Effects of acetoacetyl-CoA synthase expression on production of farnesene in Saccharomyces cerevisiae

Abstract

Efficient production of sesquiterpenes in Saccharomyces cerevisiae requires a high flux through the mevalonate pathway. To achieve this, the supply of acetyl-CoA plays a crucial role, partially because nine moles of acetyl-CoA are necessary to produce one mole of farnesyl diphosphate, but also to overcome the thermodynamic constraint imposed on the first reaction, in which acetoacetyl-CoA is produced from two moles of acetyl-CoA by acetoacetyl-CoA thiolase. Recently, a novel acetoacetyl-CoA synthase (nphT7) has been identified from Streptomyces sp. strain CL190, which catalyzes the irreversible condensation of malonyl-CoA and acetyl-CoA to acetoacetyl-CoA and, therefore, represents a potential target to increase the flux through the mevalonate pathway. This study investigates the effect of acetoacetyl-CoA synthase on growth as well as the production of farnesene and compares different homologs regarding their efficiency. While plasmid-based expression of nphT7 did not improve final farnesene titers, the construction of an alternative pathway, which exclusively relies on the malonyl-CoA bypass, was detrimental for growth and farnesene production. The presented results indicate that the overall functionality of the bypass was limited by the efficiency of acetoacetyl-CoA synthase (nphT7). Besides modulation of the expression level, which could be used as a means to partially restore the phenotype, nphT7 from Streptomyces glaucescens showed clearly higher efficiency compared to Streptomyces sp. strain CL190.Electronic supplementary materialThe online version of this article (doi:10.1007/s10295-017-1911-6) contains supplementary material, which is available to authorized users.

Enhancing 3-hydroxypropionic acid production in combination with sugar supply engineering by cell surface-display and metabolic engineering of Schizosaccharomyces pombe

“…Notably, engineering of the availability of acetyl-CoA has been reported to yield a positive effect on 3-HP production, presumably via the increase in the supply of malonyl-CoA. This strategy has been employed successfully in S. cerevisiae to improve the production of 3-HP [ 11 , 28 ], butanol [ 29 , 30 ], and isoprenoids [ 31 , 32 ]. Specifically, these laboratories enhanced the flux towards acetyl-CoA by co-expression of a Salmonella enterica gene encoding acetyl-CoA synthase harboring a point mutation (referred to here as acsSE* ) and a gene encoding an intact aldehyde dehydrogenase ( S. cerevisiae ald6 ).…”

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“…Aacts are known to play a crucial role in increasing the flux through the MVA pathway. However, it has been described that balanced activity of Aacts in relation to the other enzymes of the MVA pathway is important for the MVA pathway flux [50] , [51] , [52] . For example, overexpression of Aact promotes its thiolysis activity, which is triggered by increased levels of acetoacetyl-CoA.…”

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