A synthetic regulon enhances the fitness of yeast on non-native nutrients

Metabolic engineering has enabled production of bio-based chemicals in organisms, usually by overexpressing the genes (heterologous or native genes) involved in the biochemical pathway to debottleneck rate limiting steps. Recent studies have shown that engineered regulatory systems, such as removing feedback inhibition, can further improve the performance of engineered strains. We hypothesize that engineering a global regulatory system (regulon) could provide a new paradigm in engineering biological systems and complement current tools available for metabolic engineering. To demonstrate this, we use the assimilation of xylose by S. cerevisiae as a test case. Xylose is a non-native sugar to this yeast, but an abundant natural sugar. Currently, engineering xylose assimilation for biomass or ethanol production in S. cerevisiae has been limited to overexpression of initial genes in the pathway to convert xylose to xylulose-5-phosphate followed by expression of non-oxidative Pentose Phosphate Pathway genes to increase the flux towards glycolysis. However, growth involves coordinated control of multiple pathways involving carbon metabolism, cofactor regeneration, amino acid synthesis, nucleotide synthesis, cell cycle maintenance etc., and debottlenecking rate-limiting reactions in all of the necessary pathways required for growth in xylose would involve extensive pathway engineering. To get around this, we hypothesized that further enhancement in xylose utilization can be made by addressing the issue from a regulatory perspective rather than metabolic. To that end, we decided on a regulon engineering strategy whereby a sugar sensing regulon can be engineered to trigger transcriptional machinery when xylose is encountered thereby enhancing the growth and biocatalytic fitness in this non-native sugar. Previous studies have shown that presence of xylose weakly upregulates galactose catabolic genes (GAL). This suggested that xylose can mimic galactose as an agonist of the GAL regulon, and that this system could serve as a platform to develop a xylose-dependent regulatory system. We first engineered the signal transduction step that increases the sensitivity and response kinetics of the GAL regulon for xylose as well as its native ligand, galactose. We further show that by switching ON the regulon using a dual positive feedback loop system we could achieve growth rates comparable to current evolutionary engineered strains. In this process, we also enhanced the galactose sensing capabilities of the sensor, thereby achieving higher growth rates on galactose than the wild type strains. Finally, we also show that under non-inducing conditions, strains carrying the xylose regulon show better growth fitness than strains with constitutive expression of xylose metabolic genes. Further increase in growth rates, xylose uptake, and specific chemical (including biofuels) production can be achieved by expanding the genes under the synthetic xylose regulon. Please click Additional Files below to see the full abstract

Surpassing thermodynamic, kinetic, and stability barriers to isomerization catalysis for tagatose biosynthesis

D-Tagatose is a rare ketohexose sugar with sweetness similar to that of sucrose. However, its glycemic index and caloric value is much lower because of low bioavailability, making it an attractive GRAS (generally regarded as safe) sugar substitute. Recent studies have also demonstrated that it is anti-hyperglycemic and prebiotic, which promotes gut health. Thus, there exists a high demand in food industry for the economical production of rare sugars, like tagatose. The enzyme L-arabinose isomerase (LAI) that responsible for the reversible isomerization of the pentose L-arabinose to L-ribulose can also isomerize the hexose D-galactose to D-tagatose. LAI has thus been the enzyme of choice to produce tagatose, although, to date, few commercial bioprocesses exist. A variety of LAIs from different microorganisms have been isolated and have reported optimal activity at a range of temperatures and pH. Some of the limitations of tagatose biosynthesis using LAI that may be hindering commercial viability are, 1) unfavorable enzymatic kinetics since galactose is not the native substrate of LAI, 2) low enzyme stability, particularly in the absence of divalent metal ions, and 3) low equilibrium constant for galactose to tagatose isomerization. Few previous reports have been successful at engineering enzymatic properties of LAI for industrial application; often addressing only one of the bottlenecks to productivity. To address the kinetic issue, several groups have used enzyme engineering methods to enhance catalytic efficiency of LAI toward galactose and have shown moderate increases in productivity. To counter low-stability issues, many groups have tested the utility of thermophilic enzymes. However, most thermophilic enzymes rely on divalent metal ions (Mn2+, Co2+, Fe2+) for stability, and high reaction temperatures (≥ 80 °C) result in significant caramelization, which are all undesirable and must be removed from product, adding to processing costs. Surface-display or encapsulation in particles or whole-cells can stabilize enzymes. Finally, the thermodynamic limitations of isomerization of galactose to tagatose are severe and, arguably, the most recalcitrant issue since ΔG°rxn ≈ +1.2 kcal/mol, which indicates theoretical maximum equilibrium conversion ~ 14 % at room temperature. Several approaches have been used to overcome this limitation. Thermophilic enzymes can achieve higher conversions than mesophilic enzymes since the equilibrium shifts toward tagatose at higher temperatures. Whole-cell biocatalysts with GRAS organisms (e.g. lactic acid bacteria (LAB) and E. coli) that disproportionately partition substrate and product across their membrane has also been shown to partially circumvent this thermodynamic limitation while simultaneously enhancing enzyme stability; albeit at a kinetic penalty imposed by substrate transport limitations. Recently, cell permeabilization and sugar transport overexpression were demonstrated as methods to overcome the kinetic penalty imposed by cellular encapsulation. There have currently been no studies that look to systematically analyze all three limitations – kinetic, thermodynamic, and enzyme stability – of the enzymatic isomerization of galactose to tagatose. This work clearly demonstrates the presence of these three limitations and provides a novel approach to balance their advantages and limitations. We use the food-safe engineered probiotic bacterium Lactobacillus plantarum as the expression host due to its increasing relevance to biochemical and biomedical research. This approach enabled ~ 50 % conversion of galactose to tagatose in 4 h (productivity of ~ 38 mmol tagatose L-1 h-1) ultimately reaching ~ 85 % conversion after 48 h at high galactose loading (300 mM) in batch culture. This is among the highest conversions and productivities reported to date for tagatose production using a mesophilic enzyme. Such an approach is expected to be applicable to other biocatalytic systems where similar trade-offs between kinetics, thermodynamics, and/or stability pose hurdles to process development. This work is currently under consideration for publication: Bober & Nair (2019) Nature Communications (in revision) https://nature-research-under-consideration.nature.com/channels/1337-under-consideration/posts/44492-surpassing-thermodynamic-kinetic-and-stability-barriers-to-isomerization-catalysis-for-tagatose-biosynthesi

