The successful production of flavonoids and stilbenes depends on efficient expression of type III polyketide synthases (PKSs), responsible

for the key step of extension and cyclization of polyketides to yield flavanone and stilbene skeletons in the respective organisms. The CUS type III PKS is from rice, Oryza sativa, which is phylogenetically distinct from Zingiberales. The discovery of CUS prompted to employ it as a type III PKS at the polyketide synthesis step in the artificial biosynthesis pathway for microbial production of plant-specific curcuminoids. Rice bran pitch, an industrial waste residue from production of rice edible oil, was also successfully used as a source of ferulic acid to yield curcumin.

encoding phenylalanine ammmonia-lyase (PAL), from the yeast Rhodotorula rubra and 4CL (ScCCL), encoding 4-coumarate : CoA ligase (4CL), from Streptomyces coelicolor. In addition to this plasmid, we also constructed plasmid pRSF-ACC, in which the acetyl-CoA carboxylase (ACC) genes from Corynebacterium glutamicum were placed under the control of the T7 promoter and the ribosome-binding sequence to increase the intracellular pool of malonyl-CoA in E. coli. The successful production of flavonoid and stilbene compounds by E. coli prompted us to design an artificial gene cluster and express it for the synthesis of curcuminoids in E. coli.

vetores
At the first step, PAL converts tyrosine and phenylalanine to the corresponding phenylpropanoid acids, p-coumaric acid and cinnamic acid, respectively, which are then activated to CoA esters by 4CL. The plasmid, pCDF-PAL/LE4CL-1, for the first step contained PAL from R. rhodotorula and 4CL from L. erythrorhizon, both of which were under the control of the T7 promoter and the ribosome-binding sequence in the vector pCDFDuet-1 (Fig. 1c).

At the second step, CUS condenses two molecules of the CoA ester of the phenylpropanoid acid with one molecule of malonyl-CoA to produce curcuminoids. The plasmid, pET-CUS, for the second step contained the CUS gene under the control of the T7 promoter and the ribosome binding sequence in the vector pET16b (Fig. 1c). Both plasmids and pRSF-ACC had different replication origins and different selective markers, thus being maintained in the same E. coli cell.

the bisdemethoxycurcumin produced by E. coli were identical to those of the authentic sample

obtido

As a representative, the yield of the triketide pyrone derived from p-coumaric acid was determined. The yield of this triketide pyrone *5+ (227 mg l21) was greater than that (~6 mg l21) of the major curcuminoid *4+ in this reaction. Previous study revealed that CUS changes the product ratio between curcuminoids and pyrones depending on the substrate concentrations (Katsuyama et al., 2007b). For instance, when the reaction was started with the concentration of p-coumaroyl-CoA 10-fold higher than that of malonyl-CoA, curcuminods became predominant as a product. The low yield of curcuminoids is therefore due to low concentrations of p-coumaroyl-CoA and cinnamoyl-CoA, derived from the endogenous tyrosine and phenylalanine, respectively, in the E. coli cell by the actions of PAL and 4CL.

The E. coli cells were incubated at 26 uC for 60 h in the presence of 3 mM each of tyrosine (543 mg l21) or phenylalanine (495 mg l21), or both, plus glucose, antibiotics and IPTG in M9 minimal medium. Although CUS prefers p-coumaroyl-CoA as a substrate, approximately two times more in comparison to cinnamoyl- CoA (Katsuyama et al., 2007b), the yield of dicinnamoylmethane was greater than that of bisdemethoxycurcumin in all reactions. This may be caused by the different rates of incorporation of tyrosine and phenylalanine into the pathway, which results from the substrate preferences of PAL and 4CL.

We next tried to improve the yields of curcuminoids by directly supplying phenylpropanoid acids to E. coli cells carrying 4CL, CUS and ACCon the assumption that the removal of the PAL step converting the amino acids to the corresponding carboxylic acids would increase the pcoumaroyl-CoA concentration in the E. coli cell. the ratio of bisdemethoxycurcumin to triketide pyrone was improved: it was 200-fold higher than in the reaction starting from tyrosine, probably due to an increase of the p-coumaroylCoA concentration in the E. coli cells.

