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Triterpenoids, one of the most diverse classes of natural products, have been used for centuries as active ingredients in essential oils and Chinese medicines and are of interest for many industrial applications ranging from low-calorie sweeteners to cosmetic ingredients and vaccine adjuvants. However, not only can the extraction from plant material be cumbersome due to low concentrations of the specific triterpenoid, but concerns are also increasing regarding the sustainability of wild plant harvest while meeting market demands. The alternative is to produce triterpenoids with engineered microbes. Here, we present a generally applicable strategy for triterpenoid production in the yeast Saccharomyces cerevisiae based on a modified oxidosqualene cyclase Erg7. The modification reduces the flux into the sterol pathway while increasing the precursor supply for triterpenoid production. The minimally engineered strain was exploited for the exemplary production of the lupane triterpenoids betulin, betulin aldehyde, and betulinic acid at a total titer above 6 g/L, the highest reported so far. To further highlight the chassis concept, squalene, oleanane- and dammarane-type triterpenoids were synthesized to titers at a similar gram scale. We propose the developed baker’s yeast as a host for the thousands of triterpenoid synthesis pathways from plants, reducing the pressure on the natural resources.

Methyl ketones present a group of highly reduced platform chemicals industrially produced from petroleum-derived hydrocarbons. They find applications in the fragrance, flavor, pharmacological, and agrochemical industries, and are further discussed as biodiesel blends. In recent years, intense research has been carried out to achieve sustainable production of these molecules by re-arranging the fatty acid metabolism of various microbes. One challenge in the development of a highly productive microbe is the high demand for reducing power. Here, we engineeredPseudomonas taiwanensisVLB120 for methyl ketone production as this microbe has been shown to sustain exceptionally high NAD(P)H regeneration rates. The implementation of published strategies resulted in 2.1 g Laq-1methyl ketones in fed-batch fermentation. We further increased the production by eliminating competing reactions suggested by metabolic analyses. These efforts resulted in the production of 9.8 g Laq-1methyl ketones (corresponding to 69.3 g Lorg-1in thein situextraction phase) at 53 % of the maximum theoretical yield. This represents a 4-fold improvement in product titer compared to the initial production strain and the highest titer of recombinantly produced methyl ketones reported to date. Accordingly, this study underlines the high potential ofP. taiwanensisVLB120 to produce methyl ketones and emphasizes model-driven metabolic engineering to rationalize and accelerate strain optimization efforts.

Proteins have generally been recognized to constitute the key cellular component in shaping microbial phenotypes. Due to limited cellular resources and space, optimal allocation of proteins is crucial for microbes to facilitate maximum proliferation rates while allowing a flexible response to environmental changes. Regulatory patterns of protein allocation were utilized to account for the condition-dependent proteome in a genome-scale metabolic reconstruction of Escherichia coli by linearly linking mass concentrations of protein sectors and single metabolic enzymes to flux variables. The resulting protein allocation model (PAM) correctly approximates wildtype phenotypes and flux distributions for various substrates, even under data scarcity. Moreover, we showed the ability of the PAM to predict metabolic responses of single gene deletion mutants by additionally assuming growth-limiting, transcriptional restrictions. Thus, we promote the integration of protein allocation constraints into classical constraint-based models to foster their predictive capabilities and application for strain analysis and metabolic engineering purposes.

Frontiers in Bioengineering and Biotechnology 7, 458 (2020). doi:10.3389/fbioe.2019.00458, Published by Frontiers Media, Lausanne