Increased β-Carotene Production in Recombinant Escherichia coli Harboring an Engineered Isoprenoid Precursor Pathway with Mevalonate Addition (original) (raw)
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Biotechnology and Bioprocess Engineering, 2009
Recombinant bëÅÜÉêáÅÜá~=Åçäá engineered to contain the whole mevalonate pathway and foreign genes for β-carotene biosynthesis, was utilized for production of β-carotene in bioreactor cultures. Optimum culture conditions were established in batch and pH-stat fed-batch cultures to determine the optimal feeding strategy thereby improving production yield. The specific growth rate and volumetric productivity in batch cultures at 37 o C were 1.7-fold and 2-fold higher, respectively, than those at 28 o C. Glycerol was superior to glucose as a carbon source. Maximum β-carotene production (titer of 663 mg/L and overall volumetric productivity of 24.6 mg/L × h) resulted from the simultaneous addition of 500 g/L glycerol and 50 g/L yeast extract in pH-stat fed-batch culture. © KSBB hÉóïçêÇëW=êÉÅçãÄáå~åí Escherichia coli, ÉåÖáåÉÉêÉÇ= ïÜçäÉ= ãÉî~äçå~íÉ= é~íÜï~óI=βJÅ~êçíÉåÉI= ÄáçêÉ~Åíçê= ÅìäíìêÉI= ÑÉÇJÄ~íÅÜ= ÅìäíìêÉ=
Journal of Biotechnology, 2009
The increased synthesis of building blocks of IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate) through metabolic engineering is a way to enhance the production of carotenoids. Using E. coli as a host, IPP and DMAPP supply can be increased significantly through the introduction of foreign MVA (mevalonate) pathway into it. The MVA pathway is split into two parts with the top and bottom portions supplying mevalonate from acetyl-CoA, and IPP and DMAPP from mevalonate, respectively. The bottom portions of MVA pathway from Streptococcus pneumonia, Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes and Saccharomyces cerevisiae were compared with exogenous mevalonate supplementation for -carotene production in recombinant Escherichia coli harboring -carotene synthesis genes. The E. coli harboring the bottom MVA pathway of S. pneumoniae produced the highest amount of -carotene. The top portions of MVA pathway were also compared and the top MVA pathway of E. faecalis was found out to be the most efficient for mevalonate production in E. coli. The whole MVA pathway was constructed by combining the bottom and top portions of MVA pathway of S. pneumoniae and E. faecalis, respectively. The recombinant E. coli harboring the whole MVA pathway and -carotene synthesis genes produced high amount of -carotene even without exogenous mevalonate supplementation. When comparing various E. coli strains -MG1655, DH5␣, S17-1, XL1-Blue and BL21 -the DH5␣ was found to be the best -carotene producer. Using glycerol as the carbon source for -carotene production was found to be superior to glucose, galactose, xylose and maltose. The recombinant E. coli DH5␣ harboring the whole MVA pathway and -carotene synthesis genes produced -carotene of 465 mg/L at glycerol concentration of 2% (w/v).
Biotechnology Letters, 2012
Escherichia coli DH5a strain was selected as the recombinant host, and a chemically defined medium supplemented with amino acids was used instead of a complex medium for the efficient production of b-carotene. In a fed-batch culture using glycerol with a chemically defined medium supplemented with amino acids, the concentration, specific content, and productivity of b-carotene were 2,470 mg/l, 72 mg/g cells, and 77 mg/l h after 32 h, respectively. These values were, respectively, 43, 33, and 26 % higher than those obtained using the complex medium. This is the highest b-carotene production that has been reported among the recombinant cells to date.
Applied Microbiology and Biotechnology, 2007
The lycopene synthetic pathway was engineered in Escherichia coli using the carotenoid genes (crtE, crtB, and crtI) of Pantoea agglomerans and Pantoea ananatis. E. coli harboring the P. agglomerans crt genes produced 27 mg/l of lycopene in 2YT medium without isopropyl-beta-d-thiogalactopyranoside (IPTG) induction, which was twofold higher than that produced by E. coli harboring the P. ananatis crt genes (12 mg/l lycopene) with 0.1 mM IPTG induction. The crt genes of P. agglomerans proved better for lycopene production in E. coli than those of P. ananatis. The crt genes of the two bacteria were also compared in E. coli harboring the mevalonate bottom pathway, which was capable of providing sufficient carotenoid building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), with exogenous mevalonate supplementation. Lycopene production significantly increased using the mevalonate bottom pathway and 60 mg/l of lycopene was obtained with the P. agglomerans crt genes, which was higher than that obtained with the P. ananatis crt genes (35 mg/l lycopene). When crtE among the P. ananatis crt genes was replaced with P. agglomerans crtE or Archaeoglobus fulgidus gps, both lycopene production and cell growth were similar to that obtained with P. agglomerans crt genes. The crtE gene was responsible for the observed difference in lycopene production and cell growth between E. coli harboring the crt genes of P. agglomerans and P. ananatis. As there was no significant difference in lycopene production between E. coli harboring P. agglomerans crtE and A. fulgidus gps, farnesyl diphosphate (FPP) synthesis was not rate-limiting in E. coli.