Ferrulic acid (Production of curcumin from rice bran pitch)

developed a method to extract ferulic acid from rice bran pitch through hydrolysis of c-oryzanol by alkali. We therefore planned to make use of the ferulic acid in this rice waste as a substrate for the production of curcumin.

Katsuyama 2010
Recent studies have revealed that curcumin possess a potential role in treating Alzheimers disease and hepatoprotective activity in liver injury. DCS catalyzes the condensation of malonyl-CoA onto feruloyl-CoA to give feruloyldiketide-CoA. CURS catalyzes the formation of curcuminoids from feruloyl-CoA and the feruloyldiketide- CoA synthesized by DCS

Another E. coli system carrying 4CL on plasmid pCDF-LE4CL-1, CUS on pET-CUS, and ACC on pRSF-ACC was used in the production of curcuminoids, and exogenous supplementation of cinnamic acid (1a), p-coumaric acid (2a), and ferulic acid (6a) led to the formation of dicinnamoylmethane (1c), bisdemethoxycurcumin (2c), and curcumin (6c) respectively. In the present study, we attempted to produce unnatural curcuminoids by supplying unnatural carboxylate precursors to E. coli cells harboring pCDFLE4CL- 1, pET-CUS, and pRSF-ACC. The structures of the plasmids were described previously.9) In this system, exogenously supplemented carboxylic acids are activated by the action of 4CL into the corresponding CoA esters, which are then condensed to form the corresponding curcuminoids by the action of CUS. In total, we were able to produce 15 curcuminoids (Fig. 1). Nine of these compounds are not found in nature. Obtiveram o mesmo rendimento que em 2008 para os naturais. Trace amounts of triketide pyrones, which are byproducts of the CUS reaction, were simultaneously produced in all the reactions The difference in the production ratio was presumably due to the substrate specificities of the 4CL and CUS enzymes. Because curcuminoids are focused on as potential pharmaceutical resources, the present strategy of producing unnatural curcuminoids might provide novel drug candidates.

Zang 2013

In the present paper, the caffeic acid biosynthesis pathway was reconstituted in engineered Escherichia coli to produce caffeic acid from simple biomass sugar glucose and xylose.
There is presently a rising interest in microbial production of valuable aromatic compounds from inexpensive simple carbon sources or renewable biomass feedstocks Driving the progress is a recent development of metabolic engineering on microbes aromatic amino acid biosynthesis pathway (or shikimate pathway) in which Lphenylalanine, L-tryptophan, or L-tyrosine can be used as precursors for production of desired products. However, the reported titers and yields of these products are still low, largely due to the poor availability of the substrate aromatic amino acids Caffeic acid can be biosynthesized from tyrosine through a two-step pathway, in which tyrosine is converted to pcoumaric acid and then to caffeic acid by tyrosine ammonia lyase (TAL) and 4-coumarate 3-hydroxylase (Coum3H), respectively. Varying copy numbers of TAL and 4CL genes resulted in altered caffeic acid production on both glucose and xylose.

Daniel machado
Curcumin and other curcuminoids are secondary metabolites produced by plants of the order Zingiberales.

Katsuyama and co{workers constructed an arti_cial pathway for production of curcuminoids in E. coli using the genes of phenylalanine ammonia-lyase (PAL) from the yeast Rhodotorula rubra, 4-coumarate:CoA ligase (4CL) from Lithospermum erythrorhizon and curcuminoid synthase (CUS) from rice (Oryza sativa) (Fig. 2.3) [17]. Their results show that CUS is able to catalyze the two _nal steps: condensation of the CoA esters with malonyl-CoA to form a diketide-CoA ester; and condensation of a CoA ester and a diketide-CoA ester to form a curcuminoid.

