De Novo Biosynthesis of Curcumin in Saccharomyces cerevisiae.

Curcumin, a natural polyphenol derived from turmeric, has attracted immense interest due to its diverse pharmacological properties. Traditional extraction methods from Curcuma longa plants present limitations in meeting the growing demand for this bioactive compound, giving significance to its production by genetically modified microorganisms. Herein, we have developed an engineered Saccharomyces cerevisiae to produce curcumin from glucose. A pathway composed of the 4-hydroxyphenylacetate 3-monooxygenase oxygenase complex from Pseudomonas aeruginosa and Salmonella enterica, caffeic acid O-methyltransferase from Arabidopsis thaliana, feruloyl-CoA synthetase from Pseudomonas paucimobilis, and diketide-CoA synthase and curcumin synthase from C. longa was introduced in a p-coumaric acid overproducing S. cerevisiae strain. This strain produced 240.1 ± 15.1 µg/L of curcumin. Following optimization of phenylpropanoids conversion, a strain capable of producing 4.2 ± 0.6 mg/L was obtained. This study reports for the first time the successful de novo production of curcumin in S. cerevisiae.

Aptasensor for the Detection of Moraxella catarrhalis Adhesin UspA2.

Innovative point-of-care (PoC) diagnostic platforms are desirable to surpass the deficiencies of conventional laboratory diagnostic methods for bacterial infections and to tackle the growing antimicrobial resistance crisis. In this study, a workflow was implemented, comprising the identification of new aptamers with high affinity for the ubiquitous surface protein A2 (UspA2) of the bacterial pathogen Moraxella catarrhalis and the development of an electrochemical biosensor functionalized with the best-performing aptamer as a bioreceptor to detect UspA2. After cell-systematic evolution of ligands by exponential enrichment (cell-SELEX) was performed, next-generation sequencing was used to sequence the final aptamer pool. The most frequent aptamer sequences were further evaluated using bioinformatic tools. The two most promising aptamer candidates, Apt1 and Apt1_RC (Apt1 reverse complement), had Kd values of 214.4 and 3.4 nM, respectively. Finally, a simple and label-free electrochemical biosensor was functionalized with Apt1_RC. The aptasensor surface modifications were confirmed by impedance spectroscopy and cyclic voltammetry. The ability to detect UspA2 was evaluated by square wave voltammetry, exhibiting a linear detection range of 4.0 × 104-7.0 × 107 CFU mL-1, a square correlation coefficient superior to 0.99 and a limit of detection of 4.0 × 104 CFU mL-1 at pH 5.0. The workflow described has the potential to be part of a sensitive PoC diagnostic platform to detect and quantify M. catarrhalis from biological samples.

Design of new hydrolyzed collagen-modified magnetic nanoparticles to capture pathogens.

Enrichment and diagnosis tools for pathogens currently available are time consuming, thus the development of fast and highly sensitive alternatives is desirable. In this study, a novel approach was described that enables selective capture of bacteria expressing hydrolyzed collagen-binding adhesins with hydrolyzed collagen-coated magnetic nanoparticles (MNPs). This platform could be useful to shorten the time needed to confirm the presence of a bacterial infection. MNPs were synthesized by a simple two-step approach through a green co-precipitation method using water as solvent. These MNPs were specifically designed to interact with pathogenic bacteria by establishing a hydrolyzed collagen-adhesin linker. The bacterial capture efficacy of hydrolyzed collagen MNPs (H-Coll@MNPs) for bacteria expressing collagen binding adhesins was 1.3 times higher than that of arginine MNPs (Arg@MNPs), herein used as control. More importantly, after optimization of the MNP concentration and contact time, the H-Coll@MNPs were able to capture 95% of bacteria present in the samples. More importantly, the bacteria can be enriched within 30 min and the time for bacterial identification is effectively shortened in comparison to the "gold standard" in clinical diagnosis. These results suggest that H-Coll@MNPs can be used for the selective isolation of specific bacteria from mixed populations present, for example, in biological samples.

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.

Electrochemical Aptasensor for the Detection of the Key Virulence Factor YadA of Yersinia enterocolitica.

