Optimization of fermentation conditions for the production of curcumin by engineered Escherichia coli

Curcumin is a plant secondary metabolite with outstanding therapeutic effects. Therefore, there is a great interest in developing new strategies to produce this high-value compound in a cheaper and environmentally friendly way. Curcumin heterologous production in E. coli using artificial biosynthetic pathways was previously demonstrated using synthetic biology approaches. However, the culturing conditions to produce this compound were not optimized and so far only a two-step fermentation involving the exchange of the culture medium allowed to obtain high concentrations of curcumin, which limits its production at an industrial scale. In this study, the culturing conditions to produce curcumin were evaluated and optimized. In addition, it was concluded that E. coli BL21 allows to produce higher concentrations compared to E. coli K-12 strains. Different IPTG concentrations, time of protein expression induction and substrate type and concentration were also evaluated. The highest curcumin production obtained was 959.3 µM (95.93% of percent yield), which was 3.1-fold higher than the highest concentration previously reported. This concentration was obtained using a two-stage fermentation with LB and M9. Moreover, TB demonstrated to be a very interesting alternative medium to produce curcumin since it also led to high concentrations (817.7 µM). The use of this single fermentation medium represents an advantage at industrial scale and although the final production is lower than the one obtained with the LB-M9 combination, it leads to a significantly higher curcumin production in the first 24 h of fermentation. This study allowed obtaining the highest concentrations of curcumin reported so far in a heterologous organism and is of interest for all of those working with the heterologous production of curcuminoids, other complex polyphenolic compounds or plant secondary metabolites.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of the UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01- 0145-FEDER-006684) and under the scope of the Project MultiBiorefinery-Multi-purpose strategies for broadband agro-forest and fisheries by-products valorization: a step forward for a truly integrated biorefinery (POCI-01-0145-FEDER-016403). 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 the post-doctoral grant (UMINHO/BPD/37/2015) to J.L.R. funded by FCT.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

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

Enhancing curcuminoid production using E. coli engineered strains

Curcuminoids are natural secondary metabolites from the herb Curcuma longa. Their beneficial properties, mainly as anti-cancer agents, have been exhaustively reported but the therapeutic effect of curcumin is limited by its fast systemic elimination along with poor bioavailability. Besides, curcumin extraction from plants is very expensive and it is hard to synthetize chemically. For these reasons, the use of microorganisms to produce these remarkable compounds on large scale and with greater yields constitutes an interesting approach. In the SYNBIOBACTHER project, the aim of producing curcumin from ferulic acid using an engineered Escherichia coli was achieved adding three enzymatic steps using plant genes (4-coumarate-CoA ligase (4CL) from Arabidopsis thaliana; diketide-CoA synthase (DCS) and curcumin synthase 1 (CURS1) from C. longa). The present work aims to improve curcumin production from ferulic acid by optimizing the production medium and other operational conditions. Previously, we used a standard two-step fermentation strategy (LB + M9 minimal media) to overcome the metabolic burden associated with protein overexpression and poor growth observed in minimal medium. Although feasible at the laboratory scale, the biomass separation is much more difficult, laborious and expensive in large scale fermentations. Therefore, we intend to develop a single medium formulation more suitable for the production of curcuminoids. MOPS minimal medium, TB and also LB and M9 are being evaluated. Furthermore, previously we studied in silico which gene deletions would enhance the curcumin production by the metabolic engineered E. coli. Using a recombineering approach, we are implementing those gene knockouts to construct several E. coli mutants (Δgnd; ΔfumA,fumB,fumC; ΔfumA,fumB,fumC,ccmA; ΔfumA,fumB,fumC,ccmA,argO) that will produce curcumin from ferulic acid. The curcuminoids production by these E. coli mutants is being evaluated

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

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

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

Cloning, expression and characterization of UDP-glucose dehydrogenases

Uridine diphosphate-glucose dehydrogenase (UGD) is an enzyme that produces uridine diphosphate-glucuronic acid (UDP-GlcA), which is an intermediate in glycosaminoglycans (GAGs) production pathways. GAGs are generally extracted from animal tissues. Efforts to produce GAGs in a safer way have been conducted by constructing artificial biosynthetic pathways in heterologous microbial hosts. This work characterizes novel enzymes with potential for UDP-GlcA biotechnological production. The UGD enzymes from Zymomonas mobilis (ZmUGD) and from Lactobacillus johnsonii (LbjUGD) were expressed in Escherichia coli. These two enzymes and an additional eukaryotic one from Capra hircus (ChUGD) were also expressed in Saccharomyces cerevisiae strains. The three enzymes herein studied represent different UGD phylogenetic groups. The UGD activity was evaluated through UDP-GlcA quantification in vivo and after in vitro reactions. Engineered E. coli strains expressing ZmUGD and LbjUGD were able to produce in vivo 28.4 µM and 14.9 µM UDP-GlcA, respectively. Using S. cerevisiae as the expression host, the highest in vivo UDP-GlcA production was obtained for the strain CEN.PK2-1C expressing ZmUGD (17.9 µM) or ChUGD (14.6 µM). Regarding the in vitro assays, under the optimal conditions, E. coli cell extract containing LbjUGD was able to produce about 1800 µM, while ZmUGD produced 407 µM UDP-GlcA, after 1 h of reaction. Using engineered yeasts, the in vitro production of UDP-GlcA reached a maximum of 533 µM using S. cerevisiae CEN.PK2-1C_pSP-GM_LbjUGD cell extract. The UGD enzymes were active in both prokaryotic and eukaryotic hosts, therefore the genes and expression chassis herein used can be valuable alternatives for further industrial applications.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. The authors acknowledge FCT for funding MRC doctoral grant SFRH/BD/132998/2017.info:eu-repo/semantics/publishedVersio

Atmospheric plasma and UV polymerisation for developing sustainable anti-adhesive polyethylene terephthalate (PET) surfaces

Enhancing the hydrophilicity of polymeric materials is an important step for achieving anti-adhesiveness. Thus, in this study, atmospheric plasma as a pre-treatment was combined with a UV grafting process to obtain a durable surface modification on polyethylene terephthalate (PET). The most promising conditions for the atmospheric plasma process were found to be 15 kW power and 4 m/min speed, leading to a contact angle reduction from 70 ± 6° to approximately 30°. However, it was observed that these values increased over time due to the ageing and washing of the PET surface, ultimately causing it to recover its initial contact angle. Therefore, the plasma-pre-treated PET samples were further modified through a UV grafting process using sodium acrylate (NaAc) and 3-sulfopropyl acrylate potassium salts (KAc). The grafted acrylate PET samples exhibited contact angles of 8 ± 3° and 28 ± 13° for NaAc and KAc, respectively, while showing durability in ageing and washing tests. The dry film thicknesses for both samples were found to be 28 ± 2 μm. Finally, the anti-adhesive properties of the NaAc- and KAc-treated surfaces were evaluated using an Escherichia coli expressing YadA, an adhesive protein from Yersinia. The modified PET surfaces were highly effective in reducing bacterial adhesion by more than 90%.This work was supported by the ViBrANT project, which received funding from the EU Horizon 2020 Research and Innovation Programme under Marie Sklowdowska-Curie (grant agreement no. 765042), and the Portuguese Foundation for Science and Technology (FCT) (grant number UIDB/04469/2020).info:eu-repo/semantics/publishedVersio