Metabolic Engineering Design Strategies for Increasing Carbon Fluxes Relevant for Biosynthesis in Cyanobacteria.

Cyanobacteria are promising microbial cell factories for the direct production of biochemicals and biofuels from CO2. Through genetic and metabolic engineering, they can be modified to produce a variety of both natural and non-natural compounds. To enhance the yield of these products, various design strategies have been developed. In this chapter, strategies used to enhance metabolic fluxes towards common precursors used in biosynthesis, including pyruvate, acetyl-CoA, malonyl-CoA, TCA cycle intermediates, and aromatics, are discussed. Additionally, strategies related to cofactor availability and mixotrophic conditions for bioproduction are also summarize.

Explicit kinematic equations for degree-4 rigid origami vertices, Euclidean and non-Euclidean.

We derive algebraic equations for the folding angle relationships in completely general degree-4 rigid-foldable origami vertices, including both Euclidean (developable) and non-Euclidean cases. These equations in turn lead to elegant equations for the general developable degree-4 case. We compare our equations to previous results in the literature and provide two examples of how the equations can be used: in analyzing a family of square twist pouches with discrete configuration spaces, and for proving that a folding table design made with hyperbolic vertices has a single folding mode.

Metabolic engineering of Escherichia coli for efficient biosynthesis of butyl acetate.

BACKGROUND: Butyl acetate is a versatile compound that is widely used in the chemical and food industry. The conventional butyl acetate synthesis via Fischer esterification of butanol and acetic acid using catalytic strong acids under high temperature is not environmentally benign. Alternative lipase-catalyzed ester formation requires a significant amount of organic solvent which also presents another environmental challenge. Therefore, a microbial cell factory capable of producing butyl acetate through fermentation of renewable resources would provide a greener approach to butyl acetate production. RESULT: Here, we developed a metabolically engineered strain of Escherichia coli that efficiently converts glucose to butyl acetate. A modified Clostridium CoA-dependent butanol production pathway was used to synthesize butanol which was then condensed with acetyl-CoA through an alcohol acetyltransferase. Optimization of alcohol acetyltransferase expression and redox balance with auto-inducible fermentative controlled gene expression led to an effective titer of 22.8 ± 1.8 g/L butyl acetate produced in a bench-top bioreactor. CONCLUSION: Building on the well-developed Clostridium CoA-dependent butanol biosynthetic pathway, expression of an alcohol acetyltransferase converts the butanol produced into butyl acetate. The results from this study provided a strain of E. coli capable of directly producing butyl acetate from renewable resources at ambient conditions.

Simple parameters of synthetic MRI for assessment of bone density in patients with spinal degenerative disease.

OBJECTIVE: Good bone quality is the key to avoiding osteoporotic fragility fractures and poor outcomes after lumbar instrumentation and fusion surgery. Although dual-energy x-ray absorptiometry (DEXA) screening is the current standard for evaluating osteoporosis, many patients lack DEXA measurements before undergoing lumbar spine surgery. The present study aimed to investigate the utility of using simple quantitative parameters generated with novel synthetic MRI to evaluate bone quality, as well as the correlations of these parameters with DEXA measurements. METHODS: This prospective study enrolled patients with symptomatic lumbar degenerative disease who underwent DEXA and conventional and synthetic MRI. The quantitative parameters generated with synthetic MRI were T1 map, T2 map, T1 intensity, proton density (PD), and vertebral bone quality (VBQ) score, and these parameters were correlated with T-score of the lumbar spine. RESULTS: There were 62 patients and 238 lumbar segments eligible for analysis. PD and VBQ score moderately correlated with T-score of the lumbar spine (r = -0.565 and -0.651, respectively; both p < 0.001). T1 intensity correlated fairly well with T-score (r = -0.411, p < 0.001). T1 and T2 correlated poorly with T-score. Receiver operating characteristic curve analysis demonstrated area under the curve values of 0.808 and 0.794 for detecting osteopenia/osteoporosis (T-score ≤ -1.0) and osteoporosis (T-score ≤ -2.5) with PD (both p < 0.001). CONCLUSIONS: PD and T1 intensity values generated with synthetic MRI demonstrated significant correlation with T-score. PD has excellent ability for predicting osteoporosis and osteopenia.

Correlation of bone density to screw loosening in dynamic stabilization: an analysis of 176 patients.

