Heterologous production of curcuminoids in E. coli through an artificial biosynthetic pathway

Curcuminoids are natural pigments from plants that have been reported as potential cancer-fighting drugs. Nevertheless, they have a poor bioavailability. Cellular uptake is low, and they are quickly metabolized once inside the cell, requiring repetitive oral doses to achieve sufficient concentration inside the cell for therapeutic activity. The aim of this work is to engineer an artificial biosynthetic pathway for the production of curcuminoids by Escherichia coli. Starting from the substrate tyrosine, the curcumin pathway involves several enzymatic steps: conversion of tyrosine to p-coumaric acid; conversion of p-coumaric acid to caffeic acid; production of caffeoyl-CoA from caffeic acid; production of feruloyl-CoA from caffeoyl-CoA; and finally the production of curcumin from feruloyl-CoA and possibly other curcuminoids, due to enzyme promiscuity. The enzymes involved in the two first enzymatic steps are tyrosine ammonia lyase from Rhodotorula glutinis, P450 CYP199A2 from Rhodopseudomonas palustris, and the redox partners pdr from Pseudomonas putida and pux from R. palustris. Coumaric acid and caffeic acid were successfully produced. A coumaroyl-CoA ligase from Arabidopsis thaliana is being explored for the conversion of the different carboxylic acids into their corresponding CoA esters. Different combinations of this enzyme and caffeoyl-CoA 3-methyl transferase may lead to the production of different curcuminoids. For the last step of the pathway two approaches are being studied: the use of diketide-CoA synthase and curcuminoid synthase from Curcuma longa, and curcumin synthase from Oryza sativa that itself catalyzes both steps. Curcumin and bisdemethoxycurcumin were produced using both approaches and their production was confirmed by HPLC analysis, as well as by the yellow color of the culture supernatant. Successful construction of the curcuminoids biosynthetic pathway would mark a significant step forward in the in situ production of these poorly soluble, anti-carcinogenic compounds

Design and construction of a new biosynthetic pathway for the production of curcuminoids in Escherichia coli

Curcuminoids are produced by plants and due to their potential as novel cancer-fighting drugs they have recently attracted increased attention. Nevertheless, they have a poor bioavailability. Cellular uptake is low, and they are quickly metabolized once inside the cell, requiring repetitive oral doses to achieve sufficient concentration inside the cell for therapeutic activity. The goal of this PhD project is to engineer a synthetic pathway for curcuminoid in a model bacterium and trigger its release concurrent with ultrasound treatment. The proposed tasks involve several design and engineering steps to program Escherichia coli to execute the new synthetic pathway triggered by a temperature increase. The heat shock response machinery of E. coli will be used as a sensor in the design of the model bacterium. Afterwards, the gene sequences of the enzymes that catalyze each reaction in the curcuminoid pathway will be synthesized and introduced in the E. coli genome applying several cloning strategies. Data from several well documented experiments on E. coli in relevant conditions that have been published were analyzed to select the most expressed heat shock genes in E. coli with the strongest heat shock promoters. The ibpA, dnaK and fxsA gene promoters were chosen based on their induction rates and expression and were validated by RT-qPCR and subsequently through the construction of a stress probe using an adequate reporter gene

Heterologous production of acrylic acid: current challenges and perspectives

Acrylic acid (AA) is a chemical with high market value used in industry to produce diapers, paints, adhesives and coatings, among others. AA available worldwide is chemically produced mostly from petroleum derivatives. Due to its economic relevance, there is presently a need for innovative and sustainable ways to synthesize AA. In the past decade, several semi-biological methods have been developed and consist in the bio-based synthesis of 3-hydroxypropionic acid (3-HP) and its chemical conversion to AA. However, more recently, engineered Escherichia coli was demonstrated to be able to convert glucose or glycerol to AA. Several pathways have been developed that use as precursors glycerol, malonyl-CoA or β-alanine. Some of these pathways produce 3-HP as an intermediate. Nevertheless, the heterologous production of AA is still in its early stages compared, for example, to 3-HP production. So far, only up to 237 mg/L of AA have been produced from glucose using β-alanine as a precursor in fed-batch fermentation. In this review, the advances in the production of AA by engineered microbes, as well as the hurdles hindering high-level production, are discussed. In addition, synthetic biology and metabolic engineering approaches to improving the production of AA in industrial settings are presented.This study was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of the UIDB/04469/2020 unit.info:eu-repo/semantics/publishedVersio

