Natural sweeteners are plant-based proteins with a sweet taste that resembles sugar. Natural sweeteners are non-nutritive but have more nutrients than refined sugar. Biotechnological methods such as fermentation and genetic engineering are used in their production. In addition to being healthier than artificial sweeteners, natural sweeteners offer other health advantages. The bacterium E. coli’s mevalonate (MVA) and intrinsic methylerythritol 4-phosphate (MEP) pathways enable the eukaryotic S. cerevisiae to create isopentenyl diphosphate (IPP) and its isomer, dimethylallyl pyrophosphate (DMAPP), which are required for the manufacturing of plant-based isoprenoids. Both can consequently be used to create isoprenoid sweeteners. S. cerevisiae is regarded as a favorable host because it has an endoplasmic reticulum (ER) that permits it to perform the recombinant production of membrane-localized plant-derived P450s. As previously stated, P450s catalyze multiple steps in the synthesis of all three sweeteners. As a result, these microorganisms and enzymes facilitate the efficient synthesis of sweeteners, hence enhancing their availability. The purity, content, and thermal behavior of these sweeteners are measured via thermal analysis. The biosynthetic and biochemical procedures involved in the synthesis and characterization of naturally occurring sweetening chemicals will be the main topics of this review. We’ll also go over the most current developments in the field of biotechnology.