Although osteoporosis has negative impacts on lumbar fusion, its effects on screw loosening in dynamic stabilization remain elusive. We aimed to correlate bone mineral density (BMD) with screw loosening in Dynesys dynamic stabilization (DDS). Consecutive patients who underwent 2- or 3-level DDS for spondylosis, recurrent disc herniations, or low-grade spondylolisthesis at L3-5 were retrospectively reviewed. BMD was assessed by the Hounsfield Unit (HU) in vertebral bodies (VB) and pedicles with and without cortical bone (CB) on pre-operative computed tomography (CT). Screw loosening was assessed by radiographs and confirmed by CT. HU values were compared between the loosened and intact screws. 176 patients and 918 screws were analyzed with 78 loosened screws found in 36 patients (mean follow-up: 43.4 months). The HU values of VB were similar in loosened and intact screws (p = 0.14). The HU values of pedicles were insignificantly less in loosened than intact screws (including CB: 286.70 ± 118.97 vs. 297.31 ± 110.99, p = 0.45; excluding CB: 238.48 ± 114.90 vs. 240.51 ± 108.91, p = 0.88). All patients had clinical improvements. In conclusion, the HU values, as a surrogate for BMD, were unrelated to screw loosening in DDS. Therefore, patients with compromised BMD might be potential candidates for dynamic stabilization rather than fusion.

Iatrogenic cerebrospinal fluid leak after repeated nasal swab tests for COVID-19: illustrative case.

BACKGROUND: Nasal swab tests are one of the most essential tools for screening coronavirus disease 2019 (COVID-19). The authors report a rare case of iatrogenic cerebrospinal fluid (CSF) leak from the anterior skull base after repeated nasal swab tests for COVID-19, which was treated with endoscopic endonasal repair. OBSERVATIONS: A 41-year-old man presented with clear continuous rhinorrhea through his left nostril for 5 days after repeated nasal swabbing for COVID-19. There were no obvious risk factors for spontaneous CSF leak. Computed tomography cisternography showed contrast accumulation in the left olfactory fossa and along the left nasal cavity. Such findings aligned with a preliminary diagnosis of CSF leakage through the left cribriform plate. Magnetic resonance imaging confirmed the presence of a CSF fistula between his left cribriform plate and superior nasal concha. The patient underwent endoscopic endonasal repair. CSF rhinorrhea ceased after the surgery, and no recurrence was noted during the 12-week postoperative follow-up period. LESSONS: Although rare, iatrogenic CSF leakage can be a serious complication following COVID-19 nasal swab tests, especially when infection may cause significant neurological sequelae. Healthcare providers should become familiar with nasal cavity anatomy and be well trained in performing nasal swab tests.

Cervical disc arthroplasty at C2-3: illustrative case.

BACKGROUND: Since the beginning of the 21st century, cervical disc arthroplasty (CDA) has been accepted as an alternative to anterior cervical discectomy and fusion for surgical management of disc problems. The published clinical trials of CDA have included patients with radiculopathy or myelopathy caused by one- or two-level disc herniation at C3-7. However, it remains uncertain whether CDA is a viable option for C2-3 disc herniation. OBSERVATIONS: In this report, a 52-year-old man presented with hand numbness, arm pain, and myelopathic symptoms that were refractory to medical treatment for more than 6 months. The magnetic resonance images demonstrated herniated discs at C2-3, C3-4, and C4-5, causing stenosis. There was no ossification of posterior longitudinal ligament and the spine was mobile, so he received anterior discectomies with artificial disc replacement at each of the C2-3, C3-4, and C4-5 levels. The surgery went smoothly, and his neurological symptoms were promptly relieved. The postoperative radiographs at 24 months demonstrated a preserved range of motion at each level. LESSONS: To date, this was the first report of CDA performed at C2-3, which also involved three consecutive levels of disc replacement. The report suggested that both C2-3 and three-consecutive-level CDA may be a viable option for cervical disc disease.

Metabolic Engineering Design Strategies for Increasing Acetyl-CoA Flux.