Potential applications of the Escherichia coli heat shock response in synthetic biology

The Escherichia coli heat shock response (HSR) is a complex mechanism triggered by heat shock and by a variety of other growth-impairing stresses. We explore here the potential use of the E. coli HSR mechanism in synthetic biology approaches. Several components of the regulatory mechanism (such as heat shock promoters, proteins, and RNA thermosensors) can be extremely valuable in the creation of a toolbox of well-characterized biological parts to construct biosensors or microbial cell factories with applications in the environment, industry, or healthcare. In the future, these systems can be used for instance to detect a pollutant in water, to regulate and optimize the production of a compound with industrial relevance, or to administer a therapeutic agent in vivo.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and under the scope of the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462). The authors also acknowledge financial support from BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020–Programa Operacional Regional do Norte, and a Postdoctoral grant (UMINHO/BPD/37/2015) to J.L.R. funded by the FCT.info:eu-repo/semantics/publishedVersio

Microbial production of caffeic acid

First Online: 04 October 2022Caffeic acid is a hydroxycinnamic acid mostly produced in plants although its microbial production has also been reported. This compound presents several biological activities and potential therapeutic properties. Additionally, it can be a precursor or intermediary of various relevant compounds. Current production methods include the inefficient, expensive, and not environmentally friendly extraction from plants that accumulate this compound in very low amounts. Therefore, highly efficient and environmentally friendly methods are needed. Microbial biosynthesis can potentially produce it in a purer, faster, and greener way. Since the establishment of caffeic acid heterologous production in Streptomyces fradiae, several studies have been published regarding its production in Escherichia coli and Saccharomyces cerevisiae. These studies include the production from supplemented tyrosine or p-coumaric acid but also glucose using tyrosine-overproducing strains. Presently, there are three different pathways to produce caffeic acid that have in common the first step that is catalyzed by a microbial tyrosine ammonia lyase that converts tyrosine to p-coumaric acid. The second step that synthesizes caffeic acid from p-coumaric acid was identified as the pathway bottleneck and can be performed by 4-coumarate 3-hydroxylase, hydroxyphenylacetate 3-hydroxylase (4HPA3H) complex or a cytochrome P450 CYP199A2 system. Although all these enzymes have been identified in bacteria, and caffeic acid has only recently been produced in S. cerevisiae, the productions in this host have almost reached the maximum productions reported for E. coli (569 mg/L vs. 767 mg/L, respectively). The maximum production was obtained from glucose using the 4HPA3H pathway. These developments on caffeic acid heterologous production are very promising.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.info:eu-repo/semantics/publishedVersio

Design and construction of a new biosynthetic pathway for the production of curcuminoids in Escherichia coli

