Metabolic engineering of Ashbya gossypii for deciphering the de novo biosynthesis of γ-lactones

Abstract

Background Lactones are highly valuable cyclic esters of hydroxy fatty acids that find application as pure fragrances or as building blocks of speciality chemicals. While chemical synthesis often leads to undesired racemic mixtures, microbial production allows obtaining optically pure lactones. The production of a specific lactone by biotransformation depends on the supply of the corresponding hydroxy fatty acid, which has economic and industrial value similar to γ-lactones. Hence, the identification and exploration of microorganisms with the rare natural ability for de novo biosynthesis of lactones will contribute to the long-term sustainability of microbial production. In this study, the innate ability of Ashbya gossypii for de novo production of γ-lactones from glucose was evaluated and improved. Results Characterization of the volatile organic compounds produced by nine strains of this industrial filamentous fungus in glucose-based medium revealed the noteworthy presence of seven chemically different γ-lactones. To decipher and understand the de novo biosynthesis of γ-lactones from glucose, we developed metabolic engineering strategies focused on the fatty acid biosynthesis and the β-oxidation pathways. Overexpression of AgDES589 , encoding a desaturase for the conversion of oleic acid (C18:1) into linoleic acid (C18:2), and deletion of AgELO624 , which encodes an elongase that catalyses the formation of C20:0 and C22:0 fatty acids, greatly increased the production of γ-lactones (up to 6.4-fold; (7.6 ± 0.8) × 10 3 µg/g Cell Dry Weight ). Further substitution of AgPOX1 , encoding the exclusive acyl-CoA oxidase in A. gossypii , by a codon-optimized POX2 gene from Yarrowia lipolytica , which encodes a specific long chain acyl-CoA oxidase, fine-tuned the biosynthesis of γ-decalactone to a relative production of more than 99%. Conclusions This study demonstrates the potential of A. gossypii as a model and future platform for de novo biosynthesis of γ-lactones. By means of metabolic engineering, key enzymatic steps involved in their production were elucidated. Moreover, the combinatorial metabolic engineering strategies developed resulted in improved de novo biosynthesis of γ-decalactone. In sum, these proof-of-concept data revealed yet unknown metabolic and genetic determinants important for the future exploration of the de novo production of γ-lactones as an alternative to biotransformation processes. Electronic supplementary material The online version of this article (10.1186/s12934-019-1113-1) contains supplementary material, which is available to authorized users.

Production of a Distilled Spirit Using Cassava Flour as Raw Material: Chemical Characterization and Sensory Profile

“…Thus, high concentrations of cis / trans -oak lactone were found in the cassava spirit with application of wood, which were extracted from oak chips by the hydroalcoholic matrix. Moreover, lactones can derive from microbial de novo production or transformation of fatty acids present in the raw material [ 22 , 23 ]. For example, lactones are commonly found in the fermentation of rice or when using it as an adjunct for brewing, resulting from biotransformation of lipids in the raw material [ 24 , 25 , 26 ].…”

Sporulation in Ashbya gossypii

“…The most well-known may be the proof of the whole genome duplication in S. cerevisiae [19,20]. The molecular analysis of riboflavin production and the novel bioengineering efforts to implement Ashbya as a platform strain for proteins, lipids or flavor compounds has made Ashbya a very interesting strain for the biotech industry as an alternative and addition to S. cerevisiae and other yeasts as well as for synthetic biology approaches [3,6,7,9,10,18]. Developmental biology contributed to the analysis of polarized hyphal growth, septation, nuclear migration and asynchronous nuclear divisions in a filamentous fungus [22][23][24]27,[44][45][46][47][48][49].…”

“…Soon after its potential for riboflavin overproduction was recognized, which led to the establishment of Ashbya as a platform producer of vitamin B 2 on an industrial scale [2][3][4]. Such a platform strain has expanded uses e.g., in the production of other vitamins, flavor compounds or lipids [5][6][7][8][9]. Metabolic engineering and metabolic flux studies in Ashbya have been used to determine and improve key enzymatic reactions in pathways required for riboflavin or gamma-lactone production [9].…”

“…Such a platform strain has expanded uses e.g., in the production of other vitamins, flavor compounds or lipids [5][6][7][8][9]. Metabolic engineering and metabolic flux studies in Ashbya have been used to determine and improve key enzymatic reactions in pathways required for riboflavin or gamma-lactone production [9]. Ashbya requires sucking insects to form plant infection as it does not produce penetration pegs or lytic enzymes.…”

Multiplex genomic edition in Ashbya gossypii using CRISPR-Cpf1

“…gossypii is a filamentous fungus that is currently exploited in the industry for the production of riboflavin [1]. Furthermore, A. gossypii has been proposed as a microbial cell factory for the production of other relevant compounds such as folic acid, nucleosides, recombinant proteins, γ lactones and biolipids [2][3][4][5][6]. In this context, the development of novel molecular tools is essential for the implementation of rational system metabolic engineering approaches in A. gossypii.…”