Identification of genes affecting lycopene accumulation in <i>Escherichia coli</i> using a shot‐gun method

Kang, Min Jung; Lee, Young Mi; Yoon, Sang Hwal; Kim, Jung Heon; Ock, So Won; Jung, Kyung Hwa; Shin, Yong Chul; Keasling, Jay D.; Kim, Seon Won

doi:10.1002/bit.20539

Cited by 87 publications

(53 citation statements)

References 42 publications

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“…SigB ( B ) is the general stressresponsive alternative sigma factor in Gram-positive bacteria; RpoS ( S ) is the counterpart in Gram-negative bacteria (5, 10). It has also been reported that overexpression of rpoS can enhance lycopene production in E. coli (15). Additionally, biosynthesis of lycopene and biosynthesis of isoprene shared most of the DXP pathway.…”

Section: Discussionmentioning

confidence: 88%

“…Previous work has shown that overexpression of rpoS can increase lycopene production in E. coli (15). rpoS encodes the sigma S subunit of RNA polymerase in E. coli.…”

Section: Resultsmentioning

confidence: 99%

“…5, the DXP pathway is not regulated by SigB; the sigB deletion strain showed an isoprene production level similar to that of the wild-type strain under both control and stress conditions. The increased lycopene production caused by RpoS overexpression in E. coli might be a result of increased resistance to the challenge of lycopene accumulation (15). Heat, salt, oxidative stress, and ethanol stress factors could either directly regulate isoprene synthase or indirectly affect some regulators (such as transcriptional regulators), which could subsequently control isoprene synthase and thus govern isoprene production.…”

Section: Discussionmentioning

confidence: 99%

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Enhancing Isoprene Production by Genetic Modification of the 1-Deoxy- d -Xylulose-5-Phosphate Pathway in Bacillus subtilis

Xue

Ahring

2011

Appl Environ Microbiol

110

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To enhance the production of isoprene, a volatile 5-carbon hydrocarbon, in the Gram-positive spore-forming rod-shaped bacterium Bacillus subtilis, 1-deoxy-D-xylulose-5-phosphate synthase (Dxs) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr) were overexpressed in B. subtilis DSM 10. For the strain that overexpresses Dxs, the yield of isoprene was increased 40% over that by the wild-type strain. In the Dxr overexpression strain, the level of isoprene production was unchanged. Overexpression of Dxr together with Dxs showed an isoprene production level similar to that of the Dxs overproduction strain. The effects of external factors, such as stress factors including heat (48°C), salt (0.3 M NaCl), ethanol (1%), and oxidative (0.005% H 2 O 2 ) stress, on isoprene production were further examined. Heat, salt, and H 2 O 2 induced isoprene production; ethanol inhibited isoprene production. In addition, induction and repression effects are independent of SigB, which is the general stress-responsive alternative sigma factor of Gram-positive bacteria.Isoprene, also known as 2-methyl-1,3-butadiene, is a volatile 5-carbon pure hydrocarbon. Derived from biomass through a biochemical process, isoprene can be utilized as a fossil fuel alternative and a platform chemical for the production of highvalue biobased chemicals, such as rubber, elastomers, and isoprenoid medicines (17, 39). Isoprene has several advantages over ethanol. First, it is much easier to separate from the fermentation broth, since it is present in the upper gas phase of a fermentor due to its low boiling point (34°C) and low solubility in water. Second, bacteria are more tolerant to isoprene than to ethanol (the MIC of isoprene for Bacillus subtilis strain DSM 10 is more than 10%, which is higher than the MIC of ethanol according to our unpublished data). Third, isoprene is a highly versatile molecule that can be biochemically and thermochemically re-formed into more complicated products.Isoprene is produced both by eukaryotes and by prokaryotes, including humans, plants, yeasts, and bacteria (18). Two pathways in isoprene biosynthesis have been discovered: the mevalonate (MVA) pathway and the nonmevalonate route, or the 1-deoxy-D-xylulose-5-phosphate (DXP) pathway ( Fig. 1) (13, 39). The MVA pathway is present in eukaryotes, archaea, and the cytosol of higher plants; the DXP pathway is present in most bacteria, green algae, and the chloroplasts of higher plants (18). B. subtilis, the host in the present study, uses the DXP pathway to produce isoprene ( Fig. 1) (37). The DXP pathway initiates with the glycolytic intermediates pyruvate and glyceraldehyde-3-phosphate (G3P). Pyruvate and G3P are condensed to form DXP by 1-deoxy-D-xylulose-5-phosphate synthase (Dxs), and subsequently, 6 enzymes, encoded by the ispC (dxr), ispD, ispE, ispF, ispG, and ispH genes, catalyze sequential reactions converting DXP to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (39). IPP and its isomer DMAPP can be interconverted by isopentenyl pyrophosphat...

