A combinatorial approach to optimize the production of curcuminoids from tyrosine in Escherichia coli

Curcuminoids are well-known for their therapeutic properties. However, their extraction from natural sources is environmentally unfriendly, expensive and limited by seasonal variability, highlighting the need for alternative production processes. We propose an optimized artificial biosynthetic pathway to produce curcuminoids, including curcumin, in Escherichia coli. This pathway involves six enzymes, tyrosine ammonia lyase (TAL), 4-coumarate 3-hydroxylase (C3H), caffeic acid O-methyltransferase (COMT), 4-coumarate-CoA ligase (4CL), diketide-CoA synthase (DCS), and curcumin synthase (CURS1). Curcuminoids pathway was divided in two modules, the first module included TAL, C3H and COMT and the second one 4CL, DCS and CURS1. Optimizing the first module of the pathway, from tyrosine to ferulic acid, enabled obtaining the highest ferulic acid titer reported so far (1325.1 M). Afterward, ferulic acid was used as substrate to optimize the second module of the pathway. We achieved the highest concentration of curcumin ever reported (1529.5 M), corresponding to a 59.4% increase. Subsequently, curcumin and other curcuminoids were produced from tyrosine (using the whole pathway) in mono-culture. The production increased comparing to a previously reported pathway that used a caffeoyl-CoA O-methyltransferase enzyme (to convert caffeoyl-CoA to feruloyl-CoA) instead of COMT (to convert caffeic to ferulic acid). Additionally, the potential of a co-culture approach was evaluated to further improve curcuminoids production by reducing cells metabolic burden. We used one E. coli strain able to convert tyrosine to ferulic acid and another able to convert the hydroxycinnamic acids produced by the first one to curcuminoids. The co-culture strategies tested led to 6.6 times increase of total curcuminoids (125.8 M) when compared to the mono-culture system. The curcuminoids production achieved in this study corresponds to a 6817% improvement. In addition, by using an inoculation ratio of 2:1, although total curcuminoids production decreased, curcumin production was enhanced and reached 43.2 M, corresponding to an improvement of 160% comparing to mono-culture system. To our knowledge, these values correspond to the highest titers of curcuminoids obtained to date. These results demonstrate the enormous potential of modular co-culture engineering to produce curcumin, and other curcuminoids, from tyrosine.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-01-0145-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. LR also acknowledges her sabbatical leave fellowship (SFRH/BSAB/142991/2018) funded by the FCTinfo:eu-repo/semantics/publishedVersio

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

De novo curcuminoids production in engineered Escherichia coli

Curcuminoids are a mixture of polyphenolic compounds (curcumin, bisdemethoxycurcumin and demethoxycurcumin) produced in the plant Curcuma longa with several therapeutic properties, including anticancer, anti-inflammatory, among others. Given this great potential, the curcuminoids market size is expected to reach $369M by 2033 with 11.3% CAGR [1]. However, these compounds are present in low amounts, accumulate during long growth periods, and their purification is environmentally unfriendly, expensive and difficult originating low yields and purity [2]. Hence, their biosynthesis by engineered microbes using synthetic biology has emerged as a potential competitive alternative. In the past, we developed for the first time a biosynthetic pathway in E. coli to produce curcumin, the curcuminoid with the highest therapeutic potential, from tyrosine, obtaining the highest production titers reported (43.2 µM curcumin, 125.8 µM total curcuminoids) [3,4]. This opened the door to produce curcumin from simple carbon sources which will be key at an industrial scale. In this study, we used a tyrosine overproducing E. coli strain to produce curcuminoids from glucose. Also, the previously constructed combinatorial biosynthetic pathway [3] was modified to overexpress more efficient enzymes for specific steps [5], including 4-hydroxyphenylacetate 3-hydroxylase units B/C from Salmonella enterica and Pseudomonas aeruginosa, that convert the highly accumulated coumaric acid into caffeic acid, and feruloyl-CoA synthase from Sphingobium sp., that converts the hydroxycinnamic acids to CoA esters, having higher affinity to ferulic acid. All these modifications allowed to obtain the highest titers of curcumin reported so far from glucose with low amounts of the other curcuminoids being produced (> 250 µM curcumin, 12 µM total curcuminoids).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. Daniela Gomes acknowledges her grant SFRH/BD/04433/2020.info:eu-repo/semantics/publishedVersio

Synthetic biology approaches to engineer saccharomyces cerevisiae towards the industrial production of valuable polyphenolic compounds

Polyphenols are plant secondary metabolites with diverse biological and potential therapeutic activities such as antioxidant, anti-inflammatory and anticancer, among others. However, their extraction from the native plants is not enough to satisfy the increasing demand for this type of compounds. The development of microbial cell factories to effectively produce polyphenols may represent the most attractive solution to overcome this limitation and produce high amounts of these bioactive molecules. With the advances in the synthetic biology field, the development of efficient microbial cell factories has become easier, largely due to the development of the molecular biology techniques and by the identification of novel isoenzymes in plants or simpler organisms to construct the heterologous pathways. Furthermore, efforts have been made to make the process more profitable through improvements in the host chassis. In this review, advances in the production of polyphenols by genetically engineered Saccharomyces cerevisiae as well as by synthetic biology and metabolic engineering approaches to improve the production of these compounds at industrial settings are discussed.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. JR is recipient of a fellowship supported by a doctoral advanced training (SFRH/BD/138325/2018) funded by FCT.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

