Systematic Metabolic Engineering Combined with Iterative SCRaMbLE Boost Sweetener Thaumatin Production by Saccharomyces cerevisiae

Abstract

Thaumatin is a natural sweet protein with excellent thermal stability and high commercial value, serving as an ideal sucrose substitute in the food industry. However, its sustainable application has been limited by low natural abundance in plant sources and high extraction costs. Herein, multidimensional engineering of Saccharomyces cerevisiae was employed to significantly enhance thaumatin production. First, systematic optimization of the expression system-including the promoter, terminator, and signal peptide-enabled the engineered strain to achieve a titer of 128.5 mg•L⁻¹.Subsequently systematic metabolic engineering, targeting transcription, translation, secretion, and degradation pathways, increased the titer to 320.0 mg•L⁻¹. To further break through the productivity bottleneck, genome rearrangement was conducted using SCRaMbLE system, yielding a high-producing mutant yYPLV-4, which exhibited a 2.96-fold increase over the parental strain.Integrated multi-omics analysis revealed crucial genetic modifications and metabolic reprogramming events, including enhanced amino acid biosynthesis, reduced ubiquitination and byproduct formation.Guided by these insights, combinatorial engineering of high-yield targets further elevated the titer to 467.7 mg•L⁻¹. Following fermentation optimization, fedbatch fermentation in a 5 L bioreactor yielded a final thaumatin titer of 3.04 g•L⁻¹-the highest level reported to date. This study establishes a new benchmark for microbial thaumatin production and provides an adaptable framework that integrates systematic engineering with SCRaMbLE-based genome remodeling to build efficient heterologous protein-producing cell factories.