Reconstruction of a bacterial isoprenoid biosynthetic pathway in Saccharomyces cerevisiae

Center for Microbial Biotechnology, DTU-Biosys, Building 223, 2800 Kgs Lyngby, Denmark.
FEBS Letters (Impact Factor: 3.17). 12/2008; 582(29):4032-8. DOI: 10.1016/j.febslet.2008.10.045
Source: PubMed


A eukaryotic mevalonate pathway transferred and expressed in Escherichia coli, and a mammalian hydrocortisone biosynthetic pathway rebuilt in Saccharomyces cerevisiae are examples showing that transferring metabolic pathways from one organism to another can have a powerful impact on cell properties. In this study, we reconstructed the E. coli isoprenoid biosynthetic pathway in S. cerevisiae. Genes encoding the seven enzymatic steps of the pathway were cloned and expressed in S. cerevisiae. mRNA from the seven genes was detected, and the pathway was shown able to sustain growth of yeast in conditions of inhibition of its constitutive isoprenoid biosynthetic pathway.

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    • "galactose and glucose (Johnston et al., 1994; Lohr et al., 1995). Since the promoters of GAL1 and GAL10 in this system can be induced at least 1000-fold (Giniger and Ptashne, 1988), high-copy 2m plasmids carrying these two promoters has previously been applied for the recombinant protein production (Choi et al., 1994; Romanos et al., 1992) and biosynthetic pathway construction in S. cerevisiae (Maury et al., 2008; Shiba et al., 2007; Steen et al., 2008). Nevertheless, using plasmids to construct a biosynthetic pathway is hindered by drawbacks such as " plasmid instability, " " plasmid burden " (Karim et al., 2012; Zhang et al., 1996) and special medium requirements. "
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    ABSTRACT: Saccharomyces cerevisiae is an important platform organism for the synthesis of a great number of natural products. However, the assembly of controllable and genetically stable heterogeneous biosynthetic pathways in S. cerevisiae still remains a significant challenge. Here, we present a strategy for reconstructing controllable multi-gene pathways by employing the GAL regulatory system. A set of marker recyclable integrative plasmids (pMRI) was designed for decentralized assembly of pathways. As proof-of-principle, a controllable β-carotene biosynthesis pathway (∼16 kb) was reconstructed and optimized by repeatedly using GAL10-GAL1 bidirectional promoters with high efficiency (80%-100%). By controlling the switch time of the pathway, production of 11 mg/g DCW of total carotenoids (72.57mg/L) and 7.41mg/g DCW of β-carotene was achieved in shake-flask culture. In addition, the engineered yeast strain exhibited high genetic stability after 20 generations of subculture. The results demonstrated a controllable and genetically stable biosynthetic pathway capable of increasing the yield of target products. Furthermore, the strategy presented in this study could be extended to construct other pathways in S. cerevisisae. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.
    Full-text · Article · Jan 2014 · Biotechnology and Bioengineering
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    • "Despite this challenge, as well as other hurdles associated with constructing, testing, and balancing large networks of molecular pathways (where design complexity can be intimidating), some examples have shown success. Two notable applications include production of high butanol titers through the transfer of the Clostridium acetobutylicum butanol pathway to a variety of bacterial hosts [20] and production of valencene in yeast by transferring an isoprenoid pathway consisting of 7 E. coli genes [21]. "
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    ABSTRACT: Synthetic biology is a nascent technical discipline that seeks to enable the design and construction of novel biological systems to meet pressing societal needs. However, engineering biology still requires much trial and error because we lack effective approaches for connecting basic "parts" into higher-order networks that behave as predicted. Developing strategies for improving the performance and sophistication of our designs is informed by two overarching perspectives: "bottom-up" and "top-down" considerations. Using this framework, we describe a conceptual model for developing novel biological systems that function and interact with existing biological components in a predictable fashion. We discuss this model in the context of three topical areas: biochemical transformations, cellular devices and therapeutics, and approaches that expand the chemistry of life. Ten years after the construction of synthetic biology's first devices, the drive to look beyond what does exist to what can exist is ushering in an era of biology by design.
    Full-text · Article · Nov 2010 · BioMed Research International
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    • "Microbial production of sesquiterpenes has been investigated in the last decade by several groups (for review see Maury et al., 2005). The yeast Saccharomyces cerevisiae has been successfully used for heterologous production of sesquiterpenes (Asadollahi et al., 2008, 2009; Jackson et al., 2003; Kirby et al., 2008; Lindahl et al., 2006; Maury et al., 2008; Ro et al., 2006; Shiba et al., 2007; Takahashi et al., 2007). Although expression of heterologous sesquiterpene synthase genes in yeast allows production of the corresponding sesquiterpenes, titers are not sufficient to support a viable industrial process. "
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    ABSTRACT: The mevalonate pathway in the yeast Saccharomyces cerevisiae was deregulated in order to enhance the intracellular pool of farnesyl diphosphate (FPP), the direct precursor for the biosynthesis of sesquiterpenes. Over-expression of the catalytic domain of HMG1, both from the genome and plasmid, resulted in higher production of cubebol, a plant originating sesquiterpene, and increased squalene accumulation. Down-regulation of ERG9 by replacing its native promoter with the regulatable MET3 promoter, enhanced cubebol titers but simultaneous over-expression of tHMG1 and repression of ERG9 did not further improve cubebol production. Furtheremore, the concentrations of squalene and ergosterol were measured in the engineered strains. Unexpectedly, significant accumulation of squalene and restoring the ergosterol biosynthesis were observed in the ERG9 repressed strains transformed with the plasmids harboring cubebol synthase gene. This could be explained by a toxicity effect of cubebol, possibly resulting in higher transcription levels for the genes under control of MET3 promoter, which could lead to accumulation of squalene and ergosterol.
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