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      Lycopene overproduction in Saccharomyces cerevisiae through combining pathway engineering with host engineering

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          Abstract

          Background

          Microbial production of lycopene, a commercially and medically important compound, has received increasing concern in recent years. Saccharomyces cerevisiae is regarded as a safer host for lycopene production than Escherichia coli. However, to date, the lycopene yield (mg/g DCW) in S. cerevisiae was lower than that in E. coli and did not facilitate downstream extraction process, which might be attributed to the incompatibility between host cell and heterologous pathway. Therefore, to achieve lycopene overproduction in S. cerevisiae, both host cell and heterologous pathway should be delicately engineered.

          Results

          In this study, lycopene biosynthesis pathway was constructed by integration of CrtE, CrtB and CrtI in S. cerevisiae CEN.PK2. When YPL062W, a distant genetic locus, was deleted, little acetate was accumulated and approximately 100 % increase in cytosolic acetyl-CoA pool was achieved relative to that in parental strain. Through screening CrtE, CrtB and CrtI from diverse species, an optimal carotenogenic enzyme combination was obtained, and CrtI from Blakeslea trispora (BtCrtI) was found to have excellent performance on lycopene production as well as lycopene proportion in carotenoid. Then, the expression level of Bt CrtI was fine-tuned and the effect of cell mating types was also evaluated. Finally, potential distant genetic targets ( YJL064W, ROX1, and DOS2) were deleted and a stress-responsive transcription factor INO2 was also up-regulated. Through the above modifications between host cell and carotenogenic pathway, lycopene yield was increased by approximately 22-fold (from 2.43 to 54.63 mg/g DCW). Eventually, in fed-batch fermentation, lycopene production reached 55.56 mg/g DCW, which is the highest reported yield in yeasts.

          Conclusions

          Saccharomyces cerevisiae was engineered to produce lycopene in this study. Through combining host engineering (distant genetic loci and cell mating types) with pathway engineering (enzyme screening and gene fine-tuning), lycopene yield was stepwise improved by 22-fold as compared to the starting strain. The highest lycopene yield (55.56 mg/g DCW) in yeasts was achieved in 5-L bioreactors. This study provides a good reference of combinatorial engineering of host cell and heterologous pathway for microbial overproduction of pharmaceutical and chemical products.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s12934-016-0509-4) contains supplementary material, which is available to authorized users.

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          Most cited references43

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          Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin.

          Malaria, caused by Plasmodium sp, results in almost one million deaths and over 200 million new infections annually. The World Health Organization has recommended that artemisinin-based combination therapies be used for treatment of malaria. Artemisinin is a sesquiterpene lactone isolated from the plant Artemisia annua. However, the supply and price of artemisinin fluctuate greatly, and an alternative production method would be valuable to increase availability. We describe progress toward the goal of developing a supply of semisynthetic artemisinin based on production of the artemisinin precursor amorpha-4,11-diene by fermentation from engineered Saccharomyces cerevisiae, and its chemical conversion to dihydroartemisinic acid, which can be subsequently converted to artemisinin. Previous efforts to produce artemisinin precursors used S. cerevisiae S288C overexpressing selected genes of the mevalonate pathway [Ro et al. (2006) Nature 440:940-943]. We have now overexpressed every enzyme of the mevalonate pathway to ERG20 in S. cerevisiae CEN.PK2, and compared production to CEN.PK2 engineered identically to the previously engineered S288C strain. Overexpressing every enzyme of the mevalonate pathway doubled artemisinic acid production, however, amorpha-4,11-diene production was 10-fold higher than artemisinic acid. We therefore focused on amorpha-4,11-diene production. Development of fermentation processes for the reengineered CEN.PK2 amorpha-4,11-diene strain led to production of > 40 g/L product. A chemical process was developed to convert amorpha-4,11-diene to dihydroartemisinic acid, which could subsequently be converted to artemisinin. The strains and procedures described represent a complete process for production of semisynthetic artemisinin.
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            Carotenoids as modulators of lipid membrane physical properties.