Fixed confidence community mode estimation

Our aim is to estimate the largest community (a.k.a., mode) in a population composed of multiple disjoint communities. This estimation is performed in a fixed confidence setting via sequential sampling of individuals with replacement. We consider two sampling models: (i) an identityless model, wherein only the community of each sampled individual is revealed, and (ii) an identity-based model, wherein the learner is able to discern whether or not each sampled individual has been sampled before, in addition to the community of that individual. The former model corresponds to the classical problem of identifying the mode of a discrete distribution, whereas the latter seeks to capture the utility of identity information in mode estimation. For each of these models, we establish information theoretic lower bounds on the expected number of samples needed to meet the prescribed confidence level, and propose sound algorithms with a sample complexity that is provably asymptotically optimal. Our analysis highlights that identity information can indeed be utilized to improve the efficiency of community mode estimation.Comment: To appear in Performance Evaluatio

Engineering the microbiota to treat metabolic disorders

Inborn errors of metabolism (IEM) are a family of more than 500 potentially lethal congenital genetic disorders that cumulatively affect 1 in 1000 newborns. In many IEMs, pathologies manifest as a result of improper metabolism of nutrients in food. In Phenylketonuria (PKU) for example, elevated levels of phenylalanine and the accumulation of aberrant metabolic intermediates in the system lead to acute and chronic toxicities. Resultantly, many disorders within this group are generally treated through lifelong nutritional management due to the lack of alternative and pharmacological options. Longitudinal studies have indicated that even with strict adherence to a diet of synthetic supplements, patients experience chronic issues like frailty, delayed growth, and intellectual disabilities. Recently, enzyme-replacement therapies (ERT) have demonstrated promise in pre-clinical and clinical settings by providing a metabolic sink for phenylalanine in PKU. As an enhancement to traditional ERT, we are developing a novel therapeutic for IEMs associated with amino acids by expressing metabolic enzymes in lactic acid bacteria (LAB) that natively colonize the human gastrointestinal (GI) tract. Starting with an enzyme under clinical development for PKU, phenylalanine ammonia-lyase (PAL), and by promoting the intestinal adhesion and colonization characteristics, the engineered LAB will intervene before amino acid absorption occurs in the small intestines during digestion. To engineer new enzymes with activities required for treating IEMs, we have developed a novel facile selection and screening methodology. This can potentially be utilized to enhance enzymatic properties or identify mutants with altered substrate specificity, creating a spectrum of PALs that can be used to treat IEMs associated with other amino acids. Here we describe the methodology, development, and optimization of this method. To characterize and engineer microbial adhesion to intestinal mucus, we developed a novel assay that is able to capture the quantitative and mechanistic binding thermodynamics of cells to mucus. We will discuss the development of this assay and its implementation for engineering improved mucus binding. The platform technologies discussed here will be instrumental in realizing microbiota-based therapeutics as an emerging and urgently-needed treatment for IEMs that currently have inadequate or no options

