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

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

doi:10.1186/s12934-016-0447-1

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

Microb Cell Fact. 2016 Mar 3:15:48. doi: 10.1186/s12934-016-0447-1.

Authors

Sarah Rodriguez^{1

2}, Charles M Denby^{3

4}, T Van Vu^{5

6}, Edward E K Baidoo^{7

8}, George Wang^{9

10}, Jay D Keasling^{11

12

13

14

15}

Affiliations

¹ Joint BioEnergy Institute, Emeryville, CA, USA. srodriguez@lbl.gov.
² Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. srodriguez@lbl.gov.
³ Joint BioEnergy Institute, Emeryville, CA, USA. cmdenby@lbl.gov.
⁴ California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, CA, USA. cmdenby@lbl.gov.
⁵ Joint BioEnergy Institute, Emeryville, CA, USA. vvu@lbl.gov.
⁶ Department of Bioengineering, University of California, Berkeley, CA, USA. vvu@lbl.gov.
⁷ Joint BioEnergy Institute, Emeryville, CA, USA. eebaidoo@lbl.gov.
⁸ Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. eebaidoo@lbl.gov.
⁹ Joint BioEnergy Institute, Emeryville, CA, USA. georgewang@lbl.gov.
¹⁰ Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. georgewang@lbl.gov.
¹¹ Joint BioEnergy Institute, Emeryville, CA, USA. JDKeasling@lbl.gov.
¹² Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. JDKeasling@lbl.gov.
¹³ California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, CA, USA. JDKeasling@lbl.gov.
¹⁴ Department of Bioengineering, University of California, Berkeley, CA, USA. JDKeasling@lbl.gov.
¹⁵ Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, USA. JDKeasling@lbl.gov.

Abstract

Background: With increasing concern about the environmental impact of a petroleum based 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-CoA derived fuels and chemicals.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

ATP Citrate (pro-S)-Lyase / metabolism*
Acetyl Coenzyme A / biosynthesis*
Cytosol / drug effects
Cytosol / metabolism*
Intracellular Space / metabolism
Metabolic Networks and Pathways / drug effects
Mevalonic Acid / metabolism*
Nitrogen / pharmacology
Protein Engineering / methods*
Saccharomyces cerevisiae / drug effects
Saccharomyces cerevisiae / metabolism*
Time Factors

Substances

Acetyl Coenzyme A
ATP Citrate (pro-S)-Lyase
Nitrogen
Mevalonic Acid