-glucosidase (BGL1), as well as the enhanced green fluorescent protein (eGFP) marker protein. We designed a new chassis vector, pTrEno-4X-2A, for this work. Expression of these cellulase enzymes was confirmed by real-time quantitative reverse transcription PCR and immunoblot analysis. The activity of each cellulase was assessed using chromogenic substrates, which confirmed the functionality of the enzymes. Expression and activity of these enzymes were proportional to the level of eGFP fluorescence, thereby validating the reliability of this screening technique. An 18-fold differencein protein expression was observed between the first and third genes within the 2A-peptide construct. The availability of this new multi-gene expression and screening tool is expected to greatly impact multi-enzyme applications, such as the production of complex commercial enzyme formulations and metabolic pathway enzymes, especially those destined for cell-free applications.

Timing of Complications Following Surgery for Distal Femur Fractures in Older Adults.

Introduction: The purpose of this study was to identify the timing and nature of complications associated with distal femur fracture surgery in patients aged 65 and older using the American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) database. Methods: The ACS NSQIP database was queried for adults aged 65 and older who received surgical treatment for a distal femur fracture between 01 January 2015 and 31 December 2021. Cox regression models and risk tables adjusted for baseline clinical characteristics were created for 14 complications (Superficial Surgical Site Infection (SSI), Deep SSI, Organ/Space SSI, Pneumonia, Pulmonary Embolism (PE), Deep Venous Thrombosis (DVT), Urinary Tract Infection (UTI), Stroke/Cerebrovascular accident (CVA), Myocardial Infarction (MI), Renal Failure, Cardiac Arrest (CA), Re-operation, Sepsis, and Death within 30 days of surgery). Model summaries were used to identify significant variables with a Bonferroni correction applied. Results: A total of 3956 adults met inclusion criteria and were included in analysis. The most common complications were UTI (5.2%), death (4.1%), and pneumonia (3.4%). Complications typically occurred within 14 days after surgery, except for SSI, which occurred between post-op days 11 and 24. Conclusions: Distal femur fractures are a substantial source of morbidity and mortality in the older adult population. Our findings underscore the need for comprehensive preoperative risk assessment and patient management strategies to mitigate the impact of identified risk factors in this vulnerable population.

Phylogenetics-based identification and characterization of a superior 2,3-butanediol dehydrogenase for Zymomonas mobilis expression.

BACKGROUND: Zymomonas mobilis has recently been shown to be capable of producing the valuable platform biochemical, 2,3-butanediol (2,3-BDO). Despite this capability, the production of high titers of 2,3-BDO is restricted by several physiological parameters. One such bottleneck involves the conversion of acetoin to 2,3-BDO, a step catalyzed by 2,3-butanediol dehydrogenase (Bdh). Several Bdh enzymes have been successfully expressed in Z. mobilis, although a highly active enzyme is yet to be identified for expression in this host. Here, we report the application of a phylogenetic approach to identify and characterize a superior Bdh, followed by validation of its structural attributes using a mutagenesis approach. RESULTS: Of the 11 distinct bdh genes that were expressed in Z. mobilis, crude extracts expressing Serratia marcescens Bdh (SmBdh) were found to have the highest activity (8.89 µmol/min/mg), when compared to other Bdh enzymes (0.34-2.87 µmol/min/mg). The SmBdh crystal structure was determined through crystallization with cofactor (NAD+) and substrate (acetoin) molecules bound in the active site. Active SmBdh was shown to be a tetramer with the active site populated by a Gln247 residue contributed by the diagonally opposite subunit. SmBdh showed a more extensive supporting hydrogen-bond network in comparison to the other well-studied Bdh enzymes, which enables improved substrate positioning and substrate specificity. This protein also contains a short α6 helix, which provides more efficient entry and exit of molecules from the active site, thereby contributing to enhanced substrate turnover. Extending the α6 helix to mimic the lower activity Enterobacter cloacae (EcBdh) enzyme resulted in reduction of SmBdh function to nearly 3% of the total activity. In great contrast, reduction of the corresponding α6 helix of the EcBdh to mimic the SmBdh structure resulted in ~ 70% increase in its activity. CONCLUSIONS: This study has demonstrated that SmBdh is superior to other Bdhs for expression in Z. mobilis for 2,3-BDO production. SmBdh possesses unique structural features that confer biochemical advantage to this protein. While coordinated active site formation is a unique structural characteristic of this tetrameric complex, the smaller α6 helix and extended hydrogen network contribute towards improved activity and substrate promiscuity of the enzyme.

Within-socket myoelectric prediction of continuous ankle kinematics for control of a powered transtibial prosthesis.

OBJECTIVE: Powered robotic prostheses create a need for natural-feeling user interfaces and robust control schemes. Here, we examined the ability of a nonlinear autoregressive model to continuously map the kinematics of a transtibial prosthesis and electromyographic (EMG) activity recorded within socket to the future estimates of the prosthetic ankle angle in three transtibial amputees. APPROACH: Model performance was examined across subjects during level treadmill ambulation as a function of the size of the EMG sampling window and the temporal 'prediction' interval between the EMG/kinematic input and the model's estimate of future ankle angle to characterize the trade-off between model error, sampling window and prediction interval. MAIN RESULT: Across subjects, deviations in the estimated ankle angle from the actual movement were robust to variations in the EMG sampling window and increased systematically with prediction interval. For prediction intervals up to 150 ms, the average error in the model estimate of ankle angle across the gait cycle was less than 6°. EMG contributions to the model prediction varied across subjects but were consistently localized to the transitions to/from single to double limb support and captured variations from the typical ankle kinematics during level walking. SIGNIFICANCE: The use of an autoregressive modeling approach to continuously predict joint kinematics using natural residual muscle activity provides opportunities for direct (transparent) control of a prosthetic joint by the user. The model's predictive capability could prove particularly useful for overcoming delays in signal processing and actuation of the prosthesis, providing a more biomimetic ankle response.

Long-term juxtafoveal retinal telangiectasia.

PURPOSE: To determine the long-term natural history of idiopathic juxtafoveal telangiectasia (IJRT). METHODS: Record review in 2 university-based and 2 private vitreoretinal practices sought patients with IJRT documented by color photographs and fluorescein angiograms (FAs) during the period January 1, 1980, to December 31, 1993. Patients then had repeated examinations and FAs. RESULTS: Twenty patients with IJRT in 32 eyes had follow-up examinations. Fifteen patients had color photographs and FAs, and one had color photographs alone. Follow-up varied from 10 years to 21 years (average, 15 years). Six eyes were treated by laser photocoagulation at onset. Twenty-four of the 26 untreated eyes lost vision as measured by Snellen testing. Visual loss and morphologic progression depended on IJRT type. Six of 8 untreated eyes with type IA IJRT lost vision by >/ or = 3 lines (Snellen), 4 to 20/70 or worse. Vision loss was caused by progressive telangiectatic changes and intraretinal edema. Fifteen of 20 initially untreated eyes with type IIA IJRT developed either central retinal pigment epithelium membranes or subretinal neovascularization with loss of vision to 20/80 or less. CONCLUSION: IJRT prognosis depends on type and clinical features. Long-term prognosis for central vision is poor.