New point-of-care (POC) diagnosis of bacterial infections are imperative to overcome the deficiencies of conventional methods, such as culture and molecular methods. In this study, we identified new aptamers that bind to the virulence factor Yersinia adhesin A (YadA) of Yersinia enterocolitica using cell-systematic evolution of ligands by exponential enrichment (cell-SELEX). Escherichia coli expressing YadA on the cell surface was used as a target cell. After eight cycles of selection, the final aptamer pool was sequenced by high throughput sequencing using the Illumina Novaseq platform. The sequencing data, analyzed using the Geneious software, was aligned, filtered and demultiplexed to obtain the key nucleotides possibly involved in the target binding. The most promising aptamer candidate, Apt1, bound specifically to YadA with a dissociation constant (Kd) of 11 nM. Apt1 was used to develop a simple electrochemical biosensor with a two-step, label-free design towards the detection of YadA. The sensor surface modifications and its ability to bind successfully and stably to YadA were confirmed by cyclic voltammetry, impedance spectroscopy and square wave voltammetry. The biosensor enabled the detection of YadA in a linear range between 7.0 × 104 and 7.0 × 107 CFU mL−1 and showed a square correlation coefficient >0.99. The standard deviation and the limit of detection was ~2.5% and 7.0 × 104 CFU mL−1, respectively. Overall, the results suggest that this novel biosensor incorporating Apt1 can potentially be used as a sensitive POC detection system to aid the diagnosis of Y. enterocolitica infections. Furthermore, this simple yet innovative approach could be replicated to select aptamers for other (bacterial) targets and to develop the corresponding biosensors for their detection.

Tailoring fructooligosaccharides composition with engineered Zymomonas mobilis ZM4.

Zymomonas mobilis ZM4 is an attractive host for the development of microbial cell factories to synthesize high-value compounds, including prebiotics. In this study, a straightforward process to produce fructooligosaccharides (FOS) from sucrose was established. To control the relative FOS composition, recombinant Z. mobilis strains secreting a native levansucrase (encoded by sacB) or a mutated ß-fructofuranosidase (Ffase-Leu196) from Schwanniomyces occidentalis were constructed. Both strains were able to produce a FOS mixture with high concentration of 6-kestose. The best results were obtained with Z. mobilis ZM4 pB1-sacB that was able to produce 73.4 ± 1.6 g L-1 of FOS, with a productivity of 1.53 ± 0.03 g L-1 h-1 and a yield of 0.31 ± 0.03 gFOS gsucrose-1. This is the first report on the FOS production using a mutant Z. mobilis ZM4 strain in a one-step process. KEY POINTS: • Zymomonas mobilis was engineered to produce FOS in a one-step fermentation process. • Mutant strains produced FOS mixtures with high concentration of 6-kestose. • A new route to produce tailor-made FOS mixtures was presented.

Perspectives on the design of microbial cell factories to produce prenylflavonoids.

Prenylflavonoids are flavonoid-derived compounds characterized by the presence of a lipophilic prenylated side-chain in the flavonoid skeleton. These compounds are present in several food supplements and food products, and have a wide variety of recognized biological activities, namely estrogenic, antioxidant, anti-inflammatory and anticancer. Since these compounds are present in nature in very low amounts, their extraction from plants is not enough to fulfill the current demand, besides being an inefficient and environmentally unfriendly process. For these reasons, the use of microorganisms as microbial cell factories represents an interesting alternative to produce prenylflavonoids in a faster and cheaper way. Saccharomyces cerevisiae has been used as chassis to produce prenylflavonoids. Moreover, Escherichia coli can also emerge as an alternative chassis to produce these compounds. However, there is still a long way before prenylflavonoids can be produced by heterologous organisms with relevant yields and titers. In this review, we highlight the biosynthetic pathways involved in the production of prenylflavonoids. Additionally, we review the advances, challenges and strategies on the heterologous production of these compounds in a competitive way.

Heterologous production of chondroitin.

Chondroitin sulfate (CS) is a glycosaminoglycan with a broad range of applications being a popular dietary supplement for osteoarthritis. Usually, CS is extracted from animal sources. However, the known risks of animal products use have been driving the search for alternative methods and sources to obtain this compound. Several pathogenic bacteria naturally produce chondroitin-like polysaccharides through well-known pathways and, therefore, have been the basis for numerous studies that aim to produce chondroitin using non-pathogenic hosts. However, the yields obtained are not enough to meet the high demand for this glycosaminoglycan. Metabolic engineering strategies have been used to construct improved heterologous hosts. The identification of metabolic bottlenecks and regulation points, and the screening for efficient enzymes are key points for constructing microbial cell factories with improved chondroitin yields to achieve industrial CS production. The recent advances on enzymatic and microbial strategies to produce non-animal chondroitin are herein reviewed. Challenges and prospects for future research are also discussed.

