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

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

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

Development of a low-cost culture medium for biopolymer production by Rhizobium viscosum CECT 908 and its potential application in Microbial Enhanced Oil Recovery

Polymers are a versatile class of compounds that play an essential role in our society, being their production estimated in more than 180 million tons per year. Nowadays, the world market is dominated by synthetic and plant-derived polymers. Biopolymers of microbial origin are characterized by their better environmental compatibility and biodegradability when compared with the synthetic ones, and their production is faster than those obtained from plants. Microbial biopolymers usually exhibit excellent rheological properties, stability at a wide range of temperatures, salinities and pH values, as well as a broad variety of chemical structures, which results in different physicochemical and rheological properties. However, despite their outstanding properties, their application is still limited by their high production costs. In this work, an alternative low-cost culture medium was developed for biopolymer production by Rhizobium viscosum CECT 908, containing sugarcane molasses (60 g/L) and corn steep liquor (1%, v/v) as carbon and nitrogen sources, respectively. Using this low-cost medium, higher biopolymer production and apparent viscosity values (5.2 g/L and 6700 mPa s, respectively) were obtained comparing with the synthetic medium (2.3 g/L and 1100 mPa s), which contained glucose and yeast extract. As a result, the cost of the culture medium necessary to produce 1 Kg of biopolymer was reduced more than 20 times. The biopolymer produced in the alternative low-cost medium exhibited better rheological properties as compared to xanthan gum, including higher viscosity at the same concentration. Furthermore, it was found to be stable at temperatures up to 80ºC, NaCl concentrations as high as 200 g/L, and high shear rates (300 s-1). Polymers are widely used by the oil industry to increase the oil reservoirs productivity during the tertiary oil recovery processes. In sand-pack column assays performed using a heavy crude oil (40ºC= 170 mPa s), this biopolymer produced using the low-cost medium demonstrated a better performance than xanthan gum, recovering almost 50% of the entrapped oil. Results herein obtained highlight that the R. viscosum biopolymer is a promising candidate for application in MEOR as an alternative to the conventional microbial and synthetic polymers currently used.info:eu-repo/semantics/publishedVersio

Synthetic biology: heterologous production of bioactive agents

Book of Abstracts of CEB Annual Meeting 2017Synthetic biology provides powerful tools to design innovative products and technologies. In the medical field, examples include the elucidation of disease mechanisms, identification of potential targets, discovery of new chemotherapeutics or design of novel drugs [1]. Additionally, it enables the development of economically attractive microbial production processes for complex natural products. Plants secondary metabolites are considered high-value chemicals exhibiting interesting biological activities. However, they are present in plants in low amounts and accumulate during long growth periods. Their extraction is often problematic since their purification is difficult originating low yields, which are also consequence of environmental and regional factors. In addition, chemical synthesis is complex and environmentally unfriendly. Therefore, the biosynthesis of these 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 that exhibit very interesting pharmacological properties. Under this scope, we designed and constructed an artificial pathway using codon-optimized enzymes for the production of these compounds in Escherichia coli [2]. 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 designed and 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 pcoumaric 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. Saccharomyces cerevisae, which is also an interesting host, has only been used to produce other polyketides (e.g. resveratrol) [2]. However, it presents some unique advantages over E. coli for the design and construction of biosynthetic pathways to produce curcuminoids or coumarins. Hence, we are also exploring S. cerevisae as a potential chassis for the production of these valuable chemicals. References [1] Rodrigues, LR; Kluskens, L. Synthetic Biology & Bioinformatics: Prospects in the cancer arena, In HS Lopes; LM Cruz (eds), Computational Biology and Applied Bioinformatics, 8, 159-186, InTech, Rijeka, Croatia, 2011. [2] Rodrigues, JL; Prather, K; Kluskens, LD; Rodrigues, LR, Heterologous production of curcuminoids: A Review, Microbiology and Molecular Biology Reviews 79(1), 39-60, 2014.info:eu-repo/semantics/publishedVersio