Improved memory-rate trade-off for caching with demand privacy

We consider the demand-private coded caching problem in a noiseless broadcast network. It is known from past works that a demand-private scheme for N files and K users can be obtained from a non-private scheme for N files and NK users. We first propose a scheme that improves on this idea by removing some redundant transmissions. The memory- rate trade-off achieved using this scheme is shown to be within a multiplicative factor of 3 from the optimal for all the memory regimes when K K = 2.J. Ravi has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (Grant No. 714161). The work of B. K. Dey was supported in part by the Bharti Centre for Communication in IIT Bombay. The work of N. Karamchandani is supported in part by a Science and Engineering Research Board (SERB) grant on "Content Caching and Delivery over Wireless Networks"

Automatic Endoscopic Ultrasound Station Recognition with Limited Data

Pancreatic cancer is a lethal form of cancer that significantly contributes to cancer-related deaths worldwide. Early detection is essential to improve patient prognosis and survival rates. Despite advances in medical imaging techniques, pancreatic cancer remains a challenging disease to detect. Endoscopic ultrasound (EUS) is the most effective diagnostic tool for detecting pancreatic cancer. However, it requires expert interpretation of complex ultrasound images to complete a reliable patient scan. To obtain complete imaging of the pancreas, practitioners must learn to guide the endoscope into multiple "EUS stations" (anatomical locations), which provide different views of the pancreas. This is a difficult skill to learn, involving over 225 proctored procedures with the support of an experienced doctor. We build an AI-assisted tool that utilizes deep learning techniques to identify these stations of the stomach in real time during EUS procedures. This computer-assisted diagnostic (CAD) will help train doctors more efficiently. Historically, the challenge faced in developing such a tool has been the amount of retrospective labeling required by trained clinicians. To solve this, we developed an open-source user-friendly labeling web app that streamlines the process of annotating stations during the EUS procedure with minimal effort from the clinicians. Our research shows that employing only 43 procedures with no hyperparameter fine-tuning obtained a balanced accuracy of 90%, comparable to the current state of the art. In addition, we employ Grad-CAM, a visualization technology that provides clinicians with interpretable and explainable visualizations

Renal Manifestations of Tuberous Sclerosis Complex

Tuberous sclerosis complex (TSC) is a genetic condition caused by a mutation in either the TSC1 or TSC2 gene. Disruption of either of these genes leads to impaired production of hamartin or tuberin proteins, leading to the manifestation of skin lesions, tumors, and seizures. TSC can manifest in multiple organ systems with the cutaneous and renal systems being the most commonly affected. These manifestations can secondarily lead to the development of hypertension, chronic kidney disease, and neurocognitive declines. The renal pathologies most commonly seen in TSC are angiomyolipoma, renal cysts, and less commonly, oncocytomas. In this review, we highlight the current understanding on the renal manifestations of TSC along with current diagnosis and treatment guidelines

Prevalence of pulmonary tuberculosis in young adult patients with type 1 diabetes mellitus in India

Background: There is limited information on Prevalence of Pulmonary Tuberculosis (PTB) in patients with type-1-diabetes. We assessed the prevalence of PTB in patients with type-1-diabetes attending the outpatient-clinic in a tertiary-care hospital. Methods: 151 patients with type-1-diabetes were screened for PTB by clinical examination and chest-radiography. Sputum Acid-Fast Bacilli Test (AFB) and Mycobacterium tuberculosis (M. tb) culture were performed in patients with clinical and radiological features suggestive of a possibility of PTB and also in those with history of PTB in the past. Their average glycated haemoglobin (HbA1c) during preceding 2 years was assessed. Sputum culture positive patients were managed by a pulmonologist. Results: 5/151 patients had respiratory symptoms and radiographic findings suggestive of PTB. 20/151 patients were asymptomatic but had history of PTB. Four of the five symptomatic patients and 12 with past PTB were positive for sputum M. tb by culture, giving a prevalence of 10.6% sputum culture positive in type-1-diabetes. Average HbA1c was comparable in patients with and without positive sputum culture. ESR and Mantoux test were not discriminatory in these groups. Four clinically symptomatic M. tb culture positive and four asymptomatic patients with sputum culture positive for M. tb on two occasions (6 weeks apart) were put on Antitubercular Treatment (ATT). Patients who were culture positive for M. tb only on one occasion were kept on a close follow up. Conclusions: Patients with type-1-diabetes mellitus in India have high prevalence of PTB. They need to be actively screened for PTB by sputum M. tb culture in order to initiate early treatment and to prevent transmission in the community