Tese de doutoramento em BioengenhariaCurcuminoids are plant phenolic compounds with great anti-cancer potential. This thesis addresses the construction of an artificial biosynthetic pathway for curcuminoids production in Escherichia coli. This production was triggered by chemical or thermal induction to be used in industrial applications or bacterial therapies, respectively. To produce curcuminoids, including curcumin, from tyrosine, caffeic acid was produced as an intermediate in the pathway. The caffeic acid pathway design included tyrosine ammonia lyase (TAL) from Rhodotorula glutinis to convert tyrosine to p-coumaric acid and 4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris to convert p-coumaric acid to caffeic acid. TAL was able to efficiently convert tyrosine to p-coumaric acid and CYP199A2 exhibited higher catalytic activity towards p-coumaric acid than C3H. This is the first study that shows caffeic acid production using CYP199A2 and tyrosine as the initial precursor; 280 mg/L of caffeic acid was produced. After validating the first steps of the pathway, curcuminoids production was studied. The best results were obtained with Arabidopsis thaliana 4-coumaroyl-CoA ligase (4CL1) and Curcuma longa diketide-CoA synthase (DCS) and curcumin synthase (CURS1), yielding 70 mg/L of curcumin from ferulic acid. To produce curcumin through the caffeic acid pathway, caffeoyl-CoA O-methyltransferase from Medicago sativa was used to convert caffeoyl-CoA to feruloyl-CoA. Using caffeic acid, p-coumaric acid or tyrosine as a substrate, 3.90 mg/L, 0.26 mg/L and 0.20 mg/L of curcumin were produced, respectively. Finally, caffeic acid and curcumin production was induced by heat using the dnaK heat shock promoter. Caffeic acid was successfully produced from tyrosine using TAL, C3H or CYP199A2 and the highest production was 14.41 mg/L. Regarding curcumin, 0.37 mg/L was produced from ferulic acid using 4CL1, DCS and CURS1. This is the first report on the in vivo use of DCS and CURS1 to produce curcuminoids. It was also demonstrated that curcumin can be produced from tyrosine using a pathway through the caffeic acid production. This alternative pathway represents a significant improvement in the heterologous production of curcuminoids using E. coli. In addition, caffeic acid and curcumin production in E. coli can be triggered by heat, thus suggesting its potential for the development of new bacterial therapies.Os curcuminóides são compostos fenólicos de plantas com grande potencial anti-cancerígeno. Esta tese aborda a construção de uma via biossintética para a produção artificial de curcuminóides em Escherichia coli. Esta produção foi induzida por via química ou térmica para ser usada em aplicações industriais ou em terapias bacterianas. Para produzir os curcuminóides, incluindo a curcumina, a partir da tirosina, o ácido cafeico foi produzido como intermediário na via. A via do ácido cafeico incluiu a tirosina amónia liase (TAL) da Rhodotorula glutinis para converter a tirosina em ácido p-cumárico e o 4-cumarato 3-hidroxilase (C3H) da Saccharothrix espanaensis ou o citocromo P450 CYP199A2 da Rhodopseudomonas palustris para converter o ácido p-cumárico em cafeico. TAL foi capaz de converter de forma eficiente a tirosina em ácido p-cumárico e CYP199A2 exibiu uma maior actividade catalítica em relação ao ácido p-cumárico do que C3H. Este é o primeiro estudo que mostra a produção de ácido cafeico utilizando CYP199A2 e a tirosina como precursor; tendo sido produzidos 280 mg/L. Depois de validar os primeiros passos da via, os curcuminóides foram produzidos. Os melhores resultados foram obtidos com a 4-cumaroil-CoA ligase (4CL1) da Arabidopsis thaliana e com a dicetídeo-CoA sintase (DCS) e curcumina sintase 1 (CURS1) da Curcuma longa e 70 mg/L de curcumina foram produzidos a partir do ácido ferúlico. Para produzir curcumina pela via do ácido cafeico, a cafeoil-CoA O-metiltransferase da Medicago sativa foi utilizada para converter o cafeoil-CoA em feruloil-CoA. Utilizando o ácido cafeico, o ácido p-cumárico e a tirosina como substrato, 3.90 mg/L, 0.26 mg/L e 0.20 mg/L de curcumina foram produzidos, respectivamente. Finalmente, a produção de ácido cafeico e de curcumina foi induzida por calor utilizando o promotor de choque térmico dnaK. O ácido cafeico foi produzido com sucesso usando TAL, C3H ou CYP199A2 e a sua maior produção foi de 14,41 mg/L. Em relação à curcumina, foram produzidos 0,37 mg/L usando 4CL1, DCS e CURS1. Este é o primeiro estudo sobre a utilização in vivo de DCS e CURS1 para produzir curcuminóides. A curcumina foi também produzida pela primeira vez a partir da tirosina usando a via de produção de ácido cafeico. A produção de ácido cafeico e de curcumina pode também ser despoletada por calor, sugerindo assim o seu potencial para o desenvolvimento de novas terapias bacterianas.Esta investigação foi financiada pela Fundação para a Ciência e Tecnologia através da concessão de uma bolsa de doutoramento (SFRH/BD/51187/2010), co-financiada pelo COMPETE - POPH - QREN - Tipologia 4.1 - Formação Avançada - e pelo ON2, comparticipados pelo Fundo Social Europeu (FSE) e por fundos nacionais do Ministério da Ciência, Tecnologia e Ensino Superior (MCTES)