Du e et al
Escherichia coli E. coli is widely used as model systems and considered as the primary prokaryotic host for the expression of heterologous genes due to its extensive genetic characterization (Krings and Berger, 1998). Moreover, most of its biological processes are well understood and there are extensive genetic tools readily available for its gene manipulation (Rodriguez et al., 2003). It is also firstly chosen as host and to heterologously produce the flavonoids by designing and constructing the artificial phenylpropanoid biosynthetic pathways. Now about 0.75 mg/L of pinocembrin and 0.45 mg/L of naringenin could be produced with E. coli (Hwang et al., 2003). However, the yields of flavonoids were too low for large-scale production. This possibly resulted from the inefficient carbon flux from glucose, the amino acid precursors toward the phenylpropanoid biosynthetic pathway and the low amount of malonyl-CoA in E. coli cell. Saccharomyces cerevisiae Jiang et al. (2005) chose S. cerevisiae as the eukaryotic heterologous host to successfully produce the flavonoids after Ro and Douglas began to reconstitute the early steps of the phenylpropanoid pathway in S. cerevisiae (Ro and Douglas, 2004). In the S. cerevisiae AH22 strain that coexpressed PAL, 4CL, and CHS, approximately 7 mg/L of naringenin and 0.8 mg/L of pinocembrin could be produced. The yield in S. cerevisiae was higher than in E. coli which the phenylpropanoid pathway was firstly chosen to express. The key factor is that S. cerevisiae has some advantages over E. coli for expressing certain eukaryotic heterologous proteins. Yeast system is not only capable of performing posttranslational modifications of the eukaryotic proteins but also has many similar intracellular compartments to plant cells. In addition, yeast has been shown to be an excellent host for CYP activity in vivo (Bayoumi et al., 2008; Humphreys et al., 1999; Jiang and Morgan, 2004; Pompon et al., 1996; Szczebara et al., 2003). Other strains Streptomyces venezuelae has a rapid growth, relative ease of genetic manipulation, abundant supply of substrates (Jung et al., 2006; Park et al., 2008; Yoon et al., 2002) and produces a wide range of important secondary metabolites (Pfeifer and Khosla, 2001), so it is also used Du et al. 2569 as a robust hoterologous host for plant flavonoids production (Table 1). Phellinus igniarius is a medicinal mushroom containing many bioactive compounds, and is viewed as a attractive alternative for the efficient production of secondary metabolites (Zhong, 2005). Zhu et al. (2010) have constructed an expression vector containing Vitreoscilla hemoglobin gene, which supplies more oxygen for the aerobic organisms growth, for the first successful and significant heterologous production of flavonoids in P. igniarius (Table 1).

Identification and characterization of multiple curcumin synthases from the herb Curcuma longa (katsuyama 2009 II)
The present study has demonstrated that the herb C. longa contains at least two type III PKSs, CURS2 and CURS3, that are capable of curcuminoid biosynthesis, in addition to the previously identified CURS1 [8]. CURS1 and CURS2 showed similar substrate specificity; they preferred feruloyl-CoA (5) as a starter substrate. On the other hand, CURS3 showed substrate specificity different from that of CURS1 and CURS2; it preferred both p-coumaroyl-CoA (6) and

feruloyl-CoA (5) as a starter substrate. Although these characteristics were obtained from the kinetic analysis using cinnamoyldiketide- NAC, which is a mimic of the natural substrates, these results suggested that CURS2, like CURS1, mainly catalyzes formation of curcumin (1) and demethoxycurcumin (2) by condensing feruloyl- CoA with pcoumaroyldiketide-CoA (7) or feruloyldiketide- CoA (4) as an extender substrate (Fig. 1A). In contrast, CURS3 probably catalyzes synthesis of three curcuminoids (1, 2 and 3) by condensing feruloyl-CoA or p-coumaroyl-CoA with pcoumaroyldiketide- CoA (7) or feruloyldiketide-CoA (4) as an extender substrate