Joana Lúcia Lima Correia Rodrigues

Heterologous Production of Curcuminoids

SUMMARY Curcuminoids, components of the rhizome of turmeric, show several beneficial biological activities, including anticarcinogenic, antioxidant, anti-inflammatory, and antitumor activities. Despite their numerous pharmaceutically important properties, the low natural abundance of curcuminoids represents a major drawback for their use as therapeutic agents. Therefore, they represent attractive targets for heterologous production and metabolic engineering. The understanding of biosynthesis of curcuminoids in turmeric made remarkable advances in the last decade, and as a result, several efforts to produce them in heterologous organisms have been reported. The artificial biosynthetic pathway (e.g., in Escherichia coli) can start with the supplementation of the amino acid tyrosine or phenylalanine or of carboxylic acids and lead to the production of several natural curcuminoids. Unnatural carboxylic acids can also be supplemented as precursors and lead to the production of unnatural compounds with possibly novel therapeutic properties. In this paper, we review the natural conversion of curcuminoids in turmeric and their production by E. coli using an artificial biosynthetic pathway. We also explore the potential of other enzymes discovered recently or already used in other similar biosynthetic pathways, such as flavonoids and stilbenoids, to increase curcuminoid yield and activity.

Heterologous production of caffeic acid from tyrosine in Escherichia coli

Caffeic acid is a plant secondary metabolite and its biological synthesis has attracted increased attention due to its beneficial effects on human health. In this study, Escherichia coli was engineered for the production of caffeic acid using tyrosine as the initial precursor of the pathway. The 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. The genes were codon-optimized and different combinations of plasmids were used to improve the titer of caffeic acid. TAL was able to efficiently convert 3mM of tyrosine to p-coumaric acid with the highest production obtained being 2.62mM (472mg/L). CYP199A2 exhibited higher catalytic activity towards p-coumaric acid than C3H. The highest caffeic acid production obtained using TAL and CYP199A2 and TAL and C3H was 1.56mM (280mg/L) and 1mM (180mg/L), respectively. This is the first study that shows caffeic acid production using CYP199A2 and tyrosine as the initial precursor. This study suggests the possibility of further producing more complex plant secondary metabolites like flavonoids and curcuminoids.

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.

Selection of Escherichia coli heat shock promoters toward their application as stress probes

The mechanism of heat shock response of Escherichia coli can be explored to program novel biological functions. In this study, the strongest heat shock promoters were identified by microarray experiments conducted at different temperatures (37°C and 45°C, 5min). The promoters of the genes ibpA, dnaK and fxsA were selected and validated by RT-qPCR. These promoters were used to construct and characterize stress probes using green fluorescence protein (GFP). Cellular stress levels were evaluated in experiments conducted at different shock temperatures during several exposure times. It was concluded that the strength of the promoter is not the only relevant factor in the construction of an efficient stress probe. Furthermore, it was found to be crucial to test and optimize the ribosome binding site (RBS) in order to obtain translational efficiency that balances the transcription levels previously verified by microarrays and RT-qPCR. These heat shock promoters can be used to trigger in situ gene expression of newly constructed biosynthetic pathways.

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 β-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.

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.

Synthetic biology: perspectives in industrial biotechnology

The field of synthetic biology combines relevant expertise from various science, engineering, and computing disciplines to take the best of biology for the benefit of humans and the environment. Herein we review some of the tools and methods of synthetic biology, such as the use of engineering principles and genome editing tools. In addition, we discuss the advantages of synthetic biology and how it can be used to improve industrial biotechnology, for example, in the production of biofuels, drugs, and other value-added chemicals. Finally, we approach the perception that the population in general has about synthetic biology and how the scientists working in this field are trying to change it by creating containment measures, some of them using synthetic biology itself, to increase biosecurity and biosafety.

Synthetic biology strategies towards the development of new bioinspired technologies for medical applications

Abstract Synthetic biology in combination with systems biology uses engineering principles to leverage the power of biology. The impact and potential of systems and synthetic biology in the prevention, diagnosis and treatment of diseases are growing. In this chapter, we will revise some of the tools, methods and applications within these fields that can be useful in biomedical engineering and human health, such as genome editing and the development of quorum sensing mechanisms, among others. We will discuss remarkable advances made, for example, in vaccine development, diagnosis and treatment of cancer and infectious diseases, microbiome engineering and cell therapy. Lastly, the challenges and perspectives of these fields will be debated.

Heterologous production of plant natural polyphenolic compounds in Escherichia coli

SB 7.0 Heterologous production of plant natural polyphenolic compounds in Escherichia coli Joana L. Rodrigues, Márcia R. Couto and Lígia R. Rodrigues Centre of Biological Engineering, University of Minho, Portugal Plants secondary metabolites are important for their survival and are usually considered as high-value chemicals. However, they are generally present in low amounts and accumulate during long growth periods. Their extraction is often problematic since their purification from mixtures containing compounds with similar structures is difficult originating low yields also subject to environmental and regional factors. In addition, chemical synthesis is normally too complex and environmentally unfriendly. Therefore, the biosynthesis of high-value chemicals in engineered organisms has emerged as a competitive alternative compared to chemistry-based methods. Curcuminoids and coumarins are polyphenolic compounds produced in plants in low amounts that exhibit interesting pharmacological properties. In this study, we report the construction of an artificial pathway using codon-optimized enzymes for the production of these compounds in Escherichia coli. Both types of polyphenolic compounds can be produced from tyrosine or hydroxycinnamic acids as precursors. To produce curcumin, the most studied curcuminoid for therapeutic purposes, 4-coumaroyl-CoA ligase (4CL) from Arabidopsis thaliana, curcuminoid synthase from Oryza sativa or diketide-CoA synthase and curcumin synthase from Curcuma longa were used. Using this pathway 354 mg/L of curcumin was produced, which corresponds to the highest concentration obtained so far using a heterologous host. Curcumin was also produced for the first time using tyrosine as precursor and caffeic acid as an intermediate. Other curcuminoids, such as bisdemethoxycurcumin and demethoxycurcumin were also produced using as precursors tyrosine or hydroxycinnamic acids (p-coumaric acid or a mixture of p-coumaric and ferulic acids, respectively). Based in this pathway, a similar pathway was constructed to produce coumarins. The enzymes 4CL and p-coumaroyl-CoA 2’-hydroxylase from Ipomoea batatas were used to produce the coumarins umbelliferone, scopoletin and esculetin from p-coumaric acid, ferulic acid and caffeic acid, respectively. Approximately 20-50 mg/L of each coumarin was produced. The optimization of coumarins production from tyrosine is being conducted.