Production of curcumin from ferulic acid by an engineered Saccharomyces cerevisiae

Dissertação de mestrado em BiotecnologiaCurcumin is a secondary metabolite produced by Curcuma longa. Studies have confirmed its biological and therapeutic effects in several diseases being the anticancer activity the most documented. Since curcumin is synthetized in low amounts, its heterologous production could represent a rapid and easy method to obtain large amounts of this bioactive compound. Curcumin has already been produced by Escherichia coli. However, the curcumin biosynthetic pathway has never been engineered in Saccharomyces cerevisiae. As a eukaryotic organism, it presents unique advantages over E. coli regarding the expression of plant derived genes. This work aimed to design, construct and validate a biosynthetic pathway composed by genes from different plants to produce curcumin from ferulic acid in an engineered S. cerevisiae. The enzymes involved in the artificial pathway are: 4-coumarate-CoA ligase (4CL) and different type III polyketide synthases (PKSs). In C. longa there are two types of PKSs - diketide-CoA synthase (DCS) and curcumin synthase (CURS) - that catalyse different reactions. Curcuminoid synthase (CUS) from Oryza sativa is also a PKS and catalysis the “one-pot” synthesis of curcuminoids in E. coli. Shuttle vectors with enzymes from different organisms were constructed and transformed into two S. cerevisiae strains. The vectors carried 4CL from Arabidopsis thaliana or Lithospermum erythrorhizon, and CUS or DCS and CURS. Curcumin was visually produced with a pathway composed by 4CL from L. erythrorhizon and CUS from O. sativa. The maximal curcumin production was 233.42 ng/L representing a yield of 1.46 %. Also, three different extraction methods were tested which revealed the importance of cell lysis in curcumin extraction from yeast. In addition, CRISPR-Cas9 method was used to knockout a gene from S. cerevisiae that codifies ferulic acid decarboxylase. As expected, the mutant strain was not able to consume ferulic acid. However, no curcumin production could be detected in the preliminary assays. It is important to mention that this study allowed to produce curcumin for the first time in an engineering yeast. In the future, optimizations at genetic and operational level are needed to improve the curcumin titters.A curcumina é um metabolito secundário produzido pela Curcuma longa. Vários estudos confirmaram o seu efeito biológico e terapêutico em várias doenças sendo a atividade anticancerígena a mais documentada. Devido ao facto da curcumina ser sintetizada em pequenas quantidades, a sua produção heteróloga pode representar um método rápido e fácil para obter grandes quantidades deste composto bioativo. A curcumina já foi produzida em Escherichia coli. No entanto, a sua via biossintética nunca foi introduzida em Saccharomyces cerevisiae. Como organismo eucariótico, este apresenta vantagens únicas em relação a E. coli para expressão de genes derivados de plantas. Este trabalho teve como objetivo projetar, construir e validar uma via biossintética composta por genes de diferentes plantas para produzir curcumina a partir de ácido ferúlico em S. cerevisiae. As enzimas envolvidas na via artificial são: 4-cumárico-CoA ligase (4CL) e as policetídeo sintase tipo III (PKS). Em C. longa existem dois tipos de PKSs - diketide-CoA sintase (DCS) e curcumina sintase (CURS) - que catalisam reações diferentes. A curcuminoide sintase (CUS) da Oryza sativa é também uma PKS. Esta enzima foi capaz a sintetizar curcuminoides em E. coli num só passo. Para esse efeito, vetores “shuttle” com enzimas de diferentes organismos foram construídos e transformados em duas estirpes de S. cerevisiae. Os vetores possuíam 4CL de Arabidopsis thaliana ou de Lithospermum erythrorhizon, e CUS ou DCS e CURS. Foi possível aferir visualmente a produção de curcumina nas leveduras onde foi inserida uma via composta por 4CL de L. erythrorhizon e CUS de O. sativa. A produção máxima de curcumina foi de 233,42 ng/L, o que representa um rendimento mássico de 1,5%. Além disso, três métodos de extração diferentes foram testados, o que demostrou a importância da lise celular para extração de curcumina em levedura. Além disso, o método CRISPR-Cas9 foi usado para silenciar um gene em S. cerevisiae que codifica a ácido ferúlico descarboxilase. Como esperado, o mutante resultante não foi capaz de consumir ácido ferúlico. No entanto, em ensaios preliminares também não foi possível detetar a produção de curcumina. É de realçar que este foi o primeiro trabalho em que a curcumina foi produzida por uma levedura geneticamente modificada. No futuro, são necessárias otimizações a nível genético e operacional por forma a aumentar os rendimentos de produção

Biosynthesis of plant polyphenol compounds with therapeutic and industrial relevance

