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.
    Biotechnology and Bioengineering 01/2014; 111(1). DOI:10.1002/bit.25002 · 4.13 Impact Factor
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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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    • "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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