A General Hybrid Modeling Framework for Systems Biology Applications: Combining Mechanistic Knowledge with Deep Neural Networks under the SBML Standard

This work was supported by the Associate Laboratory for Green Chemistry—LAQV which is financed by national funds from FCT/MCTES (UIDB/50006/2020 and UIDP/50006/2020). This work has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement number 870292 (BioICEP project). J.P. acknowledges a PhD grant (SFRD/BD14610472019), Fundação para a Ciência e Tecnologia (FCT) and R.S.C. the contract CEECIND/01399/2017In this paper, a computational framework is proposed that merges mechanistic modeling with deep neural networks obeying the Systems Biology Markup Language (SBML) standard. Over the last 20 years, the systems biology community has developed a large number of mechanistic models that are currently stored in public databases in SBML. With the proposed framework, existing SBML models may be redesigned into hybrid systems through the incorporation of deep neural networks into the model core, using a freely available python tool. The so-formed hybrid mechanistic/neural network models are trained with a deep learning algorithm based on the adaptive moment estimation method (ADAM), stochastic regularization and semidirect sensitivity equations. The trained hybrid models are encoded in SBML and uploaded in model databases, where they may be further analyzed as regular SBML models. This approach is illustrated with three well-known case studies: the Escherichia coli threonine synthesis model, the P58IPK signal transduction model, and the Yeast glycolytic oscillations model. The proposed framework is expected to greatly facilitate the widespread use of hybrid modeling techniques for systems biology applications.publishersversionpublishe

Microbial production of curcumin

First Online: 01 October 2022Curcumin, a polyphenol produced by turmeric (Curcuma longa), has attracted increased attention due to its potential as a novel cancer-fighting drug. However, to satisfy the required curcumin demand for health-related studies, high purity curcumin preparations are required, which are difficult to obtain and are very expensive. Curcumin and other curcuminoids are usually obtained through plant extraction. However, these polyphenols accumulate in low amounts over long periods in the plant and their extraction process is costly and not environmentally friendly. In addition, curcumin chemical synthesis is complex. All these reasons limit the advances in studies related to the in vitro and in vivo curcumin biological activities. The microbial production of curcumin appears as a solution to overcome the limitations associated with the currently used methods. Curcumin biosynthesis begins with the conversion of the aromatic amino acids, phenylalanine and tyrosine, into phenylpropanoids, the curcuminoid precursors. The phenylpropanoids are then activated through condensation with a CoA molecule. Afterwards, curcuminoids are synthesized by the action of type III polyketide synthases (PKS) that combine two activated phenylpropanoids and a malonyl-CoA molecule. To engineer microbes to produce curcumin, the curcuminoid biosynthetic genes must be introduced as microorganisms lack the enzymatic reactions responsible to synthesize curcuminoids. In this chapter, the advances regarding the microbial production of curcumin are exposed. The heterologous production of curcumin has been mainly achieved in the bacteria Escherichia coli. However, other microorganisms have already been explored. Besides the introduction of curcumin biosynthetic genes, the optimization of the microbial chassis must also be considered to maximize the production yields. The strategies employed for this purpose are also herein presented. The maximum titer of curcumin produced by a genetically engineered E. coli was 563.4 mg/L with a substrate conversion yield of 100% from supplemented ferulic acid. Moreover, the de novo production of curcumin was accomplished in E. coli reaching 3.8 mg/L of curcumin. Overall, the recent developments on curcumin heterologous production are very encouraging.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/BIO/04469/2020 unit, and by LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechnaical Systems, LA/P/0029/2020. J.R. is recipient of a doctoral fellowship (SFRH/BD/138325/2018) supported by a doctoral advanced training funded by FCT.info:eu-repo/semantics/publishedVersio

CRISPR-Cas9: a powerful tool to efficiently engineer Saccharomyces cerevisiae

Saccharomyces cerevisiae has been for a long time a common model for fundamental biological studies and a popular biotechnological engineering platform to produce chemicals, fuels, and pharmaceuticals due to its peculiar characteristics. Both lines of research require an effective editing of the native genetic elements or the inclusion of heterologous pathways into the yeast genome. Although S. cerevisiae is a well-known host with several molecular biology tools available, a more precise tool is still needed. The clustered, regularly interspaced, short palindromic repeats–associated Cas9 (CRISPR-Cas9) system is a current, widespread genome editing tool. The implementation of a reprogrammable, precise, and specific method, such as CRISPR-Cas9, to edit the S. cerevisiae genome has revolutionized laboratory practices. Herein, we describe and discuss some applications of the CRISPR-Cas9 system in S. cerevisiae from simple gene knockouts to more complex processes such as artificial heterologous pathway integration, transcriptional regulation, or tolerance engineering.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/BIO/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund (ERDF) under the scope of Norte2020—North Portugal Regional Program. In addition, this research has been carried out at the Biomass and Bioenergy Research Infrastructure (BBRI)—LISBOA-010145- FEDER-022059, supported by Operational Program for Competitiveness and Internationalization (PORTUGAL2020), the Lisbon Portugal Regional Operational Program (Lisboa2020), and Norte2020 under the Portugal 2020 Partnership Agreement, through the ERDF. J.R. is recipient of a fellowship supported by a doctoral advanced training (SFRH/BD/138325/2018) funded by FCT.info:eu-repo/semantics/publishedVersio

