ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae

Rodriguez, Sarah; Denby, Charles M.; Van Vu, T.; Baidoo, Edward E. K.; Wang, George; Keasling, Jay D.

doi:10.1186/s12934-016-0447-1

Title: ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae

Journal Article · Thu Mar 03 00:00:00 EST 2016 · Microbial Cell Factories

DOI:https://doi.org/10.1186/s12934-016-0447-1· OSTI ID:1618802

Rodriguez, Sarah; Denby, Charles M.; Van Vu, T.; Baidoo, Edward E. K.; Wang, George; Keasling, Jay D.

© 2016 Rodriguez et al. Background: With increasing concern about the environmental impact of a petroleumbased economy, focus has shifted towards greener production strategies including metabolic engineering of microbes for the conversion of plant-based feedstocks to second generation biofuels and industrial chemicals. Saccharomyces cerevisiae is an attractive host for this purpose as it has been extensively engineered for production of various fuels and chemicals. Many of the target molecules are derived from the central metabolite and molecular building block, acetyl-CoA. To date, it has been difficult to engineer S. cerevisiae to continuously convert sugars present in biomass-based feedstocks to acetyl-CoA derived products due to intrinsic physiological constraints-in respiring cells, the precursor pyruvate is directed away from the endogenous cytosolic acetyl-CoA biosynthesis pathway towards the mitochondria, and in fermenting cells pyruvate is directed towards the byproduct ethanol. In this study we incorporated an alternative mode of acetyl-CoA biosynthesis mediated by ATP citrate lyase (ACL) that may obviate such constraints. Results: We characterized the activity of several heterologously expressed ACLs in crude cell lysates, and found that ACL from Aspergillus nidulans demonstrated the highest activity. We employed a push/pull strategy to shunt citrate towards ACL by deletion of the mitochondrial NAD + -dependent isocitrate dehydrogenase (IDH1) and engineering higher flux through the upper mevalonate pathway. We demonstrated that combining the two modifications increases accumulation of mevalonate pathway intermediates, and that both modifications are required to substantially increase production. Finally, we incorporated a block strategy by replacing the native ERG12 (mevalonate kinase) promoter with the copper-repressible CTR3 promoter to maximize accumulation of the commercially important molecule mevalonate. Conclusion: By combining the push/pull/block strategies, we significantly improved mevalonate production. We anticipate that this strategy can be used to improve the efficiency with which industrial strains of S. cerevisiae convert feedstocks to acetyl-CoAderived fuels and chemicals.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: DEAC02-05CH11231; AC02-05CH11231; MCB-1330914

OSTI ID:: 1618802

Alternate ID(s):: OSTI ID: 1257387; OSTI ID: 1379132

Journal Information:: Microbial Cell Factories, Journal Name: Microbial Cell Factories Vol. 15 Journal Issue: 1; ISSN 1475-2859

Publisher:: Springer Science + Business MediaCopyright Statement

Country of Publication:: United Kingdom

Language:: English

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Cited by: 42 works

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ATP citrate lyase
Mevalonate pathway
Saccharomyces cerevisiae
Acetyl Coenzyme A
Metabolic engineering
Isoprenoid synthesis

ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae [Supplemental Data] Rodriguez, Sarah; Denby, Charles; Vu, T. https://doi.org/10.6084/m9.figshare.c.3605225 The current record is supplemented by this figure	figure	March 2016
MOESM4 of ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae Rodriguez, Sarah; Denby, Charles; Vu, T. https://doi.org/10.6084/m9.figshare.c.3605225_d1 The current record is supplemented by this text	text	January 2016
MOESM5 of ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae Rodriguez, Sarah; Denby, Charles; Baidoo, Edward https://doi.org/10.6084/m9.figshare.c.3605225_d2.v1 The current record is supplemented by this figure	figure	March 2016
MOESM3 of ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae Rodriguez, Sarah; Denby, Charles; Vu, T. https://doi.org/10.6084/m9.figshare.c.3605225_d3.v1 The current record is supplemented by this figure	figure	March 2016
MOESM1 of ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae Rodriguez, Sarah; Denby, Charles; Vu, T. https://doi.org/10.6084/m9.figshare.c.3605225_d4 The current record is supplemented by this text	text	January 2016
MOESM6 of ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae Rodriguez, Sarah; Denby, Charles; Vu, T. https://doi.org/10.6084/m9.figshare.c.3605225_d5.v1 The current record is supplemented by this figure	figure	March 2016
MOESM2 of ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae Rodriguez, Sarah; Denby, Charles; Baidoo, Edward https://doi.org/10.6084/m9.figshare.c.3605225_d6.v1 The current record is supplemented by this figure	figure	March 2016
ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae [Supplemental Data] Rodriguez, Sarah; Denby, Charles; Vu, T. https://doi.org/10.6084/m9.figshare.c.3605225.v1 The current record is supplemented by this figure	figure	March 2016
MOESM4 of ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae Rodriguez, Sarah; Denby, Charles; Vu, T. https://doi.org/10.6084/m9.figshare.c.3605225_d1.v1 The current record is supplemented by this dataset	dataset	March 2016
MOESM1 of ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae Rodriguez, Sarah; Denby, Charles; Vu, T. https://doi.org/10.6084/m9.figshare.c.3605225_d4.v1 The current record is supplemented by this figure	figure	March 2016
MOESM5 of ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae Rodriguez, Sarah; Denby, Charles; Vu, T. https://doi.org/10.6084/m9.figshare.c.3605225_d2 The current record is supplemented by this text	text	January 2016
MOESM6 of ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae Rodriguez, Sarah; Denby, Charles; Vu, T. https://doi.org/10.6084/m9.figshare.c.3605225_d5 The current record is supplemented by this text	text	January 2016
MOESM2 of ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae Rodriguez, Sarah; Denby, Charles; Vu, T. https://doi.org/10.6084/m9.figshare.c.3605225_d6 The current record is supplemented by this text	text	January 2016