Production of curcumin from ferulic acid by an engineered Saccharomyces cerevisiae

II Congress in Health Sciences Research: Towards Innovation and Entrepreneurship - Trends in Biotechnology for Biomedical ApplicationsCurcumin is a secondary metabolite produced in the rhizome of Curcuma longa that represents the most active and studied naturally-derived curcuminoid product. 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 in engineered Escherichia coli although with low yields. However, the curcumin biosynthetic pathway has never been engineered in Saccharomyces cerevisiae. As a eukaryotic organism, it presents unique advantages over E. coli that facilitate the expression of plant derived genes. This work aimed to design an artificial biosynthetic pathway for the production of curcumin by S. cerevisiae. The principal enzymes involved in the artificial pathway are: 4-coumarate-CoA ligase (4CL) and the type III polyketide synthases (PKSs). In C. longa there are two types of PKSs - diketide-CoA (DCS) and curcumin synthase (CURS) - that catalyse different reactions. Curcuminoid synthase (CUS) from Oryza sativa is also a PKS able of catalysing the one-pot synthesis of curcuminoids in E. coli. Herein, we intended to produce curcumin using ferulic acid as precursor. For that purpose, shuttle vectors with enzymes from different organisms were constructed and transformed in S. cerevisiae CENPK2-1C. The vectors carry 4CL from Arabidopsis thaliana or Lithospermum erythrorhizon, and DCS and CURS or CUS. DCS and CURS were codon-optimized for S. cerevisiae. In addition, CRISPRCas9 method was used to knockout a gene from S. cerevisiae that codifies ferulic acid decarboxylase that is responsible for the ferulic acid decarboxylation as a detoxification process. The engineered strains are currently being tested for curcumin production.info:eu-repo/semantics/publishedVersio

Combinatorial approaches for de novo production of flavonoids inEscherichia coli

Polyphenols are plant secondary metabolites that are produced in response to biotic and abiotic stress. Flavonoids, one of the most representative group of these metabolites, includes at least 9000 compounds. Among them, naringenin has been widely studied due to its interesting biological activities, namely anticancer, anti-inflammatory, and antioxidant. Due to its potential applications and to the attempt to satisfy the industrial demand, there has been an increased interest in the heterologous production of this compound in a microbial chassis. The production of naringenin by a microorganism involves a first step of conversion of tyrosine into coumaric acid by tyrosine ammonia-lyase (TAL). Afterwards, coumaric acid is converted into coumaroyl-CoA by 4-coumarate-CoA ligase (4CL). Coumaroyl-CoA is further converted into naringenin chalcone by chalcone synthase (CHS) and then into naringenin by chalcone isomerase (CHI). In this work, we aimed to design, construct and validate an efficient biosynthetic pathway to produce naringenin in Escherichia coli by performing a step-by-step optimization. To construct the biosynthetic pathway to produce naringenin, TAL, 4CL, CHS, and CHI genes from different organisms were selected. Initially, TAL from Rhodotorula glutanis (RgTAL) and TAL from Flavobacterium johnsoniae (FjTAL) were cloned into the pRSFDuet-1 vector and were further expressed in three different E. coli strains (E. coli BL21, K12 MG1655 and M-PAR-121) to select the best enzyme and strain to produce coumaric acid. The highest production was obtained in the E. coli M-PAR-121 strain expressing FjTAL (2.54 g/L of coumaric acid from 40 g/L glucose). E. coli M-PAR-121 is a tyrosine-overproducing strain and it can produce high amounts of tyrosine from glucose that can be converted into coumaric acid. Thus, this strain and enzyme were chosen to construct the complete biosynthetic pathway. Afterwards, 4CL and CHS steps were constructed and validated. Specifically, 4CL from Arabidopsis thaliana (At4CL), 4CL from Vitis Vinifera (Vv4CL), and 4CL from Petroselinum crispum (Pc4CL) were cloned into the pACYCDuet-1 vector. Moreover, CHS from A. thaliana (AtCHS), CHS from Petunia hybrida (PhCHS), and CHS from Curcubita maxima (CmCHS) were cloned into pCDFDuet-1 vector. Twelve different combinations of the 4CL and CHS genes were expressed in the best strain able to produce coumaric acid and the naringenin chalcone production from glucose was evaluated. The best naringenin chalcone production was obtained in the E. coli M-PAR-121 strain expressing pRSFDuet_FjTAL, pACYCDuet_At4CL and pCDFDuet_CmCHS (311.0 mg/L). Aiming to increase the productivity of the engineered strain, the metabolic burden of the cells was reduced by cloning the three genes in only two plasmids. From the different combinations tested, E. coli M-PAR-121 strain holding pRSFDuet_FjTAL_CmCHS and pACYCDuet_At4CL has reached the highest production of naringenin chalcone (560.2 mg/L). Afterwards, the last step of the biosynthetic pathway was validated. At this point, CHI from A. thaliana, CHI from M. sativa and CHI from C. maxima were tested. The maximum production was achieved in the E. coli M-PAR-121 strain expressing pRSFDuet_FjTAL_CmCHS and pACYCDuet_At4CL_AtCHI (366.6 mg/L). This production was one of the highest reported so far in shake-flask experiments using glucose as substrate.Daniela Gomes acknowledges SFRH/BD/04433/2020 grant, from Portuguese Foundation for Science and Technology (FCT). This study was supported by the 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