            Carotenoids are a group of pigments present both in the plant and animal kingdoms, which play several important physiological functions. The protection against active oxygen species, realised via the quenching of excited states of photosensitizing molecules, quenching of singlet oxygen and scavenging of free radicals, is one of the main biological functions of carotenoids. Several recent research indicate that the protection of biomembranes against oxidative damage can be also realised via the modification of the physical properties of the lipid phase of the membranes. This work presents an overview of research on an effect of carotenoids on the structural and dynamic properties of lipid membranes carried out with the application of different techniques such as Electron Paramagnetic Resonance, Nuclear Magnetic Resonance, Differential Scanning Calorimetry, X-ray diffractometry, monomolecular layer technique and other techniques. It appears that, in most cases, polar carotenoids span lipid bilayer and have their polar groups anchored in the opposite polar zones of the membrane. Owing to the van der Waals interactions of rigid rod-like molecules of carotenoid and acyl chains of lipids, pigment molecules rigidify the fluid phase of the membranes and limit oxygen penetration to the hydrophobic membrane core susceptible to oxidative degradation.
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              Identifying gene targets for the metabolic engineering of lycopene biosynthesis in Escherichia coli.

              The identification of genetic targets that are effective in bringing about a desired phenotype change is still an open problem. While random gene knockouts have yielded improved strains in certain cases, it is also important to seek the guidance of cell-wide stoichiometric constraints in identifying promising gene knockout targets. To investigate these issues, we undertook a genome-wide stoichiometric flux balance analysis as an aid in discovering putative genes impacting network properties and cellular phenotype. Specifically, we calculated metabolic fluxes such as to optimize growth and then scanned the genome for single and multiple gene knockouts that yield improved product yield while maintaining acceptable overall growth rate. For the particular case of lycopene biosynthesis in Escherichia coli, we identified such targets that we subsequently tested experimentally by constructing the corresponding single, double and triple gene knockouts. While such strains are suggested (by the stoichiometric calculations) to increase precursor availability, this beneficial effect may be further impacted by kinetic and regulatory effects not captured by the stoichiometric model. For the case of lycopene biosynthesis, the so identified knockout targets yielded a triple knockout construct that exhibited a nearly 40% increase over an engineered, high producing parental strain.
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                Author and article information

                Contributors
                cyan@tju.edu.cn
                86-22-60973987 , wenhai.xiao@tju.edu.cn
                ying.wang@tju.edu.cn
                season_l@sina.com
                lixia01@tju.edu.cn
                yjyuan@tju.edu.cn
                Journal
                Microb Cell Fact
                Microb. Cell Fact
                Microbial Cell Factories
                BioMed Central (London )
                1475-2859
                21 June 2016
                21 June 2016
                2016
                : 15
                : 113
                Affiliations
                [ ]Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, 300072 People’s Republic of China
                [ ]SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072 People’s Republic of China
                Author information
                http://orcid.org/0000-0001-9427-6881
                Article
                509
                10.1186/s12934-016-0509-4
                4915043
                27329233
                f4480e28-081a-44da-b660-3e0de360a80d
                © The Author(s) 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 28 February 2016
                : 6 June 2016
                Funding
                Funded by: the Ministry of Science and Technology of China ("863"Program)
                Award ID: 2012AA02A701
                Award ID: 2012AA02A701
                Award ID: 2012AA02A701
                Award ID: 2012AA02A701
                Award ID: 2012AA02A701
                Award Recipient :
                Funded by: the International S and T Cooperation Program of China
                Award ID: 2015DFA00960
                Award ID: 2015DFA00960
                Award ID: 2015DFA00960
                Award ID: 2015DFA00960
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2016

                Biotechnology
                metabolic engineering,lycopene,saccharomyces cerevisiae,heterologous pathway,synthetic biology

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