Acetyl-CoA is a key metabolite precursor for the biosynthesis of lipids, polyketides, isoprenoids, amino acids, and numerous other bioproducts which are used in various industries. Metabolic engineering efforts aim to increase carbon flux towards acetyl-CoA in order to achieve higher productivities of its downstream products. In this review, we summarize the strategies that have been implemented for increasing acetyl-CoA flux and concentration, and discuss their effects. Furthermore, recent works have developed synthetic acetyl-CoA biosynthesis routes that achieve higher stoichiometric yield of acetyl-CoA from glycolytic substrates.

A balanced ATP driving force module for enhancing photosynthetic biosynthesis of 3-hydroxybutyrate from CO2.

Using engineered photoautotrophic microorganisms for the direct chemical synthesis from CO2 is an attractive direction for both sustainability and CO2 mitigation. However, the behaviors of non-native metabolic pathways may be difficult to control due to the different intracellular contexts between natural and heterologous hosts. While most metabolic engineering efforts focus on strengthening driving forces in pathway design to favor biochemical production in these organisms, excessive driving force may be detrimental to product biosynthesis due to imbalanced cellular intermediate distribution. In this study, an ATP-hydrolysis based driving force module was engineered into cyanobacterium Synechococcus elongatus PCC 7942 to produce 3-hydroxybutyrate (3HB), a valuable chemical feedstock for the synthesis of biodegradable plastics and antibiotics. However, while the ATP driving force module is effective for increasing product formation, uncontrolled accumulation of intermediate metabolites likely led to metabolic imbalance and thus to cell growth inhibition. Therefore, the ATP driving force module was reengineered by providing a reversible outlet for excessive carbon flux. Upon expression of this balanced ATP driving force module with 3HB biosynthesis, engineered strain produced 3HB with a cumulative titer of 1.2 g/L, a significant increase over the initial strain. This result highlighted the importance of pathway reversibility as an effective design strategy for balancing driving force and intermediate accumulation, thereby achieving a self-regulated control for increased net flux towards product biosynthesis.

Renewable synthesis of n-butyraldehyde from glucose by engineered Escherichia coli.

BACKGROUND: n-Butyraldehyde is a high-production volume chemical produced exclusively from hydroformylation of propylene. It is a versatile chemical used in the synthesis of diverse C4-C8 alcohols, carboxylic acids, esters, and amines. Its high demand and broad applications make it an ideal chemical to be produced from biomass. RESULTS: An Escherichia coli strain was engineered to produce n-butyraldehyde directly from glucose by expressing a modified Clostridium CoA-dependent n-butanol production pathway with mono-functional Coenzyme A-acylating aldehyde dehydrogenase (Aldh) instead of the natural bifunctional aldehyde/alcohol dehydrogenase. Aldh from Clostridium beijerinckii outperformed the other tested homologues. However, the presence of native alcohol dehydrogenase led to spontaneous conversion of n-butyraldehyde to n-butanol. This problem was addressed by knocking out native E. coli alcohol dehydrogenases, significantly improving the butyraldehyde-to-butanol ratio. This ratio was further increased reducing media complexity from Terrific broth to M9 media containing 2% yeast extract. To increase production titer, in situ liquid-liquid extraction using dodecane and oleyl alcohol was investigated. Results showed oleyl alcohol as a better extractant, increasing the titer of n-butyraldehyde produced to 630 mg/L. CONCLUSION: This study demonstrated n-butyraldehyde production from glucose. Through sequential strain and condition optimizations, butyraldehyde-to-butanol ratio was improved significantly compared to the parent strain. Results from this work may serve as a basis for further development of renewable n-butyraldehyde production.

Closed-loop learning control of bio-networks.

A general goal of systems biology is to acquire a detailed quantitative understanding of the life-sustaining interactions between genes and proteins. There arises an interesting question of whether these network dynamics can be controlled externally. In the open-loop approach to experimental biology, a control design would be chosen based on a desired target response and modeling with all the available knowledge about the system. If the system is not completely understood or disturbances occur, then unexpected deviations from the desired response can arise. A means to circumvent this difficulty is to optimize the controls in a closed-loop operation by modifying successive input controls based on the performance of previous controls. This paper presents a simulation of closed-loop learning control applied to biological systems in order to generate a desired response. The most powerful advantage of this technique is that the controls are deduced based on experimental results and the process can operate without a model for the underlying biochemical network. This feature eliminates the problem of faulty predictions as well as the need for a detailed understanding of the underlying molecular pathways, suggesting that biological systems can be controlled even before the post-systems biology era.