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

Skin-Integrated wearable systems and implantable biosensors: a comprehensive review

Biosensors devices have attracted the attention of many researchers across the world. They have the capability to solve a large number of analytical problems and challenges. They are future ubiquitous devices for disease diagnosis, monitoring, treatment and health management. This review presents an overview of the biosensors field, highlighting the current research and development of bio-integrated and implanted biosensors. These devices are micro- and nano-fabricated, according to numerous techniques that are adapted in order to offer a suitable mechanical match of the biosensor to the surrounding tissue, and therefore decrease the body’s biological response. For this, most of the skin-integrated and implanted biosensors use a polymer layer as a versatile and flexible structural support, combined with a functional/active material, to generate, transmit and process the obtained signal. A few challenging issues of implantable biosensor devices, as well as strategies to overcome them, are also discussed in this review, including biological response, power supply, and data communication.This research was funded by FCT- FUNDAÇÃO PARA A CIÊNCIA E TECNOLOGIA, grant numbers: PTDC/EMD-EMD/31590/2017 and PTDC/BTM-ORG/28168/2017

Cryopreservation of cell laden natural origin hydrogels for cartilage regeneration strategies

Statement of Purpose: An increase of scientific published literature and clinical experience supports the requirement of providing products like cultured cells and tissues to the market. One of the main prospects of cartilage tissue engineering is the possibility of developing custom-made regenerative medicine solutions on an individual patient basis. The efficient preservation and storage procedure will provide products available as needed which could be adapted to an autologous immediate solution. Thus, the aim of this study was to examine the effects of the cryopreservation on the chondrogenic differentiation characteristics of human mesenchymal stem cells isolated from adipose tissue (hASCs). Furthermore, we also propose to determine hASCs-hydrogel stability and confirm the potential of these bioengineered constructs to be applied in cartilage regeneration. The results obtained show that the hydrogels withstand the cryopreservation process maintaining their structural integrity, with good cell content after cryopreservation. Thus, cell encapsulation systems of natural based hydrogels may be an interesting approach for the long term preservation of cartilage tissue engineered products. Methods: The κ-carrageenan (κCR) hydrogels were produced using an ionotropic gelation method. Then, stem cells, namely human adipose derived stem cells (hASCs), were encapsulated in κCR discs (5 mm dia. x 3 mm height) at a density of 5x106 cell/cm3 and cultured for 21 days in standard basal (BM) or chondrogenic media (ChM). The cell hydrogels were cryopreserved in liquid nitrogen for up to 30 days. The overall morphology of κCR discs with encapsulated hASCs was observed under light microscope. hASCs viability and proliferation rate was determined by double stand DNA quantification. Additionally, chondrogenic differentiation of hASCs encapsulated in the hydrogels is being characterized by histological staining for selective cartilage staining and real time PCR analysis (Sox9, aggrecan, and collagen: type I, type II and type X). DMA analysis allowed determining the mechanical properties of κ-carrageenan hydrogels, namely storage (elastic) and loss (viscous) while immersed in wet state at 24 °C and throughout a physiological relevant range of frequencies. The described characterization assays were performed both before (BC) and after cryopreservation/freezing (AC) time points. Results: The cell morphology, distribution and appearance of the hydrogels are clearly observed from the microscopic light images (Figure 1A). It is possible to observe the smooth and homogeneous surface, the well defined and stable shape before and after the freezing process. Encapsulated hASCs were able to maintain cellular content, despite an expected decrease observed upon cryopreservation (Figure 1B), which is associated to a recovery time after thawing. The microscopic images and biological evaluation of κCR hydrogel revealed that the cryopreservation process did not change the cellular morphology; the surface and integrity of the hydrogel disc and enables maintenance of hASCs after exposure to low temperatures environments. Figure 1. (A) Representative optical micrographs of hASCs encapsulated in κCR hydrogels and cultured in ChM and BM before and after cryopreservation and (B) cell proliferation results, based on the quantification of dsDNA content. Scale bar represent 100 μm. Conclusions: The results obtained so far indicated the feasibility of hASCs-κCR system in cartilage tissue engineering regeneration strategies due to its ability to support hASCs viability before and after cryopreservation. Ongoing studies on the assessment of chondrogenic features of these cryopreserved systems will provide information on the effect of cryopreservation indicative of a stable chondrocyte phenotype. In summary, this study provided information on the potential of ASCs-hydrogel constructs for a long term storage and ready to use bioengineered tissue substitutes for cartilage regeneration strategies. References: (Popa EG. Biomacromolecules 2011;12:3952-3961