Disrupted short chain specific β‐oxidation and improved synthase expression increase synthesis of short chain fatty acids in Saccharomyces cerevisiae

Abstract

Biologically derived fatty acids have gained tremendous interest as an alternative to petroleum-derived fuels and chemical precursors. We previously demonstrated the synthesis of short chain fatty acids in Saccharomyces cerevisiae by introduction of the Homo sapiens fatty acid synthase (hFAS) with heterologous phosphopantetheine transferases and heterologous thioesterases. In this study, short chain fatty acid production was improved by combining a variety of novel enzyme and metabolic engineering strategies. The use of a H. sapiens-derived thioesterase and phosphopantetheine transferase were evaluated. In addition, strains were engineered to disrupt either the full β-oxidation (by deleting FAA2, PXA1, and POX1) or short chain-specific β-oxidation (by deleting FAA2, ANT1, and PEX11) pathways. Prohibiting full β-oxidation increased hexanoic and octanoic acid levels by 8- and 79-fold relative to the parent strain expressing hFAS. However, by targeting only short chain β-oxidation, hexanoic and octanoic acid levels increased further to 31- and 140-fold over the parent. In addition, an optimized hFAS gene increased hexanoic, octanoic, decanoic and total short chain fatty acid levels by 2.9-, 2.0-, 2.3-, and 2.2-fold, respectively, relative to the non-optimized counterpart. By combining these unique enzyme and metabolic engineering strategies, octanoic acid was increased more than 181-fold over the parent strain expressing hFAS.

A Yeast-Based Biosensor for Screening of Short- and Medium-Chain Fatty Acid Production

“…This is of particular importance since the highest SMCFA titers produced in yeast so far were reached in complex medium. 23,27,28 To avoid possible stability problems by using a multicopy plasmid-based biosensor, we integrated the sensor construct into the S. cerevisiae CEN.PK113−11C genome resulting in strain LBY27. This sensor strain, however, only showed a barely detectable increase in the intensity of the signal upon C 8 FA addition in comparison to the control CEN.PK113−11C strain ( Figure 2C).…”

Designing and Creating a Synthetic Omega Oxidation Pathway in Saccharomyces cerevisiae Enables Production of Medium-Chain α, ω-Dicarboxylic Acids

“…Thioesterase exhibits the activity of splitting of an ester into acid and alcohol. Hence, overexpression of thioesterase often facilitate the production of fatty acid in yeast ( Chen et al, 2014 ; Leber and Da Silva, 2014 ; Leber et al, 2016 ). For example, ‘TesA in E. coli exhibits both acyl-ACP and acyl-CoA thioesterase and ‘TesA could be actively expressed in S.cerevisiae to produce free fatty acids ( Runguphan and Keasling, 2014 ).…”

Engineering xylose utilization in Yarrowia lipolytica by understanding its cryptic xylose pathway

“…Metabolic engineering has made significant advancements in the last decade toward engineering microbes that produce a variety of industrially useful products [ 1 ]. A large emphasis has been placed on developing downstream metabolic pathways to produce novel products, including alkanes [ 2 ], alcohols [ 3 – 5 ], aldehydes [ 6 – 8 ], esters [ 4 , 9 ], fatty acids [ 4 , 10 , 11 ], and lipid-based products [ 4 , 12 ] from traditional carbon feedstocks such as glucose. Advancements in lignocellulose degradation have made unconventional sugars abundantly available [ 13 , 14 ].…”