Engineering thioesterase as a driving force for novel itaconate production via its degradation scheme.

Incorporation of irreversible steps in pathway design enhances the overall thermodynamic favorability and often leads to better bioconversion yield given functional enzymes. Using this concept, here we constructed the first non-natural itaconate biosynthesis pathway driven by thioester hydrolysis. Itaconate is a commercially valuable platform chemical with wide applications in the synthetic polymer industry. Production of itaconate has long relied on the decarboxylation of TCA cycle intermediate cis-aconitate as the only biosynthetic route. Inspired by nature's design of itaconate detoxification, here we engineered a novel itaconate producing pathway orthogonal to native metabolism with no requirement of auxotrophic knock-out. The reversed degradation pathway initiates with pyruvate and acetyl-CoA condensation forming (S)-citramalyl-CoA, followed by its dehydration and isomerization into itaconyl-CoA then hydrolysis into itaconate. Phenylacetyl-CoA thioesterase (PaaI) from Escherichia coli was identified via screening to deliver the highest itaconate formation efficiency when coupled to the reversible activity of citramalate lyase and itaconyl-CoA hydratase. The preference of PaaI towards itaconyl-CoA hydrolysis over acetyl-CoA and (S)-citramalyl-CoA also minimized the inevitable precursor loss due to enzyme promiscuity. With acetate recycling, acetyl-CoA conservation, and condition optimization, we achieved a final itaconate titer of 1 g/L using the thioesterase driven pathway, which is a significant improvement compared to the original degradation pathway based on CoA transferase. This study illustrates the significance of thermodynamic favorability as a design principle in pathway engineering.

Comparison of Ultrasound-Derived Muscle Thickness With Computed Tomography Muscle Cross-Sectional Area on Admission to the Intensive Care Unit: A Pilot Cross-Sectional Study.

INTRODUCTION: The development of bedside methods to assess muscularity is an essential critical care nutrition research priority. We aimed to compare ultrasound-derived muscle thickness at 5 landmarks with computed tomography (CT) muscle area at intensive care unit (ICU) admission. Secondary aims were to (1) combine muscle thicknesses and baseline covariates to evaluate correlation with CT muscle area and (2) assess the ability of the best-performing ultrasound model to identify patients with low CT muscle area. METHODS: Adult patients who underwent CT scanning at the third lumbar area <72 hours after ICU admission were prospectively recruited. Muscle thickness was measured at mid-upper arm, forearm, abdomen, and thighs. Low CT muscle area was determined using published cutoffs. Pearson correlation compared ultrasound-derived muscle thickness and CT muscle area. Linear regression was used to develop ultrasound prediction models. Bland-Altman analyses compared ultrasound-predicted and CT-measured muscle area. RESULTS: Fifty ICU patients were enrolled, aged 52 ± 20 years. Ultrasound-derived muscle thickness at each landmark correlated with CT muscle area (P < .001). The sum of muscle thickness at mid-upper arm and bilateral thighs, including age, sex, and the Charlson Comorbidity Index, improved the correlation with CT muscle area (r = 0.85; P < .001). Mean difference between ultrasound-predicted and CT-measured muscle area was -2 cm2 (95% limits of agreement, -40 cm2 to +36 cm2 ). The best-performing ultrasound model demonstrated good ability to identify 14 patients with low CT muscle area (area under curve = 0.79). CONCLUSION: Ultrasound shows potential for assessing muscularity at ICU admission (Clinicaltrials.gov NCT03019913).