Utilization of a styrene-derived pathway for 2-phenylethanol production in budding yeast.

2-Phenylethanol (2-PE) is an important flavor ingredient and is widely applied in the fields of food, cosmetics, and pharmaceuticals. Despite that Saccharomyces cerevisiae has the ability to naturally synthesize 2-PE via the Ehrlich pathway, de novo synthesis of 2-PE in high titer still remains a huge challenge. In this study, a non-native styrene degradation pathway was introduced into S. cerevisiae, which represents the first time to demonstrate the functional expression of "styrene-derived" 2-PE synthesis in yeast. Using a host strain engineered with L-phenylalanine (L-Phe) overproduction, the heterologous 2-PE pathway coupled with endogenous Ehrlich pathway produced 233 mg/L 2-PE under shake flasks. Additionally, we further engineered the permease transporters to improve the intracellular L-Phe availability, and further improved the 2-PE titer to 680 mg/L. Taken together, our work represents one of the pioneering reports to explore "styrene-derived" pathway in S. cerevisiae. The synthetic yeast described here might be used as a platform for the future development of next-generation high-yielding 2-PE yeast strains.Key Points• A styrene-derived pathway was established in yeast for 2-phenylethanol productions; membrane-associated styrene oxide isomerase was functional in yeast.• Transporter engineering to improve the L-phenylalanine importation with enhanced 2-phenylethanol productions.

Minimal aromatic aldehyde reduction (MARE) yeast platform for engineering vanillin production.

BACKGROUND: Vanillin represents one of the most widely used flavoring agents in the world. However, microbial synthesis of vanillin is hindered by the host native metabolism that could rapidly degrade vanillin to the byproducts. RESULTS: Here, we report that the industrial workhorse Saccharomyces cerevisiae was engineered by systematic deletion of oxidoreductases to improve the vanillin accumulation. Subsequently, we harnessed the minimal aromatic aldehyde reduction (MARE) yeast platform for de novo synthesis of vanillin from glucose. We investigated multiple coenzyme-A free pathways to improve vanillin production in yeast. The vanillin productivity in yeast was enhanced by multidimensional engineering to optimize the supply of cofactors (NADPH and S-adenosylmethionine) together with metabolic reconfiguration of yeast central metabolism. The final yeast strain with overall 24 genetic modifications produced 365.55 ± 7.42 mg l-1 vanillin in shake-flasks, which represents the best reported vanillin titer from glucose in yeast. CONCLUSIONS: The success of vanillin overproduction in budding yeast showcases the great potential of synthetic biology for the creation of suitable biocatalysts to meet the requirement in industry. Our work lays a foundation for the future implementation of microbial production of aromatic aldehydes in budding yeast.

Multidimensional Optimization of Saccharomyces cerevisiae for Carotenoid Overproduction.

Microbial synthesis of carotenoids is a highly desirable alternative to plant extraction and chemical synthesis. In this study, we investigated multidimensional strategies to improve the carotenoid synthesis in the industrial workhorse of Saccharomyces cerevisiae. First, we rewired the yeast central metabolism by optimizing non-oxidative glycolysis pathway for an improved acetyl-CoA supply. Second, we restricted the consumption of farnesyl pyrophosphate (FPP) by the down-regulation of squalene synthase using the PEST degron. Third, we further explored the human lipid binding/transfer protein saposin B (hSapB)-mediated metabolic sink for an enhanced storage of lipophilic carotenoids. Last, the copper-induced GAL expression system was engineered to function in the yeast-peptone-dextrose medium for an increased biomass accumulation. By combining the abovementioned strategies, the final engineered yeast produced 166.79 ± 10.43 mg/l ß-carotene in shake flasks, which was nearly 5-fold improvement of the parental carotenoid-producing strain. Together, we envision that multidimensional strategies reported here might be applicable to other hosts for the future industrial development of carotenoid synthesis from renewable feedstocks.

Engineering Saccharomyces cerevisiae-based biosensors for copper detection.