Engineering Saccharomyces cerevisiae towards the production of curcumin

Curcumin, a polyphenol produced by turmeric (Curcuma longa), has attracted increased attention due to its potential as a novel cancer-fighting drug. However, to satisfy the required curcumin demand for health-related studies, high purity curcumin preparations are required, which are difficult to obtain and extremely expensive. Curcumin accumulates in low amounts over long periods in the plant and its extraction process is costly and not environmentally friendly. In addition, its chemical synthesis is complex. All these reasons limit the advances related to the in vitro and in vivo curcumin biological activities. Herein, we intend to develop a genetically engineered Saccharomyces cerevisiae capable of producing pure curcumin from simple carbon sources such as glucose. The curcumin biosynthetic pathway in plants starts with the phenylpropanoid pathway, whose reactions convert the aromatic amino acids (phenylalanine/tyrosine) to the curcumin precursor ferulic acid. Afterwards, curcumin is produced under the catalysis of 4-coumarate-CoA ligase (4CL) and type III polyketide synthases (PKSs) with the involvement of one malonyl-CoA molecule. As starting point for the development of a yeast cell factory, we tested the curcumin production using 2-micron plasmid vectors carrying the 4CL1 from Arabidopsis thaliana and the PKS curcuminoid synthase (CUS) from Oryza sativa in a wild-type S. cerevisiae strain. This modified strain was able to produce 111 µg/L of curcumin from supplemented ferulic acid.info:eu-repo/semantics/publishedVersio

Synthetic biology strategies for the production of plant polyphenolic compounds

[Excerpt] Polyphenols are secondary metabolites naturally produced in plants with an estimated market size of USD 2.26 billion by 2027 . These compounds have several biological and potential therapeutic activities such as anti-inflammatory, antioxidant and anticancer. [...]This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund (ERDF) under the scope of Norte2020 - Programa Operacional Regional do Norte. JR is recipient of a fellowship supported by a doctoral advanced training (SFRH/BD/138325/2018) funded by FCT.info:eu-repo/semantics/publishedVersio

Alternative models for the calculation of the RMR and Q indexes for granite rock masses

Empirical classification systems like the RMR and Q are often used in current practice of geotechnical structures design built in rock masses. They allow obtaining an overall description of the rock mass and the calculation, through analytical solutions, of strength and deformability parameters which are determinant in design. To be applied these systems need a set of geomechanical information that may not be available or can be difficult to obtain. In this work it is intended to develop new alternative regression models for the calculation of the RMR and Q indexes using less data than the original formulations and keeping a high accuracy level. It is also intended to have an insight of which parameters are the most important for the prediction of the indexes and in the rock masses behaviour. This study was carried out applying Data Mining techniques to a database of the empirical classification systems applications in a granite rock mass. Data Mining is a relatively new area of computer science which concerns with automatically find, simplify and summarize patterns and relationships within large databases. The used Data Mining techniques were the multiple regression and artificial neural networks. The developed models are able to predict the two geomechanical indexes using less information that in the original formulations with a good predictive capacity.Fundação para a Ciência e a Tecnologia (FCT) - projecto POCI/ECM/57495/2004 "Geotechnical Risk in Tunnels for High Speed Trains

Tracking features with Kalman filtering, Mahalanobis distance and a management model

Feature tracking is a complex problem whose automatic detection and execution evolved considerably in the recent past, especially due its wide range of applications, for instance in surveillance, object deformation analysis, traffic monitoring. Many of such applications require simultaneous tracking of multiple features, and also involve problems related to their appearance and disappearance of the image scene.A key component of a successful tracking system is its ability to search efficiently for the feature [1]. For the tracking system to perform properly, the most likely measured potential target location should be used to update the targets state estimator. This is generally known as the data association problem. The probability of the given measurement being correct can be a distance function between the predicted state of the target and the measured state. This becomes especially important for targets that may come close to each other or even overlap, such as people.An approach uses assignment algorithms, which were recently shown to be effective in data association for target tracking in the presence of noise. In this case, data association is formulated as a constrained optimization problem, where the cost function to be minimized is, usually, a combined likelihood function evaluated using the results from the state estimator. The state is usually constrained by position and/or velocity and the assignment by physical constraints, for example, only one measurement for a track and vice-versa [2].On the other hand, to track the captured movement the Kalman filter is a widespread technique for feature tracking, but it has recently been substituted by particle filters [3]. The Kalman filter rests on the assumption that the disturbances and initial state vector are normally distributed, and it is shown that the obtained mean of the conditional distribution of a state is an optimal estimator in the sense that it minimizes the mean square error. If the normality assumption is dropped there is no guarantee that the Kalman filter will give the conditional mean of the state vector. So, other filters have been proposed, but they reveal some problems too as well as high computational cost [3]

