Background: Limonene is an important biologically active natural product widely used in the food, cosmetic, nutraceutical and pharmaceutical industries. However, the low abundance of limonene in plants renders their isolation from plant sources non-economically viable. Therefore, engineering microbes into microbial factories for producing limonene is fast becoming an attractive alternative approach that can overcome the aforementioned bottleneck to meet the needs of industries and make limonene production more sustainable and environmentally friendly.Results: In this proof-of-principle study, the oleaginous yeastYarrowia lipolyticawas successfully engineered to produce bothd-limonene andl-limonene by introducing the heterologousd-limonene synthase fromCitrus limonandl-limonene synthase fromMentha spicata, respectively. However, only 0.124 mg/Ld-limonene and 0.126 mg/Ll-limonene were produced. To improve the limonene production by the engineered yeastY. lipolyticastrain, ten genes involved in the mevalonate-dependent isoprenoid pathway were overexpressed individually to investigate their effects on limonene titer. Hydroxymethylglutaryl-CoA reductase (HMGR) was found to be the key rate-limiting enzyme in the mevalonate (MVA) pathway for the improving limonene synthesis inY. lipolytica. Through the overexpression ofHMGRgene, the titers ofd-limonene andl-limonene were increased to 0.256 mg/L and 0.316 mg/L, respectively. Subsequently, the fermentation conditions were optimized to maximize limonene production by the engineeredY. lipolyticastrains from glucose, and the final titers ofd-limonene andl-limonene were improved to 2.369 mg/L and 2.471 mg/L, respectively. Furthermore, fed-batch fermentation of the engineered strains Po1g KdHR and Po1g KlHR was used to enhance limonene production in shake flasks and the titers achieved ford-limonene andl-limonene were 11.705 mg/L (0.443 mg/g) and 11.088 mg/L (0.385 mg/g), respectively. Finally, the potential of using waste cooking oil as a carbon source for limonene biosynthesis from the engineeredY. lipolyticastrains was investigated. We showed thatd-limonene andl-limonene were successfully produced at the respective titers of 2.514 mg/L and 2.723 mg/L under the optimal cultivation condition, where 70% of waste cooking oil was added as the carbon source, representing a 20-fold increase in limonene titer compared to that before strain and fermentation optimization.Conclusions: This study represents the first report on the development of a new and efficient process to convert waste cooking oil intod-limonene andl-limonene by exploiting metabolically engineeredY. lipolyticastrains for fermentation. The results obtained in this study lay the foundation for more future applications ofY. lipolyticain converting waste cooking oil into various industrially valuable products.

2,3-Butanediol (2,3-BD) is a high-value chemical usually produced petrochemically but which can also be synthesized by some bacteria. To date, Klebsiella pneumoniae is the most powerful 2,3-BD producer which can utilize a wide range of substrates. However, many by-products are also produced by K. pneumoniae, such as ethanol, lactate, and acetate, which negatively regulate the 2,3-BD yield and increase the costs of downstream separation and purification.

Acetate esters and higher alcohols greatly influence the quality and flavor profiles of Chinese Baijiu (Chinese liquor). Various mutants have been constructed to investigate the interactions of ATF1 overexpression, IAH1 deletion, and BAT2 deletion on the production of acetate esters and higher alcohols. The results showed that the overexpression of ATF1 under the control of the PGK1 promoter with BAT2 and IAH1 double-gene deletion led to a higher production of acetate esters and a lower production of higher alcohols than the overexpression of ATF1 with IAH1 deletion or overexpression of ATF1 with BAT2 deletion. Moreover, deletion of IAH1 in ATF1 overexpression strains effectively increased the production of isobutyl acetate and isoamyl acetate by reducing the hydrolysis of acetate esters. The decline in the production of higher alcohol by the ATF1 overexpression strains with BAT2 deletion is due to the interaction of ATF1 overexpression and BAT2 deletion. Mutants with varying abilities of producing acetate esters and higher alcohols were developed by genetic engineering. These strains have great potential for industrial application.