Engineering a biosynthetic pathway for high-value glycosaminoglycans production

info:eu-repo/semantics/publishedVersio

Saccharomyces cerevisiae as a host for chondroitin production

Chondroitin is a glycosaminoglycan that has gained widespread use in nutraceuticals and pharmaceuticals, mainly for treating osteoarthritis. Traditionally, it has been extracted from animal cartilage but recently, biotechnological processes have emerged as a commercial alternative to avoid the risk of viral or prion contamination and offer a vegan-friendly source. Typically, these methods involve producing the chondroitin backbone using pathogenic bacteria and then modifying it enzymatically through the action of sulfotransferases. Despite the challenges of expressing active sulfotransferases in bacteria, the use of eukaryotic microorganisms is still limited to a few works using Pichia pastoris. To create a safer and efficient biotechnological platform, we constructed a biosynthetic pathway for chondroitin production in S. cerevisiae as a proof-of-concept. Up to 125 mg/L and 200 mg/L of intracellular and extracellular chondroitin were produced, respectively. Furthermore, as genome-scale models are valuable tools for identifying novel targets for metabolic engineering, a stoichiometric model of chondroitin-producing S. cerevisiae was developed and used in optimization algorithms. Our research yielded several novel targets, such as uridine diphosphate (UDP)-Nacetylglucosamine pyrophosphorylase (QRI1), glucosamine-6-phosphate acetyltransferase (GNA1), or N-acetylglucosamine-phosphate mutase (PCM1) overexpression, that might enhance chondroitin production and guide future experimental research to develop more efficient host organisms for the biotechnological production process.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 with DOI 10.54499/UIDB/04469/2020. The authors acknowledge FCT for funding MRC doctoral grant SFRH/BD/132998/2017 and further extension COVID/BD/152454/2022.info:eu-repo/semantics/publishedVersio

Stoichiometric genome-scale models for the chondroitin production in Escherichia coli

Chondroitin is a natural-occurring glycosaminoglycan with applications as a nutraceutical and pharmaceutical ingredient. It can be extracted from animal tissues, though chondroitin-like polysaccharides using microorganisms emerged as a safer and more sustainable alternative source. However, chondroitin yields using either natural or recombinant microorganisms are still far from meeting the increasing demand. In this work, stoichiometric models containing the heterologous pathway necessary for producing chondroitin in E. coli were constructed and investigated for mutant predictions that would potentially improve chondroitin yields. Four models of E. coli BL21 (BIGG ID: iECBD_1354, iECD_1391, iEC1356_Bl21DE3, iB21_1397) and one of E. coli K12 (BIGG ID: iJO1366), from which the other models were derived, were used to insert the heterologous pathway composed by two enzymatic steps catalyzed by UDP-Nacetylglucosamine 4-epimerase (UAE) and chondroitin synthase/polymerase (CHSY). The models were imported in Optflux, and the evolutionary optimization was then performed for gene deletion predictions using Strength Pareto Evolutionary Algorithm 2 (SPEA2) and the parsimonious Flux Balance Analysis (pFBA) as the simulation method. Chondroitin production was not predicted to improve by combining gene deletions, probably because the competing pathways that use the intermediates are critical for cell growth. However, gene over and underexpression search allowed to identify several targets. Most of the resulting solutions were composed by the overexpression of one of the genes responsible for the production of the heterologous pathway precursor (either glmU or glmM encoding glucosamine-1-phosphate Nacetyltransferase/UDP-N-acetylglucosamine diphosphorylase and phosphoglucosamine mutase, respectively) combined with the underexpression of one of the genes associated with cell wall recycling pathways (such as membrane-bound lytic transglycosylases mltA, mltB and mltC, or the anhydromuropeptide permease ampG), which contain reactions known to consume such precursors. The solutions herein obtained will be further validated in vivo by constructing the E. coli mutants predicted to improve chondroitin production.info:eu-repo/semantics/publishedVersio

Heterologous production of plant natural polyphenolic compounds in Escherichia coli

SB7.0: The Seventh International Meeting on Synthetic BiologyPlants 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.info:eu-repo/semantics/publishedVersio

Sustainable exopolysaccharide production by rhizobium viscosum CECT908 using corn steep liquor and sugarcane molasses as sole substrates

Microbial exopolysaccharides (EPS) are promising alternatives to synthetic polymers in a variety of applications. Their high production costs, however, limit their use despite their outstanding properties. The use of low-cost substrates such as agro-industrial wastes in their production, can help to boost their market competitiveness. In this work, an alternative low-cost culture medium (CSLM) was developed for EPS production by Rhizobium viscosum CECT908, containing sugarcane molasses (60 g/L) and corn steep liquor (10 mL/L) as sole ingredients. This medium allowed the production of 6.1 ± 0.2 g EPS/L, twice the amount produced in the standard medium (Syn), whose main ingredients were glucose and yeast extract. This is the first report of EPS production by R.viscosum using agro-industrial residues as sole substrates. EPSCSLM and EPSSyn exhibited a similar carbohydrate composition, mainly 4-linked galactose, glucose and mannuronic acid. Although both EPS showed a good fit to the HerschelBulkley model, EPSCSLM displayed a higher yield stress and flow consistency index when compared with EPSSyn, due to its higher apparent viscosity. EPSCSLM demonstrated its potential use in Microbial Enhanced Oil Recovery by enabling the recovery of nearly 50% of the trapped oil in sand-pack column experiments using a heavy crude oil.info:eu-repo/semantics/publishedVersio