Reconstructing curcumin biosynthesis in yeast reveals the implication of caffeoyl-shikimate esterase in phenylpropanoid metabolic flux.

Curcumin is a polyphenolic natural product from the roots of turmeric (Curcuma longa). It has been a popular coloring and flavoring agent in food industries with known health benefits. The conventional phenylpropanoid pathway is known to proceed from phenylalanine via p-coumaroyl-CoA intermediate. Although hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase (HCT) plays a key catalysis in the biosynthesis of phenylpropanoid products at the downstream of p-coumaric acid, a recent discovery of caffeoyl-shikimate esterase (CSE) showed that an alternative pathway exists. Here, the biosynthetic efficiency of the conventional and the alternative pathway in producing feruloyl-CoA was examined using curcumin production in yeast. A novel modular multiplex genome-edit (MMG)-CRISPR platform was developed to facilitate rapid integrations of up to eight genes into the yeast genome in two steps. Using this MMG-CRISPR platform and metabolic engineering strategies, the alternative CSE phenylpropanoid pathway consistently showed higher titers (2-19 folds) of curcumin production than the conventional pathway in engineered yeast strains. In shake flask cultures using a synthetic minimal medium without phenylalanine, the curcumin production titer reached up to 1.5 mg/L, which is three orders of magnitude (∼4800-fold) improvement over non-engineered base strain. This is the first demonstration of de novo curcumin biosynthesis in yeast. Our work shows the critical role of CSE in improving the metabolic flux in yeast towards the phenylpropanoid biosynthetic pathway. In addition, we showcased the convenience and reliability of modular multiplex CRISPR/Cas9 genome editing in constructing complex synthetic pathways in yeast.

Improved protein glycosylation enabled heterologous biosynthesis of monoterpenoid indole alkaloids and their unnatural derivatives in yeast.

With over 3000 reported structures, monoterpenoid indole alkaloids (MIAs) constitute one of the largest alkaloid groups in nature, including the clinically important anticancer drug vinblastine and its semi-synthetic derivatives from Catharanthus roseus (Madagascar's periwinkle). With the elucidation of the complete 28-step biosynthesis for anhydrovinblastine, it is possible to investigate the heterologous production of vinblastine and other medicinal MIAs. In this study, we successfully expressed the flavoenzyme O-acetylstemmadenine oxidase in Saccharomyces cerevisiae (baker's yeast) by signal peptide modification, which is a vinblastine biosynthetic gene that has not been functionally expressed in this system. We also reported the simultaneous integration of ∼18 kb MIA biosynthetic gene cassettes as single copies into four genomic loci of baker's yeast by CRISPR-Cas9, which enabled the biosynthesis of vinblastine precursors catharanthine and tabersonine from the feedstocks secologanin and tryptamine. We further demonstrated the biosynthesis of fluorinated and hydroxylated catharanthine and tabersonine derivatives using our yeasts, which showed that the MIA biosynthesis accommodates unnatural substrates, and the system can be further explored to produce other complex MIAs.

Multiplex Genome Editing in Yeast by CRISPR/Cas9 - A Potent and Agile Tool to Reconstruct Complex Metabolic Pathways.

Numerous important pharmaceuticals and nutraceuticals originate from plant specialized metabolites, most of which are synthesized via complex biosynthetic pathways. The elucidation of these pathways is critical for the applicable uses of these compounds. Although the rapid progress of the omics technology has revolutionized the identification of candidate genes involved in these pathways, the functional characterization of these genes remains a major bottleneck. Baker's yeast (Saccharomyces cerevisiae) has been used as a microbial platform for characterizing newly discovered metabolic genes in plant specialized metabolism. Using yeast for the investigation of numerous plant enzymes is a streamlined process because of yeast's efficient transformation, limited endogenous specialized metabolism, partially sharing its primary metabolism with plants, and its capability of post-translational modification. Despite these advantages, reconstructing complex plant biosynthetic pathways in yeast can be time intensive. Since its discovery, CRISPR/Cas9 has greatly stimulated metabolic engineering in yeast. Yeast is a popular system for genome editing due to its efficient homology-directed repair mechanism, which allows precise integration of heterologous genes into its genome. One practical use of CRISPR/Cas9 in yeast is multiplex genome editing aimed at reconstructing complex metabolic pathways. This system has the capability of integrating multiple genes of interest in a single transformation, simplifying the reconstruction of complex pathways. As plant specialized metabolites usually have complex multigene biosynthetic pathways, the multiplex CRISPR/Cas9 system in yeast is suited well for functional genomics research in plant specialized metabolism. Here, we review the most advanced methods to achieve efficient multiplex CRISPR/Cas9 editing in yeast. We will also discuss how this powerful tool has been applied to benefit the study of plant specialized metabolism.

The yeast platform engineered for synthetic gRNA-landing pads enables multiple gene integrations by a single gRNA/Cas9 system.

Saccharomyces cerevisiae is a versatile microbial platform to build synthetic metabolic pathways for production of diverse chemicals. To expedite the construction of complex metabolic pathways by multiplex CRISPR-Cas9 genome-edit, eight desirable intergenic loci, located adjacent to highly expressed genes selected from top 100 expressers, were identified and fully characterized for three criteria after integrating green fluorescent protein (GFP) gene - CRISPR-mediated GFP integration efficiency, expression competency assessed by levels of GFP fluorescence, and assessing growth rates of GFP integrated strains. Five best performing intergenic loci were selected to build a multiplex CRISPR platform, and a synthetic 23-bp DNA comprised of 20-bp synthetic DNA with a protospacer adjacent motif (PAM) was integrated into the five loci using CRISPR-Cas9 in a sequential manner. This process resulted in five different yeast strains harbouring 1-5 synthetic gRNA-binding sites in their genomes. Using these pre-engineered yeast strains, simultaneous integrations of 2-, 3-, 4-, or 5-genes to the targeted loci were demonstrated with efficiencies from 85% to 98% using beet pigment betalain (3-gene pathway), hygromycin and geneticin resistance markers. Integrations of the multiple, foreign genes in the targeted loci with 100% precision were validated by genotyping. Finally, we further developed the strain to have 6th synthetic gRNA-binding site, and the resulting yeast strain was used to generate a yeast strain producing a sesquiterpene lactone, kauniolide by simultaneous 6-gene integrations. This study demonstrates the effectiveness of a single gRNA-mediated CRISPR platform to build complex metabolic pathways in yeast.

Biodiesel production from Scenedesmus bijuga grown in anaerobically digested food wastewater effluent.

Microalgae, Scenedesmus bijuga, was cultivated in anaerobically digested food wastewater effluent (FWE) to treat the wastewater and produce biodiesel simultaneously. Three different mixing ratios with municipal wastewater were compared for finding out proper dilution ratio in biodiesel production. Of these, 1/20 diluted FWE showed the highest biomass production (1.49 g/L). Lipid content was highest in 1/10 diluted FWE (35.06%), and the lipid productivity showed maximum value in 1/20 diluted FWE (15.59 mg/L/d). Nutrient removal was also measured in the cultivation. FAME compositions were mainly composed of C16-C18 (Over 98.94%) in S. bijuga. In addition, quality of FAMEs was evaluated by Cetane Number (CN) and Bis-allylic Position Equivalent (BAPE).