Challenges in the heterologous production of furanocoumarins in Escherichia coli

Coumarins and furanocoumarins are plant secondary metabolites with known biological activities. As they are present in low amounts in plants, their heterologous production emerged as a more sustainable and efficient approach to plant extraction. Although coumarins biosynthesis has been positively established, furanocoumarin biosynthesis has been far more challenging. This study aims to evaluate if Escherichia coli could be a suitable host for furanocoumarin biosynthesis. The biosynthetic pathway for coumarins biosynthesis in E. coli was effectively constructed, leading to the production of umbelliferone, esculetin and scopoletin (128.7, 17.6, and 15.7 µM, respectively, from tyrosine). However, it was not possible to complete the pathway with the enzymes that ultimately lead to furanocoumarins production. Prenyltransferase, psoralen synthase, and marmesin synthase did not show any activity when expressed in E. coli. Several strategies were tested to improve the enzymes solubility and activity with no success, including removing potential N-terminal transit peptides and expression of cytochrome P450 reductases, chaperones and/or enzymes to increase dimethylallylpyrophosphate availability. Considering the results herein obtained, E. coli does not seem to be an appropriate host to express these enzymes. However, new alternative microbial enzymes may be a suitable option for reconstituting the furanocoumarins pathway in E. coli. Nevertheless, until further microbial enzymes are identified, Saccharomyces cerevisiae may be considered a preferred host as it has already been proven to successfully express some of these plant enzymes.This study was supported by the 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. D.G. acknowledges her grant SFRH/BD/04433/2020.info:eu-repo/semantics/publishedVersio

Rice industry residues potential for curcumin production by engineered Saccharomyces cerevisiae

The use of curcumin, a polyphenol derived from turmeric, as a fighting cancer drug has been raising significant interest. However, obtaining curcumin-rich preparations can be challenging, and its microbial production provides a promising solution. The use of microorganisms comprises low-cost cultivation methods and fast production cycles. To this end, we have developed an engineered Saccharomyces cerevisiae strain that can produce curcumin from simple carbon sources. Curcumin biosynthesis requires several enzymatic steps, including reactions catalyzed by the phenylpropanoid pathway and by type III polyketide enzymes, none of them naturally present in yeast. To enable curcumin production, we introduced the necessary genes into the S. cerevisiae genome and confirmed the de novo production of curcumin using standard media. Rice production is a major global food industry, and its processing generates large amounts of non-food biomass. Rice residues are rich in cellulose polymer, which can be broken down to obtain free glucose. Our aim is to use extracts from rice residues as the substrate to produce curcumin with our engineered yeast. These extracts also contain intermediates of the curcumin pathway such as ferulic acid, which can enhance curcumin biosynthesis. We performed hydrolysis on rice husk and bran residues to obtain the extracts and quantified the presence of carbon sources, fermentation inhibitors, and curcumin precursors in both extracts. We are currently using these extracts in yeast fermentation for curcumin production.info:eu-repo/semantics/publishedVersio