Curcumin biosynthesis from ferulic acid by engineered Saccharomyces cerevisiae.

BACKGROUND AND GOAL: Curcumin is a polyphenolic compound found in Curcuma longa. This bioactive molecule has several reported health-benefit effects, being the anticarcinogenic activity among the most promising ones. However, curcumin extraction from natural sources is hampered by impure products obtained from harsh chemicals and limited by plant seasonality and high prices. Therefore, curcumin heterologous production emerged as an interesting alternative. Escherichia coli has been explored as chassis but the implementation of the pathway in Saccharomyces cerevisiae can have several advantages, including its generally regarded as safe status. Hence, S. cerevisiae was engineered for the first time to produce curcumin from its precursor ferulic acid. METHODS AND RESULTS: The enzymes 4-coumarate-CoA ligase (4CL1) from Arabidopsis thaliana or feruloyl-CoA synthetase (FerA) from Pseudomonas paucimobilis and type III polyketide synthases (PKSs) from Oryza sativa or C. longa were expressed in BY4741 strain. To avoid ferulic acid deviation, the gene FDC1 coding a ferulic acid decarboxylase was deleted. The maximum curcumin titer was obtained with FerA combined with C. longa PKSs (2.7 mg L-1 ). CONCLUSIONS AND IMPLICATIONS: Up to our knowledge, this is the first work reporting the expression of a feruloyl-CoA synthase and also curcuminoid biosynthetic enzymes in S. cerevisiae, and consequently, curcumin production.

Improved method for the extraction of high-quality DNA from lignocellulosic compost samples for metagenomic studies.

The world economy is currently moving towards more sustainable approaches. Lignocellulosic biomass has been widely used as a substitute for fossil sources since it is considered a low-cost bio-renewable resource due to its abundance and continuous production. Compost habitats presenting high content of lignocellulosic biomass are considered a promising source of robust lignocellulose-degrading enzymes. Recently, several novel biocatalysts from different environments have been identified using metagenomic techniques. A key point of the metagenomics studies is the extraction and purification of nucleic acids. Nevertheless, the isolation of high molecular weight DNA from soil-like samples, such as compost, with the required quality for metagenomic approaches remains technically challenging, mainly due to the complex composition of the samples and the presence of contaminants like humic substances. In this work, a rapid and cost-effective protocol for metagenomic DNA extraction from compost samples composed of lignocellulosic residues and containing high content of humic substances was developed. The metagenomic DNA was considered as representative of the global environment and presented high quality (> 99% of humic acids effectively removed) and sufficient quantity (10.5-13.8 µg g-1 of compost) for downstream applications, namely functional metagenomic studies. The protocol takes about 4 h of bench work, and it can be performed using standard molecular biology equipment and reagents available in the laboratory. KEY POINTS/HIGHLIGHTS: • Metagenomic DNA was successfully extracted from compost samples rich in humic acids • The improved protocol was established by optimizing the cell lysis method and buffer • Complete removal of humic acids was achieved through the use of activated charcoal • The suitability of the DNA was proven by the construction of a metagenomic library.

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.

Novel Biorecognition Elements against Pathogens in the Design of State-of-the-Art Diagnostics.

Infectious agents, especially bacteria and viruses, account for a vast number of hospitalisations and mortality worldwide. Providing effective and timely diagnostics for the multiplicity of infectious diseases is challenging. Conventional diagnostic solutions, although technologically advanced, are highly complex and often inaccessible in resource-limited settings. An alternative strategy involves convenient rapid diagnostics which can be easily administered at the point-of-care (POC) and at low cost without sacrificing reliability. Biosensors and other rapid POC diagnostic tools which require biorecognition elements to precisely identify the causative pathogen are being developed. The effectiveness of these devices is highly dependent on their biorecognition capabilities. Naturally occurring biorecognition elements include antibodies, bacteriophages and enzymes. Recently, modified molecules such as DNAzymes, peptide nucleic acids and molecules which suffer a selective screening like aptamers and peptides are gaining interest for their biorecognition capabilities and other advantages over purely natural ones, such as robustness and lower production costs. Antimicrobials with a broad-spectrum activity against pathogens, such as antibiotics, are also used in dual diagnostic and therapeutic strategies. Other successful pathogen identification strategies use chemical ligands, molecularly imprinted polymers and Clustered Regularly Interspaced Short Palindromic Repeats-associated nuclease. Herein, the latest developments regarding biorecognition elements and strategies to use them in the design of new biosensors for pathogens detection are reviewed.

