Presentation Information
[P01-076]Construction of the Heterologous Biosynthetic Pathway for Grifolic Acid Production in Yarrowia lipolytica
Areum Lee2, ○Suhyun Yang1, Mihyeon Ryu1, Hyeoncheol Francis Son1,2,3,4 (1. School of Biological Sciences and Technology, Chonnam National University (Korea), 2. School of Biological Sciences and Technology, Graduate School Chonnam National University (Korea), 3. Institute of Synthetic Biology for Carbon Neutralization, Chonnam National University (Korea), 4. Institute of Systems Biology and Life Science Informatics, Chonnam National University (Korea))
Keywords:
Grifolic acid (GRA),Yarrowia lipolytica,Natural product synthesis,Metabolic engineering,Heterologous biosynthesis
[Purpose]
Grifolic acid (GRA), originally identified from plants of the Ericaceae family, is a naturally occurring compound with diverse biological and pharmacological activities, including reported antiviral activity against HIV. As a high-value precursor for various bioactive molecules such as daurichromenic acid (DCA), GRA possesses considerable industrial and medicinal potential. However, the plant-based production of GRA is hindered by low yield, complex extraction processes, and high impurity levels, leading to growing interest in developing more efficient and sustainable microbial production systems. GRA biosynthesis proceeds through the prenylation of orsellinic acid (ORA) with farnesyl pyrophosphate (FPP), both of which are derived from acetyl-CoA via the polyketide and mevalonate pathways. Given these metabolic requirements, the oleaginous yeast Yarrowia lipolytica was selected as a heterologous host. This organism is notable for its high acetyl-CoA pool associated with lipid accumulation, an active MVA pathway for FPP biosynthesis, strong metabolic tolerance, efficient heterologous expression, and suitability for industrial-scale fermentation.
[Method]
To establish the GRA biosynthetic pathway in Y. lipolytica, three heterologous genes were introduced. These genes were delivered using two plasmids: two genes involved in ORA biosynthesis were arranged as a single expression cassette linked by a T2A sequence, while the gene responsible for converting ORA and FPP into GRA was introduced on a separate plasmid. After cultivation, culture supernatants and cell pellets were collected at 24 h intervals, and GRA levels in each fraction were analyzed by high-performance liquid chromatography (HPLC).
[Results]
No GRA peak was detected in the wild-type strain, whereas a distinct peak corresponding to GRA was observed in the strain harboring all three genes. Interestingly, GRA was detected in both fractions, with a noticeably higher level in the extracellular fraction, suggesting active secretion. This secretion behavior may facilitate downstream purification and recovery, offering an advantage for future large-scale production.
[Conclusion]
This study demonstrates that Y. lipolytica can serve as a platform for the microbial production of aromatic natural products such as GRA. Future metabolic engineering strategies—such as enhancement of the FPP biosynthetic pathway, optimization of enzyme expression and activity, expansion of the acetyl-CoA pool, and downregulation of competing pathways—are expected to further increase GRA yields. Thus, this study establishes the feasibility of GRA biosynthesis in Y. lipolytica and provides a foundation for developing a microbial production platform for high-value bioactive natural compounds.
Grifolic acid (GRA), originally identified from plants of the Ericaceae family, is a naturally occurring compound with diverse biological and pharmacological activities, including reported antiviral activity against HIV. As a high-value precursor for various bioactive molecules such as daurichromenic acid (DCA), GRA possesses considerable industrial and medicinal potential. However, the plant-based production of GRA is hindered by low yield, complex extraction processes, and high impurity levels, leading to growing interest in developing more efficient and sustainable microbial production systems. GRA biosynthesis proceeds through the prenylation of orsellinic acid (ORA) with farnesyl pyrophosphate (FPP), both of which are derived from acetyl-CoA via the polyketide and mevalonate pathways. Given these metabolic requirements, the oleaginous yeast Yarrowia lipolytica was selected as a heterologous host. This organism is notable for its high acetyl-CoA pool associated with lipid accumulation, an active MVA pathway for FPP biosynthesis, strong metabolic tolerance, efficient heterologous expression, and suitability for industrial-scale fermentation.
[Method]
To establish the GRA biosynthetic pathway in Y. lipolytica, three heterologous genes were introduced. These genes were delivered using two plasmids: two genes involved in ORA biosynthesis were arranged as a single expression cassette linked by a T2A sequence, while the gene responsible for converting ORA and FPP into GRA was introduced on a separate plasmid. After cultivation, culture supernatants and cell pellets were collected at 24 h intervals, and GRA levels in each fraction were analyzed by high-performance liquid chromatography (HPLC).
[Results]
No GRA peak was detected in the wild-type strain, whereas a distinct peak corresponding to GRA was observed in the strain harboring all three genes. Interestingly, GRA was detected in both fractions, with a noticeably higher level in the extracellular fraction, suggesting active secretion. This secretion behavior may facilitate downstream purification and recovery, offering an advantage for future large-scale production.
[Conclusion]
This study demonstrates that Y. lipolytica can serve as a platform for the microbial production of aromatic natural products such as GRA. Future metabolic engineering strategies—such as enhancement of the FPP biosynthetic pathway, optimization of enzyme expression and activity, expansion of the acetyl-CoA pool, and downregulation of competing pathways—are expected to further increase GRA yields. Thus, this study establishes the feasibility of GRA biosynthesis in Y. lipolytica and provides a foundation for developing a microbial production platform for high-value bioactive natural compounds.
Comment
To browse or post comments, you must log in.Log in