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Section: Discussionmentioning

confidence: 88%

“…Previous work has shown that overexpression of rpoS can increase lycopene production in E. coli (15). rpoS encodes the sigma S subunit of RNA polymerase in E. coli.…”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Enhancing Isoprene Production by Genetic Modification of the 1-Deoxy- d -Xylulose-5-Phosphate Pathway in Bacillus subtilis

Xue

Ahring

2011

Appl Environ Microbiol

110

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“…This overlap validates our methodology in identifying gene targets that improve galactose fermentation using a genomic library. Similar approaches have been successfully applied to identify gene targets eliciting many phenotypes of biotechnological importance, such as improved xylose utilization , higher tolerance to ethanol and isobutanol (Hong et al, 2010) and enhanced lycopene production (Jin and Stephanopoulos, 2007;Kang et al, 2005). All of the studies were able to discover novel genetic perturbations and to confirm existing or known genetic perturbations responsible for desired phenotypes.…”

Section: Discussionmentioning

confidence: 99%

Improved galactose fermentation of Saccharomyces cerevisiae through inverse metabolic engineering

Lee

Hong

Jung

et al. 2010

Biotech & Bioengineering

102

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Although Saccharomyces cerevisiae is capable of fermenting galactose into ethanol, ethanol yield and productivity from galactose are significantly lower than those from glucose. An inverse metabolic engineering approach was undertaken to improve ethanol yield and productivity from galactose in S. cerevisiae. A genome-wide perturbation library was introduced into S. cerevisiae, and then fast galactose-fermenting transformants were screened using three different enrichment methods. The characterization of genetic perturbations in the isolated transformants revealed three target genes whose overexpression elicited enhanced galactose utilization. One confirmatory (SEC53 coding for phosphomannomutase) and two novel targets (SNR84 coding for a small nuclear RNA and a truncated form of TUP1 coding for a general repressor of transcription) were identified as overexpression targets that potentially improve galactose fermentation. Beneficial effects of overexpression of SEC53 may be similar to the mechanisms exerted by overexpression of PGM2 coding for phosphoglucomutase. While the mechanism is largely unknown, overexpression of SNR84, improved both growth and ethanol production from galactose. The most remarkable improvement of galactose fermentation was achieved by overexpression of the truncated TUP1 (tTUP1) gene, resulting in unrivalled galactose fermentation capability, that is 250% higher in both galactose consumption rate and ethanol productivity compared to the control strain. Moreover, the overexpression of tTUP1 significantly shortened lag periods that occurs when substrate is changed from glucose to galactose. Based on these results we proposed a hypothesis that the mutant Tup1 without C-terminal repression domain might bring in earlier and higher expression of GAL genes through partial alleviation of glucose repression. mRNA levels of GAL genes (GAL1, GAL4, and GAL80) indeed increased upon overexpression of tTUP. The results presented in this study illustrate that alteration of global regulatory networks through overexpression of the identified targets (SNR84 and tTUP1) is as effective as overexpression of a rate limiting metabolic gene (PGM2) in the galactose assimilation pathway for efficient galactose fermentation in S. cerevisiae. In addition, these results will be industrially useful in the biofuels area as galactose is one of the abundant sugars in marine plant biomass such as red seaweed as well as cheese whey and molasses.

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“…This is the first demonstration of a link between activity of an enzyme in the DOXP pathway and transcriptional regulation of the pathway. Dxs has been previously implicated as the rate-limiting step for the synthesis of IPP in E. coli strains engineered to overproduce lycopene (16,20). In contrast, we found that GcpE is the bottleneck in M. tuberculosis and that Dxs appears to regulate flux through the pathway by mediating transcriptional control of GcpE, once again, indicating how mycobacterial systems differ from those of other bacteria (3).…”

Section: Discussionmentioning

confidence: 60%

The Nonmevalonate Pathway of Isoprenoid Biosynthesis in Mycobacterium tuberculosis Is Essential and Transcriptionally Regulated by Dxs

et al. 2010

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Mycobacterium tuberculosis synthesizes isoprenoids via the nonmevalonate or DOXP pathway. Previous work demonstrated that three enzymes in the pathway (Dxr, IspD, and IspF) are all required for growth in vitro. We demonstrate the essentiality of the key genes dxs1 and gcpE, confirming that the pathway is of central importance and that the second homolog of the synthase (dxs2) cannot compensate for the loss of dxs1. We looked at the effect of overexpression of Dxr, Dxs1, Dxs2, and GcpE on viability and on growth in M. tuberculosis. Overexpression of dxs1 or dxs2 was inhibitory to growth, whereas overexpression of dxr or gcpE was not. Toxicity is likely to be, at least partially, due to depletion of pyruvate from the cells. Overexpression of dxs1 or gcpE resulted in increased flux through the pathway, as measured by accumulation of the metabolite 4-hydroxy-3-methyl-but-2-enyl pyrophosphate. We identified the functional translational start site and promoter region for dxr and demonstrated that it is expressed as part of a polycistronic mRNA with gcpE and two other genes. Increased expression of this operon was seen in cells overexpressing Dxs1, indicating that transcriptional control is effected by the first enzyme of the pathway via an unknown regulator.Mycobacterium tuberculosis, the causative agent of human tuberculosis, poses an increasing threat to health. The escalating incidence of drug resistant strains provides a new impetus to understand this complex pathogen and to identify metabolic targets for novel therapeutics. As a first step toward this aim, we have been directing our efforts to identifying metabolic pathways essential for bacterial viability.

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Identification of genes affecting lycopene accumulation in Escherichia coli using a shot‐gun method

Cited by 87 publications

References 42 publications

Enhancing Isoprene Production by Genetic Modification of the 1-Deoxy- d -Xylulose-5-Phosphate Pathway in Bacillus subtilis

Enhancing Isoprene Production by Genetic Modification of the 1-Deoxy- d -Xylulose-5-Phosphate Pathway in Bacillus subtilis

Improved galactose fermentation of Saccharomyces cerevisiae through inverse metabolic engineering

The Nonmevalonate Pathway of Isoprenoid Biosynthesis in Mycobacterium tuberculosis Is Essential and Transcriptionally Regulated by Dxs

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