Current progress in production of flavonoids using systems and synthetic biology platforms

Flavonoid is an industrially-important compound due to its high pharmaceutical and cosmeceutical values. However, conventional methods in extracting and synthesizing flavonoids are costly, laborious and not sustainable due to small amount of natural flavonoids, large amounts of chemicals and space used. Biotechnological production of flavonoids represents a viable and sustainable route especially through the use of metabolic engineering strategies in microbial production hosts. In this review, we will highlight recent strategies for the improving the production of flavonoids using synthetic biology approaches in particular the innovative strategies of genetically-encoded biosensors for in vivo metabolite analysis and high-throughput screening methods using fluorescence-activated cell sorting (FACS). Implementation of transcription factor based-biosensor for microbial flavonoid production and integration of systems and synthetic biology approaches for natural product development will also be discussed

Rapid Assembly of yeast expression cassettes for phenylpropanoid biosynthesis in Saccharomyces cerevisiae

Microbial production of natural products using metabolic engineering and synthetic biology approaches often involves the assembly of multiple gene fragments including regulatory elements, especially when using eukaryotes as hosts. Traditional cloning strategy using restriction enzyme digestion and ligation are laborious and inflexible owing to the high number of sequential cloning steps, limited cutting sites and generation of undesired ‘scar’ sequences. In this study, a homology-based isothermal DNA assembly method was carried out for one-step simultaneous assembly of multiple DNA fragments to engineer plant phenylpropanoid biosynthesis in Saccharomyces cerevisiae. Rapid construction of yeast plasmid harboring dual gene expression cassettes was achieved via isothermal assembly of four DNA fragments designed with 20 bp overlapping sequences. The rate-limiting enzyme of phenylpropanoid pathway, cinnamate 4-hydroxylase encoded by C4H gene from Polygonum minus was cloned in tandem with yeast promoter and terminator elements of S. cerevisiae for efficient construction of phenylpropanoid biosynthetic pathway in recombinant yeast. The assembled pAG-CAT (C4H-ADH1t-TEF1p) shuttle plasmid and transformation of S. cerevisiae with the plant C4H gene were confirmed via PCR analysis. Based on these findings, the yeast shuttle plasmid harboring P. minus phenylpropanoid biosynthesis gene was efficiently constructed to be the starting platform for the production of plant natural products in genetically-engineered S. cerevisiae