Integrated engineering of β-oxidation reversal and ω-oxidation pathways for the synthesis of medium chain ω-functionalized carboxylic acids

Clomburg, James M.; Blankschien, Matthew D.; Vick, Jacob E.; Chou, Alexander; Kim, Seohyoung; González, Ramón

doi:10.1016/j.ymben.2015.01.007

Cited by 132 publications

(114 citation statements)

References 45 publications

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“…A range of enzymes and pathways have been heterologously expressed to convert fatty acid thioesters-produced by endogenous fatty acid biosynthesis-into ethyl esters, acids, alcohols, alkanes, methyl ketones, dicarboxylic acids, etc. (Clomburg et al, 2015;Goh et al, 2012;Zhou et al, 2016). Among these products, long-chain fatty alcohols in the C12-C18 range have recently received intense attention due to their value and broad applications in laundry detergents, industrial lubricants and surfactants, medicines and personal care products, and potentially as biofuels (Feng et al, 2015;Liu et al, 2016;Pfleger et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Engineering high-level production of fatty alcohols by Saccharomyces cerevisiae from lignocellulosic feedstocks

d’Espaux

Ghosh

Runguphan

et al. 2017

Metabolic Engineering

104

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A B S T R A C TFatty alcohols in the C12-C18 range are used in personal care products, lubricants, and potentially biofuels. These compounds can be produced from the fatty acid pathway by a fatty acid reductase (FAR), yet yields from the preferred industrial host Saccharomyces cerevisiae remain under 2% of the theoretical maximum from glucose. Here we improved titer and yield of fatty alcohols using an approach involving quantitative analysis of protein levels and metabolic flux, engineering enzyme level and localization, pull-push-block engineering of carbon flux, and cofactor balancing. We compared four heterologous FARs, finding highest activity and endoplasmic reticulum localization from a Mus musculus FAR. After screening an additional twenty-one single-gene edits, we identified increasing FAR expression; deleting competing reactions encoded by DGA1, HFD1, and ADH6; overexpressing a mutant acetyl-CoA carboxylase; limiting NADPH and carbon usage by the glutamate dehydrogenase encoded by GDH1; and overexpressing the Δ9-desaturase encoded by OLE1 as successful strategies to improve titer. Our final strain produced 1.2 g/L fatty alcohols in shake flasks, and 6.0 g/L in fed-batch fermentation, corresponding to~20% of the maximum theoretical yield from glucose, the highest titers and yields reported to date in S. cerevisiae. We further demonstrate high-level production from lignocellulosic feedstocks derived from ionic-liquid treated switchgrass and sorghum, reaching 0.7 g/L in shake flasks. Altogether, our work represents progress towards efficient and renewable microbial production of fatty acidderived products.

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Section: Introductionmentioning

confidence: 99%

Engineering high-level production of fatty alcohols by Saccharomyces cerevisiae from lignocellulosic feedstocks

d’Espaux

Ghosh

Runguphan

et al. 2017

Metabolic Engineering

104

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“…This resulted in the production of different hydroxyl fatty acids with chain lengths of 10 to 18 carbons at a final titer of 58.7 and 548 mg/L in culture broth and fed-batch fermentation, respectively. The engineered reversal of the β-oxidation cycle, in combination with the expression of heterologous thioesterase and monooxygenase systems, has led to the biosynthesis of medium chain ω-hydroxy fatty acids and dicarboxylic acids at a titer of 0.8 and 0.5 g/L, respectively, from glycerol in E. coli [71]. …”

Section: Discussionmentioning

confidence: 99%

Combinatorial Engineering of Yarrowia lipolytica as a Promising Cell Biorefinery Platform for the de novo Production of Multi-Purpose Long Chain Dicarboxylic Acids

2017

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This proof-of-concept study establishes Yarrowia lipolytica (Y. lipolytica) as a whole cell factory for the de novo production of long chain dicarboxylic acid (LCDCA-16 and 18) using glycerol as the sole source of carbon. Modification of the fatty acid metabolism pathway enabled creating a pool of fatty acids in a β-oxidation deficient strain. We then selectively upregulated the native fatty acid ω-oxidation pathway for the enhanced terminal oxidation of the endogenous fatty acid precursors. Nitrogen-limiting conditions and leucine supplementation were employed to induce fatty acid biosynthesis in an engineered Leu -modified strain. Our genetic engineering strategy allowed a minimum production of 330 mg/L LCDCAs in shake flask. Scale up to a 1-L bioreactor increased the titer to 3.49 g/L. Our engineered yeast also produced citric acid as a major by-product at a titer of 39.2 g/L. These results provide basis for developing Y. lipolytica as a safe biorefinery platform for the sustainable production of high-value LCDCAs from non-oily feedstock.

