Molecular study on the role of vacuolar transporters in glycyrrhetinic acid production in engineered Saccharomyces cerevisiae.

Glycyrrhetinic acid (GA) is one of the major bioactive components of the leguminous plant, Glycyrrhiza spp. (Chinese licorice). Owing to GA's complicated chemical structure, its production by chemical synthesis is challenging and requires other efficient strategies such as microbial synthesis. Earlier investigations employed numerous approaches to improve GA yield by refining the synthetic pathway and improving the metabolic flux. Nevertheless, the metabolic role of transporters in GA biosynthesis in microbial cell factories has not been studied so far. In this study, we investigated the role of yeast ATP binding cassette (ABC) vacuolar transporters in GA production. Molecular docking of GA and its precursors, ß-Amyrin and 11-oxo-ß-amyrin, was performed with five vacuolar ABC transporters (Bpt1p, Vmr1p, Ybt1p, Ycf1p and Nft1p). Based on docking scores, two top scoring transporters were selected (Bpt1p and Vmr1p) to investigate transporters' functions on GA production via overexpression and knockout experiments in one GA-producing yeast strain (GA166). Results revealed that GA and its precursors exhibited the highest predicted binding affinity towards BPT1 (ΔG = -10.9, -10.6, -10.9 kcal/mol for GA, ß-amyrin and 11-oxo-ß-amyrin, respectively). Experimental results showed that the overexpression of BPT1 and VMR1 restored the intracellular as well as extracellular GA production level under limited nutritional conditions, whereas knockout of BPT1 resulted in a total loss of GA production. These results suggest that the activity of BPT1 is required for GA production in engineered Saccharomyces cerevisiae.

Transporter Engineering in Microbial Cell Factory Boosts Biomanufacturing Capacity.

Microbial cell factories (MCFs) are typical and widely used platforms in biomanufacturing for designing and constructing synthesis pathways of target compounds in microorganisms. In MCFs, transporter engineering is especially significant for improving the biomanufacturing efficiency and capacity through enhancing substrate absorption, promoting intracellular mass transfer of intermediate metabolites, and improving transmembrane export of target products. This review discusses the current methods and strategies of mining and characterizing suitable transporters and presents the cases of transporter engineering in the production of various chemicals in MCFs.

Refining Metabolic Mass Transfer for Efficient Biosynthesis of Plant Natural Products in Yeast.

Plant natural products are important secondary metabolites with several special properties and pharmacological activities, which are widely used in pharmaceutical, food, perfume, cosmetic, and other fields. However, the production of these compounds mainly relies on phytoextraction from natural plants. Because of the low contents in plants, phytoextraction has disadvantages of low production efficiency and severe environmental and ecological problems, restricting its wide applications. Therefore, microbial cell factory, especially yeast cell factory, has become an alternative technology platform for heterologous synthesis of plant natural products. Many approaches and strategies have been developed to construct and engineer the yeast cells for efficient production of plant natural products. Meanwhile, metabolic mass transfer has been proven an important factor to improve the heterologous production. Mass transfer across plasma membrane (trans-plasma membrane mass transfer) and mass transfer within the cell (intracellular mass transfer) are two major forms of metabolic mass transfer in yeast, which can be modified and optimized to improve the production efficiency, reduce the consumption of intermediate, and eliminate the feedback inhibition. This review summarized different strategies of refining metabolic mass transfer process to enhance the production efficiency of yeast cell factory (Figure 1), providing approaches for further study on the synthesis of plant natural products in microbial cell factory.

Compatible solutes adaptive alterations in Arthrobacter simplex during exposure to ethanol, and the effect of trehalose on the stress resistance and biotransformation performance.

Ethanol-tolerant Arthrobacter simplex is desirable since ethanol facilitates hydrophobic substrates dissolution on an industrial scale. Herein, alterations in compatible solutes were investigated under ethanol stress. The results showed that the amount of trehalose and glycerol increased while that of glutamate and proline decreased. The trehalose protectant role was verified and its concentration was positively related to the degree of cell tolerance. otsA, otsB and treS, three trehalose biosynthesis genes in A. simplex, also enhanced Escherichia coli stress tolerance, but the increased tolerance was dependent on the type and level of the stress. A. simplex strains accumulating trehalose showed a higher productivity in systems containing more ethanol and substrate because of better viability. The underlying mechanisms of trehalose were involved in better cell integrity, higher membrane stability, stronger reactive oxygen species scavenging capacity and higher energy level. Therefore, trehalose was a general protectant and the upregulation of its biosynthesis by genetic modification enhanced cell stress tolerance, consequently promoted productivity.

Novel trends for producing plant triterpenoids in yeast.

Triterpenoids possess versatile biological activities including antiviral, anticancer, and hepatoprotective activities. They are widely used in medicine and other health-related fields. However, current production of such compounds relies on plant culture and extraction, which brings about concerns for environmental, ecological, and infield problems. With increasing awareness of environmental sustainability, various microbes have been engineered to produce natural products, in which yeast turned out to be feasible for the heterologous biosynthesis of triterpenoids on account of its inherent advantages such as the robustness, safety, and sufficient precursor supplementation. This review has focused on recent progress regarding the biosynthesis of triterpenoids in yeast. The key enzymes to reconstruct the triterpenoid pathways in yeast, include: oxidosqualene cyclases, cytochrome P450s and UDP-glycosyltransferases are systematically presented. We then discuss recent metabolic engineering strategies and future prospects of protein engineering, pathway compartmentalization, product transportation, and other aspects for triterpenoid production in yeast.

IrrE Improves Organic Solvent Tolerance and Δ1-Dehydrogenation Productivity of Arthrobacter simplex.

During steroid bioconversion, organic solvents are widely used for facilitating hydrophobic substrate dissolution in industry. Thus, strains that tolerate organic solvents are highly desirable. IrrE, a global transcriptional factor, was introduced into Arthrobacter simplex with Δ1-dehydrogenation ability. The results evidenced that IrrE did not affect cell biological traits and biotransformation performance under non-stress conditions. However, the recombinant strain achieved a productivity higher than that of the control strain in systems containing more ethanol and substrate, which coincided with cell viability under ethanol stress, the major stress factor during biotransformation. It also demonstrated that IrrE caused genome-wide transcriptional perturbation, and several defense proteins or systems were linked with higher organic solvent tolerance. IrrE simultaneously enhanced cell resistance to various stresses, and its horizontal impacts showed strain and stress dependence. In conclusion, the introduction of exogenous global regulators is an efficient approach to enhance organic solvent tolerance in steroid-transforming strains, resulting in higher productivity.