Programmable Biosynthesis of Plant-Derived 4'-Deoxyflavone Glycosides by an Unconventional Yeast Consortium.

Previous data established 4'-deoxyflavone glycosides (4'-DFGs) as important pharmaceutical components in the roots of rare medical plants like Scutellaria baicalensis Georgi. Extracting these compounds from plants involves land occupation and is environmentally unfriendly. Therefore, a modular ("plug-and-play") yeast-consortium platform is developed to synthesize diverse 4'-DFGs de novo. By codon-optimizing glycosyltransferase genes from different organisms for Pichia pastoris, six site-specific glycosylation chassis are generated to be capable of biosynthesizing 18 different 4'-DFGs. Cellular factories showed increased 4'-DFG production (up to 18.6-fold) due to strengthened synthesis of UDP-sugar precursors and blocked hydrolysis of endogenous glycosides. Co-culturing upstream flavone-synthesis-module cells with downstream glycoside-transformation-module cells alleviated the toxicity of 4'-deoxyflavones and enabled high-level de novo synthesis of 4'-DFGs. Baicalin is produced at the highest level (1290.0 mg L-1) in a bioreactor by controlling the consortium through carbon-source shifting. These results provide a valuable reference for biosynthesizing plant-derived 4'-DFGs and other glycosides with potential therapeutic applications.

[Biosynthesis of natural products by non-conventional yeasts].

Non-conventional yeasts such as Yarrowia lipolytica, Pichia pastoris, Kluyveromyces marxianus, Rhodosporidium toruloides and Hansenula polymorpha have proven to be efficient cell factories in producing a variety of natural products due to their wide substrate utilization spectrum, strong tolerance to environmental stresses and other merits. With the development of synthetic biology and gene editing technology, metabolic engineering tools and strategies for non-conventional yeasts are expanding. This review introduces the physiological characteristics, tool development and current application of several representative non-conventional yeasts, and summarizes the metabolic engineering strategies commonly used in the improvement of natural products biosynthesis. We also discuss the strengths and weaknesses of non-conventional yeasts as natural products cell factories at current stage, and prospects future research and development trends.

Investigating Fungal Biosynthetic Pathways Using Pichia pastoris as a Heterologous Host.

Fungal natural products have extensive biological activities, and thus have been largely commercialized in the pharmaceutical, agricultural, and food industries. Recently, heterologous expression has become an irreplaceable technique to functionalize fungal biosynthetic gene clusters and synthesize fungal natural products in various chassis organisms. This chapter describes the general method of using Pichia pastoris as a chassis host to investigate fungal biosynthetic pathways.

De Novo Production of Plant 4'-Deoxyflavones Baicalein and Oroxylin A from Ethanol in Crabtree-Negative Yeast.

Baicalein and oroxylin A are well-known medicinal 4'-deoxyflavones found mainly in the roots of traditional medicinal plant Scutellaria baicalensis Georgi. However, extraction from plants is time-consuming, environmentally unfriendly, and insufficient. Although microbial synthesis of flavonoids has been extensively reported, synthesis of downstream modified 4'-deoxyflavones has not, and their yields are extremely low. Here, we reassembled the S. baicalensis 4'-deoxyflavone biosynthetic pathway in a Crabtree-negative yeast, Pichia pastoris, with activity analysis and combinatorial expression of eight biosynthetic genes, allowing production of 4'-deoxyflavones like baicalein, oroxylin A, wogonin, norwogonin, 6-methoxywogonin, and the novel 6-methoxynorwogonin. De novo baicalein synthesis was then achieved by complete pathway assembly. Toxic intermediates highly impaired the cell production capacity; hence, we alleviated cinnamic acid growth inhibition by culturing the cells at near-neutral pH and using alcoholic carbon sources. To achieve pathway balance and improve baicalein and oroxylin A synthesis, we further divided the pathway into five modules. A series of ethanol-induced and constitutive transcriptional amplification devices were constructed to adapt to the modules. This fine-tuning pathway control considerably reduced byproduct and intermediate accumulation and achieved high-level de novo baicalein (401.9 mg/L with a total increase of 1182-fold, the highest titer reported) and oroxylin A (339.5 mg/L, for the first time) production from ethanol. This study provides new strategies for the microbial synthesis of 4'-deoxyflavones and other flavonoids.

Engineered ethanol-driven biosynthetic system for improving production of acetyl-CoA derived drugs in Crabtree-negative yeast.

Many natural drugs use acetyl-CoA as the key biosynthetic precursor. While in eukaryotic chassis host like yeast, efficient biosynthesis of these drugs is often hampered by insufficient acetyl-CoA supply because of its compartmentalized metabolism. Reported acetyl-CoA engineering commonly modifies central carbon metabolism to pull and push acetyl-CoA into cytosol from sugars or redirects biosynthetic pathways in organelles, involving complicated metabolic engineering strategies. We constructed a new biosynthetic system based on a Crabtree-negative yeast, which grew exceptionally on ethanol and assimilated ethanol directly in cytosol to acetyl-CoA (3 steps). A glucose-repressed and ethanol-induced transcriptional signal amplification device (ESAD) with 20-fold signal increase was constructed by rewiring native transcriptional regulation circuits. This made ethanol the sole and fast-growing substrate, acetyl-CoA precursor, and strong biosynthetic pathway inducer simultaneously. The ESAD was used for biosynthesis of a commercial hypolipidemic drug intermediate, monacolin J. A strain producing dihydromonacolin L was firstly constructed and systematically engineered. We further developed a coculture system equipped with this upstream strain and a downstream strain with dihydromonacolin L-to-monacolin J module controlled by a synthetic constitutive transcriptional signal amplification device (CSAD). It produced a high monacolin J titre of 2.2 g/L on ethanol in bioreactor. Engineering glucose-supported and ethanol-repressed fatty acids biosynthesis in the upstream strain contributed more acetyl-CoA for monacolin J and improved its titre to 3.2 g/L, far surpassing other reported productions in yeasts. This study provides a new paradigm for facilitating the high-yield production of acetyl-CoA derived pharmaceuticals and value-added molecules.