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“…After early work on muconic acid production in the mid 1990s (Frost & Draths, 1996, Frost & Draths, 1997) the target adipic acid has recently moved back into the spotlight with several companies (Genomatica, Verdezyne) filing patents (Boussie et al, 2010, Raemakers-Franken et al, 2012 and the metabolic engineering research community re-focussing on this compound (Niu et al, 2002, IHS Chemical, 2012, Weber et al, 2012, Curran et al, 2013, Sun et al, 2013, Yu et al, 2014, Babu et al, 2015, Clomburg et al, 2015, Deng & Mao, 2015, Horwitz et al, 2015, Reizman et al, 2015, Sengupta et al, 2015, Vardon et al, 2015. Considering the extensive work that has been done it is not surprising that there is a wide variety of proclaimed metabolic routes to produce adipic acid or the precursors muconic or glucaric acid (Figure 4).…”

Section: Bio-based Production Of Adipic Acid and Potential Precursorsmentioning

confidence: 99%

“…Titers were 12 µg/L (Babu et al, 2015) and 0.5 g/L (combined C 6 , C 8 , and C 10 dicarboxylic acids) (Clomburg et al, 2015).…”

Section: Bio-based Production Of Adipic Acid and Potential Precursorsmentioning

confidence: 99%

“…Six fundamentally different pathways featuring unique intermediates and enzymatic conversions were compiled from patents (Frost & Draths, 1996, Frost & Draths, 1997, Boussie et al, 2010, Raemakers-Franken et al, 2012 and scientific publications (Draths & Frost, 1994, Weber et al, 2012, Curran et al, 2013, Sun et al, 2013, Yu et al, 2014, Babu et al, 2015, Clomburg et al, 2015, Deng & Mao, 2015, Sengupta et al, 2015, Vardon et al, 2015. In total 16 different main routes for carbon-flow were considered.…”

Section: Biochemical Pathways To Adipic Acidmentioning

confidence: 99%

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Engineering yeast shikimate pathway towards production of aromatics: rational design of a chassis cell using systems and synthetic biology

Averesch¹

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Aromatic building blocks are amongst the most important bulk feedstocks in the chemical industry. As these compounds are commonly derived from petrochemistry, obtaining them is becoming more and more a matter of costs and sustainability. Biochemistry gives rise to a wealth of compounds that can potentially replace current petroleum-based chemicals or be used for novel materials. The aromatic compounds para-aminobenzoic acid (pABA) and para-hydroxybenzoic acid (pHBA) and the aromatics derived compound cis,cismuconic acid (ccMA) can be precursors for, but are not limited to, terephthalic or/and adipic acid. These are essential feedstocks for the production of PET and nylon. The three compounds can be derived from the shikimate pathway, an anabolic pathway leading to the biosynthesis of aromatic amino acids, present in certain prokaryotes and eukaryotes, including fungi. By combination of metabolic modelling with genetic engineering, a microbial production system based on the yeast Saccharomyces cerevisiae can be designed, which effectively channels flux into the target compounds.In order to develop a competitive bio-based process, yields, titers and rates need to be maximized. While productivity or rates in a process can be altered using genetic engineering, carbon yield, and pathway feasibility are stoichiometrically and thermodynamically predetermined. Both limitations need to be considered when designing a microbial production system. For formation of adipic acid and precursors many bio-based routes exists. More rational than just picking one for in vivo studies rather all available biochemical pathways were examined in silico using metabolic modelling. To compare theoretical yields and reaction thermodynamics an interface that allowed network-embedded thermodynamic analysis of elementary flux modes was developed. This allowed distinguishing between thermodynamically feasible and infeasible flux distributions. Feasible maximum theoretical product carbon yields were substantially different in E. coli and S. cerevisiae metabolic models and ranged from 32% to 92%. Further, many pathways appeared to be restricted by a thermodynamic equilibrium lying on the substrate side, some even infeasible.The only routes that deliver significant product yields and were thermodynamically favoured were shikimate pathway based. Being currently of strong scientific interest, recent implementations of these pathways in E. coli and S. cerevisiae were evaluated and strain construction strategies optimized, using the concept of constrained minimal cut-sets.Especially in S. cerevisiae a single non-obvious knock-out target allowed coupling of growth to product formation; in particular, the deletion of the pyruvate kinase reaction resulted in a minimum yield constraint of 28%.Though unique to shikimate pathway, this strategy is transferrable to other products which are derived from chorismate and also involve the formation of pyruvate as a by-product. This applies to pHBA. With further optimizations, the strategy was applied in...

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Integrated engineering of β-oxidation reversal and ω-oxidation pathways for the synthesis of medium chain ω-functionalized carboxylic acids

Cited by 132 publications

References 45 publications

Engineering high-level production of fatty alcohols by Saccharomyces cerevisiae from lignocellulosic feedstocks

Engineering high-level production of fatty alcohols by Saccharomyces cerevisiae from lignocellulosic feedstocks

Combinatorial Engineering of Yarrowia lipolytica as a Promising Cell Biorefinery Platform for the de novo Production of Multi-Purpose Long Chain Dicarboxylic Acids

Engineering yeast shikimate pathway towards production of aromatics: rational design of a chassis cell using systems and synthetic biology

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