Biosynthesis and heterologous production of furanocoumarins: perspectives and current challenges.

Covering: up to October 2020 Furanocoumarins are plant secondary metabolites used to treat several skin disorders, such as psoriasis and vitiligo, and also with other potential therapeutic activities. Furanocoumarins are extracted from plants where they accumulate in low amounts over long growth periods. In addition, their extraction and purification are difficult in an environmentally unfriendly and expensive process. Hence, new sustainable and greener production schemes able to overcome such limitations ought to be developed. While the heterologous production of simple coumarins has been demonstrated, the biosynthesis of more complex furanocoumarins remains greatly unexplored. Although several important steps of the pathway have been elucidated in the last decade, the complete pathway has not been completely unravelled. In this paper, we review the natural conversion of amino acids into furanocoumarins, as well as the heterologous expression of each enzyme of the pathway. We also explore the challenges that need to be addressed so that their heterologous production can become a viable alternative.

Zymomonas mobilis as an emerging biotechnological chassis for the production of industrially relevant compounds.

Zymomonas mobilis is a well-recognized ethanologenic bacterium with outstanding characteristics which make it a promising platform for the biotechnological production of relevant building blocks and fine chemicals compounds. In the last years, research has been focused on the physiological, genetic, and metabolic engineering strategies aiming at expanding Z. mobilis ability to metabolize lignocellulosic substrates toward biofuel production. With the expansion of the Z. mobilis molecular and computational modeling toolbox, the potential of this bacterium as a cell factory has been thoroughly explored. The number of genomic, transcriptomic, proteomic, and fluxomic data that is becoming available for this bacterium has increased. For this reason, in the forthcoming years, systems biology is expected to continue driving the improvement of Z. mobilis for current and emergent biotechnological applications. While the existing molecular toolbox allowed the creation of stable Z. mobilis strains with improved traits for pinpointed biotechnological applications, the development of new and more flexible tools is crucial to boost the engineering capabilities of this bacterium. Novel genetic toolkits based on the CRISPR-Cas9 system and recombineering have been recently used for the metabolic engineering of Z. mobilis. However, they are mostly at the proof-of-concept stage and need to be further improved.

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.

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.

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 µM). 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 µM), 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 µM) 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 µM, 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.

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.

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 Escherichia 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 process involving the exchange of culture medium allowed high concentrations of curcumin to be obtained, 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 higher concentrations of curcumin to be produced than E. coli K-12 strains. Different isopropyl ß-d-thiogalactopyranoside 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 per cent yield), which was 3.1-fold higher than the highest concentration previously reported. This concentration was obtained using a two-stage fermentation with lysogeny broth (LB) and M9. Moreover, terrific broth was also demonstrated to be a very interesting alternative medium to produce curcumin because 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 that obtained with the LB-M9 combination, it leads to a significantly higher production of curcumin 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.

Production of curcuminoids from tyrosine by a metabolically engineered Escherichia coli using caffeic acid as an intermediate.

Curcuminoids are phenylpropanoids with high pharmaceutical potential. Herein, we report an engineered artificial pathway in Escherichia coli to produce natural curcuminoids through caffeic acid. Arabidopsis thaliana 4-coumaroyl-CoA ligase and Curcuma longa diketide-CoA synthase (DCS) and curcumin synthase (CURS1) were used to produce curcuminoids and 70 mg/L of curcumin was obtained from ferulic acid. Bisdemethoxycurcumin and demethoxycurcumin were also produced, but in lower concentrations, by feeding p-coumaric acid or a mixture of p-coumaric acid and ferulic acid, respectively. Additionally, curcuminoids were produced from tyrosine through the caffeic acid pathway. To produce caffeic acid, tyrosine ammonia lyase from Rhodotorula glutinis and 4-coumarate 3-hydroxylase from Saccharothrix espanaensis were used. Caffeoyl-CoA 3-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, 3.9, 0.3, and 0.2 mg/L of curcumin were produced, respectively. This is the first time DCS and CURS1 were used in vivo to produce curcuminoids and that curcumin was produced by feeding tyrosine. We have shown that curcumin can be produced using a pathway involvoing caffeic acid. This alternative pathway represents a step forward in the heterologous production of curcumin using E. coli.