Agro-industrial wastes as alternative substrates for the production of prebiotic with Zymomonas mobilis

Fructooligosaccharides (FOS) are promising prebiotics in the relevant and increasing market of functional food. However, to achieve a more sustainable process, the industrial production of FOS should use cheap substrates. Zymomonas mobilis (ZM) has great potential for the production of FOS due to the presence of native enzymes (levansucrase) capable of metabolizing sucrose. In addition, ZM can use different carbon sources, such as molasses and sugarcane juice, which make the FOS production process cost-effective. In this study, sugarcane molasses (a potential replacement of sucrose) and corn step liquor (CSL) (a potential replacement of yeast extract (YE)), were used as nutrients for FOS production using ZM in an in vivo bioprocess approach. FOS production process from sucrose was first optimized and 52 g/L of FOS with a yield of 0.16 g/g was obtained. Afterwards, molasses and CSL were used as alternative nutrients. After studying different combinations of CSL and YE, the highest amount of FOS (54 g/L, with a yield of 0.18 g/g) was obtained with 12 g/L of CSL and 8 g/L of YE. In addition, 45 g/L of FOS were produced from molasses containing 200 g/L of sucrose, with a yield of 0.3 g/g. With this approach, it was possible to reduce around 5.5-times the cost associated with the FOS production medium. Moreover, this study proposed a sustainable process for the valorization of agro-industrial wastes contributing to the future Circular (Bio)Economy and the EU Green Deal.Cláudia Amorim, João Rainha, Beatriz B. Cardoso and Daniela Gomes acknowledge their grants (2020.0029.CEECIND, SFRH/BD/138325/2018, SFRH/BD/132324/2017 and SFRH/BD/04433/2020, respectively) from Portuguese Foundation for Science and Technology (FCT). The study received financial support from Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and by LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020.info:eu-repo/semantics/publishedVersio

Development of a cost-effective media for biosurfactants production by Pseudomonas aeruginosa

In the last years, the textile industry has shown a growing interest in biosurfactants due to their biocompatibility , biodegradability , and versatility at various pH and temperature ranges . These compounds have found applications as softeners, wetting agents, lubricants, foam stabilizers, and even in the scouring of wool. This study aims to develop an economically efficient medium for biosurfactant production by Pseudomonas aeruginosa #112. Firstly , waste cooking oils after treatment (WCOT), a residue rich in lipids, was evaluated as an inducer of biosurfactants production . Different concentrations of these substrates (1, 2.5, 5, and 10 % w/v) were tested, and glucose was used as a carbon source. In the experiments with 1 % of WCOT it was observed a significant (p 0.05) reduction in the surface tension from 48.4 mN/m to 34.8 mN/m, suggesting the biosurfactant production . Furthermore , rice husk (RH) and vine pruning (VP) residues were identified as alternative carbon sources for biosurfactants production, when combined with WCOT . Both residues are rich in cellulose, which can be broken down into free glucose. An enzymatic pre- treatment that combines xylanase and cellulase was used to hydrolyze residues and release free glucose . The obtained results demonstrate that the combination of 1 % OUAT with hydrolyzed RH or VP resulted in a substantial (53 %) reduction in surface tension. At the end of the fermentation, 1.65 g/L and 0.26 g/L of biosurfactant were recovered for the experiments with hydrolyzed PV and RH, respectively. Additionally, the critical micelle dilution results demonstrate that the two tested media allow biosurfactant production and effective reduction of the surface tension of distilled water , even at low concentrations . This is the first report of biosurfactant production using a mixture of these three agro-industrial residues , which can be very useful for the sustainable production of these promising molecules.The authors acknowledge the financial support from integrated project be@t – Textile Bioeconomy (TCC12-i01, Sustainable Bioeconomy No. 02/C12-i01/2022), promoted by the Recovery and Resilience Plan (RRP), Next Generation EU, for the period 2021 – 2026. The authors also acknowledge the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit.info:eu-repo/semantics/publishedVersio

Life history of the common pandora, Pagellus erythrinus (Linnaeus, 1758) (Actinopterygii: Sparidae) from southern Portugal

The common pandora, Pagellus erythrinus, is a commercially important seabream for coastal fisheries in the north and central eastern Atlantic. Age, growth and reproductive aspects of the southern Portuguese population were estimated by analysing 386 specimens ranging in total length (TL) from 12.0 to 44.8 cm. Ages were estimated by counting growth bands on otoliths and verified by marginal increment analysis, with specimens ranging in age from 1 to 21 years. The von Bertalanffy growth function was selected as the most adequate model to fit this species' growth, with the estimated parameters being L-inf = 47.14 cm TL, k = 0.084 year(-1) and t(0) = - 4.42 year. The gonads were analyzed macroscopically over a one-year period and the reproductive spawning season found to occur from March to July. The length at first maturity for males occurs at 17.58 cm TL and 1.15 years while females mature at slightly smaller sizes (17.29 cm TL) and younger ages (1.04 years). The results presented in this study are important for comparing this population's parameters with those of other populations of the same species occurring in other areas, as well as for future studies, for assessing eventual changes in population parameters over time