2023
industrial manufacturing. This article explores the production of carotenoids by microbial consortia. This study aims to update and consolidate the fundamental aspects of carotenoid production from bacteria, algae, and Beta-carotene, a carotenoid found in plants, fungi, and algae, is a crucial antioxidant and anticancer agent. It is primarily derived from plants, algae, and microbes, but this method has drawbacks like high costs and low productivity. The growing demand for carotenoids has led to large-scale industrial manufacturing. However, extracting and synthesizing these chemicals can be costly and technical. Microbial synthesis offers a cost-effective alternative. Synthetic biology and metabolic engineering technologies have been used in various studies for the optimization of pathways for the overproduction of carotenoids. Four metabolic components are involved in carotenoid biosynthesis, central carbon (C), isoprene supplement, and cofactor metabolism. Metabolic engineering is a potential solution to enhance β-carotene production. This article explores the biochemical routes, methods used by natural microbial species, and metabolic engineering potential of microbial organisms for β-carotenoids production. Currently, Escherichia coli, certain euglena and yeast species are the primary microorganisms used in metabolic engineering, offering minimal environmental impact, cost-effective manufacturing, and high yield.
Strain-Dependent Carotenoid Productions in Metabolically Engineered Escherichia coli
Applied Biochemistry and Biotechnology, 2010
Seven Escherichia coli strains, which were metabolically engineered with carotenoid biosynthetic pathways, were systematically compared in order to investigate the strain-specific formation of carotenoids of structural diversity. C30 acyclic carotenoids, diaponeurosporene and diapolycopene were well produced in all E. coli strains tested. However, the C30 monocyclic diapotorulene formation was strongly strain dependent. Reduced diapotorulene formation was observed in the E. coli strain Top10, MG1655, and MDS42 while better formation was observed in the E. coli strain JM109, SURE, DH5a, and XL1-Blue. Interestingly, C40 carotenoids, which have longer backbones than C30 carotenoids, also showed strain dependency as C30 diapotorulene did. Quantitative analysis showed that the SURE strain was the best producer for C40 acyclic lycopene, C40 dicyclic β-carotene, and C30 monocyclic diapotorulene. Of the seven strains examined, the highest volumetric productivity for most of the carotenoids structures was observed in the recombinant SURE strain. In conclusion, we showed that recombinant hosts and carotenoid structures influenced carotenoid productions significantly, and this information can serve as the basis for the subsequent development of microorganisms for carotenoids of interest.
An update on microbial carotenoid production: application of recent metabolic engineering tools
Applied Microbiology and Biotechnology, 2007
Carotenoids are ubiquitous pigments synthesized by plants, fungi, algae, and bacteria. Industrially, carotenoids are used in pharmaceuticals, neutraceuticals, and animal feed additives, as well as colorants in cosmetics and foods. Scientific interest in dietary carotenoids has increased in recent years because of their beneficial effects on human health, such as lowering the risk of cancer and enhancement of immune system function, which are attributed to their antioxidant potential. The availability of carotenoid genes from carotenogenic microbes has made possible the synthesis of carotenoids in non-carotenogenic microbes. The increasing interest in microbial sources of carotenoid is related to consumer preferences for natural additives and the potential cost effectiveness of creating carotenoids via microbial biotechnology. In this review, we will describe the recent progress made in metabolic engineering of non-carotenogenic microorganisms with particular focus on the potential of Escherichia coli for improved carotenoid productivity.
Synthesis of atypical cyclic and acyclic hydroxy carotenoids in Escherichia coli transformants
Journal of Biotechnology, 1997
Pathway engineering of E. coli and higher plants for carotenoid biosynthesis View project Functional identification and biofortification of carotenoids biosynthesis genes in sweet potato (Ipomoea batatas L.) and establishment of a rapid transformation system for autumn-olive (Elaeagnus umbellata Thunb.) View project Abstract A total of eight different hydroxy carotenoids were produced in transformants of the non-carotenogenic bacterium Escherichia coli. They include the acyclic 1-hydroxyneurosporene, 1-hydroxylycopene, 1,1%-dihydroxylycopene and demethylspheroidene as well as the cyclic 3-hydroxy-i-zeacarotene, 7,8-dihydrozeaxanthin, 3 or 3%-7,8-dihydro-icarotene and 1%-hydroxy-k-carotene. Most of these uncommon carotenoids are found only in trace amounts in natural sources. For the synthesis of all the carotenoids mentioned above, E. coli was transformed with a combination of up to three compatible plasmids, which contained several carotenogenic genes from Erwinia uredo6ora and two Rhodobacter species. Their function in the pathway leading to the individual carotenoids was outlined. Finally, growth conditions were optimized for production of the hydroxy carotenoids in amounts which are suitable for their isolation and purification. © 1997 Elsevier Science B.V.
Biotechnology and Bioengineering, 2006
To increase expression of lycopene synthetic genes crtE, crtB, crtI, and ipiHP1, the four exogenous genes were cloned into a high copy pTrc99A vector with a strong trc promoter. Recombinant Escherichiacoli harboring pT-LYCm4 produced 17 mg/L of lycopene. The mevalonate lower pathway, composed of mvaK1, mvaK2, mvaD, and idi, was engineered to produce pSSN12Didi for an efficient supply of the lycopene building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Mevalonate was supplied as a substrate for the mevalonate lower pathway. Lycopene production in E. coli harboring pT-LYCm4 and pSSN12Didi with supplementation of 3.3 mM mevalonate was more than threefold greater than bacteria with pT-LYCm4 only. Lycopene production was dependent on mevalonate concentration supplied in the culture. Clump formation was observed as cells accumulated more lycopene. Further clumping was prevented by adding the surfactant Tween 80 0.5% (w/v), which also increased lycopene production and cell growth. When recombinant E. coli harboring pT-LYCm4 and pSSN12Didi was cultivated in 2YT medium containing 2% (w/v) glycerol as a carbon source, 6.6 mM mevalonate for the mevalonate lower pathway, and 0.5% (w/v) Tween 80 to prevent clump formation, lycopene production was 102 mg/L and 22 mg/g dry cell weight, and cell growth had an OD600 value of 15 for 72 h. © 2006 Wiley Periodicals, Inc.