Polyphenols are naturally produced in plants and have several biological and potential therapeutic activities such as anti inflammatory, antioxidant and anticancer They have an estimated market size of USD 2 26 billion by 2027 However, polyphenols are extracted from plants where they accumulate in low amounts over long growth periods In addition, their purification is difficult and expensive since it requires the separation of other compounds with similar chemical structures in an environmentally unfriendly process Heterologous microbial production has several benefits as it is not limited by plant availability or environmental factors and it is a renewable, environmentally friendly and sustainable approach Herein, we report the construction of artificial pathways for the production of curcuminoids and furanocoumarins using Escherichia coli as chassis These compounds can be produced from tyrosine or hydroxycinnamic acids as precursors and have in common the phenylpropanoids pathway Pure curcumin production from ferulic acid achieved 563 mg/L Curcuminoids were also produced from tyrosine 42 mg/L) using a modular pathway combining synthetic biologic and co culture engineering To our knowledge, these are the highest titers of curcuminoids obtained to date CRISPR Cas 9 was used to disrupt the lacZ gene in order to follow co culture population compositionPortuguese 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. In addition, the authors acknowledge 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. JR is recipient of a fellowship supported by a doctoral advanced training (SFRH/BD/138325/2018) funded by FCTinfo:eu-repo/semantics/publishedVersio

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

Pathway expression optimization using the Ribosome Binding Site (RBS) Calculator tool

Hydroxycinnamic acids and curcumin are plant metabolites with great therapeutic potential, including anti-inflammatory and anticancer activities. In this study, p-coumaric acid, caffeic acid and curcumin were produced in Escherichia coli using an artificial biosynthetic pathway [1]. Their production was induced by heat using the dnaK and ibpA heat shock promoters [2]. The ribosome binding sites (RBSs) used were tested and further optimized for each gene to assure an efficient translation. To optimize the RBSs we used the bioinformatic design tool RBS Calculator (v1.1) developed by Salis Lab (Penn State University) [3]. This tool predicts the translation initiation rate (TIR) of mRNAs and designs synthetic RBS with specific TIRs. This allows to improve the translation efficiency and to reach a desired response and therefore obtain the expected production using novel genes or biosynthetic pathways. Tyrosine ammonia lyase from Rhodotorula glutinis was used to produce p-coumaric acid from tyrosine. p-Coumaric acid was converted to caffeic acid using 4-coumarate 3-hydroxylase from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris. Curcumin was produced from ferulic acid using 4-coumarate-CoA ligase from Arabidopsis thaliana, diketide-CoA synthase and curcumin synthase from Curcuma longa. The optimization of the RBSs lead to an increase in the production of p-coumaric acid, caffeic acid and curcumin up to 97.8, 11.7 and 14.4 times, respectively. The highest p-coumaric acid, caffeic acid and curcumin production obtained were 2.5 mM, 370 µM and 17 µM, respectively. These results demonstrate that it is of utmost importance to consider the strength of the RBS when designing a biosynthetic pathway and user-friendly bioinformatic tools such as RBS Calculator can be very useful for that purpose. References: [1] J. L. Rodrigues, M. R. Couto, R. G. Araújo, K. L. J. Prather, L. D. Kluskens, L. R. Rodrigues. Hydroxycinnamic acids and curcumin production in engineered Escherichia coli using heat shock promoters, Biochemical Engineering Journal, 125, 41-49, 2017. [2] J. L. Rodrigues, M. Sousa, K. L. J. Prather, L. D. Kluskens, L. R. Rodrigues. Selection of Escherichia coli heat shock promoters toward their application as stress probes, Journal of Biotechnology, 188, 61-71, 2014. [3] A. E. Borujeni, A. S. Channarasappa, H. M. Salis. Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites, Nucleic Acids Research, 42, 26462659, 2014.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

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

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

Synthetic biology approaches to engineer polyphenols microbial cell factories

Polyphenols are secondary metabolites isolated from plants that can be divided into flavonoids, stilbenoids, curcuminoids, coumarins, polyphenolic amides and lignans. These exhibit diverse biological and potential therapeutic activities including antioxidant, anti-inflammatory and anticancer, among others. Despite all this potential, extracting polyphenols from plants is not straightforward given the low yields of the process. The extracted amounts are not sufficient to respond to the increasing demand for polyphenols, the process is expensive and unfriendly for the environment. Hence, developing microbial cell factories to effectively produce polyphenols arises as an attractive way to address the mentioned limitations and produce high amounts of these compounds. Advances in the metabolic engineering and synthetic biology fields have been key in the design of efficient and robust microbial cell factories, mainly due to the development of proper molecular biology tools, as well as to the unravelling of new enzymes in plants or other organisms to better engineer such heterologous pathways. Several hosts have been explored as potential polyphenols microbial cell factories. However, there is still a long way before this production at an industrial scale can become a reality. The perspectives and current challenges resulting from these developments will be discussed.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

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