Metabolic engineering of Ashbya gossypii for limonene production from xylose

Abstract

Background Limonene is a cyclic monoterpene that has applications in the food, cosmetic, and pharmaceutical industries. The industrial production of limonene and its derivatives through plant extraction presents important drawbacks such as seasonal and climate issues, feedstock limitations, low efficiency and environmental concerns. Consequently, the implementation of efficient and eco-friendly bioprocesses for the production of limonene and other terpenes constitutes an attractive goal for microbial biotechnology. In this context, novel biocatalysts with the ability to produce limonene from alternative carbon sources will help to meet the industrial demands of limonene. Results Engineered strains of the industrial fungus Ashbya gossypii have been developed to produce limonene from xylose. The limonene synthase (LS) from Citrus limon was initially overexpressed together with the native HMG1 gene (coding for HMG-CoA reductase) to establish a limonene-producing platform from a xylose-utilizing A. gossypii strain. In addition, several strategies were designed to increase the production of limonene. Hence, the effect of mutant alleles of ERG20 (erg20F95W and erg20F126W) were evaluated together with a synthetic orthogonal pathway using a heterologous neryl diphosphate synthase. The lethality of the A. gossypii double mutant erg20F95W−F126W highlights the indispensability of farnesyl diphosphate for the synthesis of essential sterols. In addition, the utilization of the orthogonal pathway, bypassing the Erg20 activity through neryl diphosphate, triggered a substantial increase in limonene titer (33.6 mg/L), without critically altering the fitness of the engineered strain. Finally, the overexpression of the native ERG12 gene further enhanced limonene production, which reached 336.4 mg/L after 96 h in flask cultures using xylose as the carbon source. Conclusions The microbial production of limonene can be carried out using engineered strains of A. gossypii from xylose-based carbon sources. The utilization of a synthetic orthogonal pathway together with the overexpression of ERG12 is a highly beneficial strategy for the production of limonene in A. gossypii. The strains presented in this work constitute a proof of principle for the production of limonene and other terpenes from agro-industrial wastes such as xylose-rich hydrolysates in A. gossypii.

D-Limonene: Promising and Sustainable Natural Bioactive Compound

“…Natural plant extraction and biotransformation are the main methods for D-limonene production [27][28][29].…”

“…These methods are safer than chemical pyrolysis, but the yield is affected by differences in the extraction methods, solvent dosages, and even the plant raw materials, which can be subject to seasonal, climatic, and geographical factors [36,[39][40][41][42][43][44]. Therefore, the production of D-limonene via efficient bioconversion is an attractive strategy [29].…”

“…For example, using the methylerythritol 4-phosphate pathway or the mevalonate-dependent pathway [60], the introduction of a specific gene fragment into the test bacterium and induced expression for the synthesis of D-limonene resulted in D-limonene production up to 0.15 g/(L•h) under ideal conditions [61]. The yield of D-limonene obtained by metabolic engineering depends on the strain, the type of exogenous gene introduced, the incubation temperature, and other factors [29,62,63]. In addition to the methylerythritol 4-phosphate pathway and mevalonatedependent pathways, the isopentenol utilization pathway and the isoprenoid alcohol pathway have also been used to produce D-limonene, with reported maximum yields of approximately 0.7 µg/(L•h) and 10.42 µg/(L•h), respectively [59,[64][65][66].…”

Efficient Synthesis of Limonene in Saccharomyces cerevisiae Using Combinatorial Metabolic Engineering Strategies

“…Microorganisms, including bacteria, yeasts, and molds, have been successfully used for the biosynthesis and derivation of limonene. − Limonene synthase encoded by LimS plays an essential role in the biosynthesis of limonene from geranyl diphosphate (GPP), and the truncated mutants of limonene synthase ( tLimS ) showed higher catalytic activity . In Saccharomyces cerevisiae, an endogenous bifunctional enzyme that has both dimethylallyltranstransferase and geranyltranstransferase activity encoded by ERG20 catalyzes the synthesis of GPP from dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) and subsequently promotes the formation of farnesyl diphosphate (FPP) from GPP and IPP.…”

Single-Cell Oil Production by Engineered Ashbya gossypii from Non-Detoxified Lignocellulosic Biomass Hydrolysate

“…gossypii is a filamentous hemiascomycete industrially used to produce riboflavin, a process in which downstream product recovery is facilitated due to the ability of this fungus to undergo autolysis during the late stationary phase of growth or at low temperatures [16]. In addition to riboflavin, engineered A. gossypii strains are capable of producing other compounds of interest for the food and feed industry, such as the nucleosides inosine and guanosine [17], folic acid [18], polyunsaturated fatty acids (PUFAs) (up to 35% of total SCOs) [19] and other microbial oils [20], γ-lactones [21], orotic acid [22], and limonene [23].…”