Heavy metals, that is Cu(II), are harmful to the environment. There is an increasing demand to develop inexpensive detection methods for heavy metals. Here, we developed a yeast biosensor with reduced-noise and improved signal output for potential on-site copper ion detection. The copper-sensing circuit was achieved by employing a secondary genetic layer to control the galactose-inducible (GAL) system in Saccharomyces cerevisiae. The reciprocal control of the Gal4 activator and Gal80 repressor under copper-responsive promoters resulted in a low-noise and sensitive yeast biosensor for copper ion detection. Furthermore, we developed a betaxanthin-based colorimetric assay, as well as 2-phenylethanol and styrene-based olfactory outputs for the copper ion detection. Notably, our engineered yeast sensor confers a narrow range switch-like behaviour, which can give a 'yes/no' response when coupled with a betaxanthin-based visual phenotype. Taken together, we envision that the design principle established here might be applicable to develop other sensing systems for various chemical detections.

New Set of Yeast Vectors for Shuttle Expression in Escherichia coli.

Promoters that play an essential role in the gene regulation are of particular interest to the synthetic biology communities. Recent advances in high-throughput DNA sequencing have greatly increased the breadth of new genetic parts. The development of promoters with broad host properties could enable rapid phenotyping of genetic constructs in different hosts. In this study, we discovered that the GAL1/10 bidirectional promoter from Saccharomyces cerevisiae could be used for shuttle expression in Escherichia coli. Further investigation revealed that the GAL1/10 bidirectional promoter is subjected to catabolite repression in E. coli. We next constructed a set of Golden-Gate assembly vectors for shuttle expression between S. cerevisiae and E. coli. The utility of shuttle vectors was demonstrated for rapid phenotyping of a multigene pathway for cinnamyl alcohol production. Taken together, our work opens a new avenue for the future development of broad host expression systems between prokaryotic and eukaryotic hosts.

High-Yielding Terpene-Based Biofuel Production in Rhodobacter capsulatus.

Energy crisis and global climate change have driven an increased effort toward biofuel synthesis from renewable feedstocks. Herein, purple nonsulfur photosynthetic bacterium (PNSB) of Rhodobacter capsulatus was explored as a platform for high-titer production of a terpene-based advanced biofuel-bisabolene. A multilevel engineering strategy such as promoter screening, improving the NADPH availability, strengthening the precursor supply, suppressing the side pathways, and introducing a heterologous mevalonate pathway, was used to improve the bisabolene titer in R. capsulatus. The above strategies enabled a 35-fold higher titer of bisabolene than that of the starting strain, reaching 1089.7 mg/L from glucose in a shake flask. The engineered strain produced 9.8 g/L bisabolene with a yield of >0.196 g/g-glucose under the two-phase fed-batch fermentation, which corresponds to >78% of theoretical maximum. Taken together, our work represents one of the pioneering studies to demonstrate PNSB as a promising platform for terpene-based advanced biofuel production.

One-Pot Cascade Biotransformation for Efficient Synthesis of Benzyl Alcohol and Its Analogs.

Benzyl alcohol is a naturally occurring aromatic alcohol and has been widely used in the cosmetics and flavor/fragrance industries. The whole-cell biotransformation for synthesis of benzyl alcohol directly from bio-based L-phenylalanine (L-Phe) was herein explored using an artificial enzyme cascade in Escherichia coli. Benzaldehyde was first produced from L-Phe via four heterologous enzymatic steps that comprises L-amino acid deaminase (LAAD), hydroxymandelate synthase (HmaS), (S)-mandelate dehydrogenase (SMDH) and benzoylformate decarboxylase (BFD). The subsequent reduction of benzaldehyde to benzyl alcohol was achieved by a broad substrate specificity phenylacetaldehyde reductase (PAR) from Solanum lycopersicum. We found the designed enzyme cascade could efficiently convert L-Phe into benzyl alcohol with conversion above 99%. In addition, we also examined L-tyrosine (L-Tyr) and m-fluoro-phenylalanine (m-f-Phe) as substrates, the cascade biotransformation could also efficiently produce p-hydroxybenzyl alcohol and m-fluoro-benzyl alcohol. In summary, the developed biocatalytic pathway has great potential to produce various high-valued fine chemicals.