Presentation Information
[P02-285]Enhanced Lipid Production from Waste-Derived Mixed VFAs by Engineered Yarrowia lipolytica
○Seungbeom Choi1, Sun-Mi Lee1 (1. Korea university (Korea))
Keywords:
Yarrowia lipolytica,Volatile fatty acid,Diacylglycerol acyltransferase,Acetyl-CoA synthetase
[Purpose]Waste-derived volatile fatty acids (VFAs) from anaerobic digestion are promising carbon feedstocks for circular biorefineries. However, the valorization of mixed VFAs remains limited by low conversion efficiency. Here, we evaluated stepwise metabolic engineering of Yarrowia lipolytica for lipid production from mixed VFAs (acetate, butyrate, and hexanoate) to generate microbial lipid feedstocks as sustainable aviation fuel (SAF) precursors and identify trade-offs among lipid accumulation, cell growth, and VFA utilization.
[Method]A mixed VFA medium containing acetate, butyrate, and hexanoate was prepared to mimic waste-derived VFA streams. Y. lipolytica strains were evaluated for growth, VFA utilization, and lipid production in this medium. Y. lipolytica PO1f was used as the parental background. Stepwise engineering included: (i) DGA1 overexpression to enhance triacylglycerol (TAG) synthesis, (ii) PEX10 deletion to assess its effect on lipid accumulation and VFA utilization, and (iii) co-expression of a heterologous acetyl-CoA synthetase (seACS) with DGA1 to reinforce acetyl-CoA supply. Total lipid production, VFA consumption, and lipid profiles were compared among strains.
[Results]Under high-VFA condition, the overexpression of DGA1 increased total lipid production from 0.294 g/L to 0.538 g/L, a 1.83-fold increase relative to the parental PO1f strain. In contrast, PEX10 deletion severely impaired growth and mixed-VFA consumption with acetate, butyrate, and hexanoate remained largely unconsumed, indicating poor conversion efficiency. Furthermore, in a separate experimental set under the same VFA condition, co-expression of seACS with DGA1 further increased total lipid production from 0.632 g/L in the DGA1-overexpressing strain to 0.815 g/L, corresponding to a 1.29-fold increase relative to DGA1 alone. Collectively, these results demonstrate that stepwise metabolic engineering improved lipid production from mixed VFAs, with the highest lipid titer reaching 0.815 g/L.
[Consideration]The result confirm that enhancing TAG formation through DGA1 overexpression consistently improves lipid accumulation from mixed VFAs, In contrast, PEX10 deletion reduced growth and mixed-VFA consumption, highlighting the critical role of functional peroxisomes in the efficient utilization of mixed-acid substrates. The co-expression of seACS with DGA1 further suggests that increasing acetyl-CoA flux can promote lipid accumulation under mixed-VFA conditions.
[Conclusion]Stepwise metabolic engineering of Y. lipolytica demonstrated that improving TAG synthesis and acetyl-CoA supply can significantly increases lipid titers from waste-derived mixed VFA substrates, while peroxisome disruption hinders mixed-VFA utilization and are therefore unsuitable for this conversion context. These findings provide a viable strategy for valorizing mixed VFAs into microbial lipid feedstocks, supporting the development of waste-to-SAF and circular bioeconomy applications.
[Method]A mixed VFA medium containing acetate, butyrate, and hexanoate was prepared to mimic waste-derived VFA streams. Y. lipolytica strains were evaluated for growth, VFA utilization, and lipid production in this medium. Y. lipolytica PO1f was used as the parental background. Stepwise engineering included: (i) DGA1 overexpression to enhance triacylglycerol (TAG) synthesis, (ii) PEX10 deletion to assess its effect on lipid accumulation and VFA utilization, and (iii) co-expression of a heterologous acetyl-CoA synthetase (seACS) with DGA1 to reinforce acetyl-CoA supply. Total lipid production, VFA consumption, and lipid profiles were compared among strains.
[Results]Under high-VFA condition, the overexpression of DGA1 increased total lipid production from 0.294 g/L to 0.538 g/L, a 1.83-fold increase relative to the parental PO1f strain. In contrast, PEX10 deletion severely impaired growth and mixed-VFA consumption with acetate, butyrate, and hexanoate remained largely unconsumed, indicating poor conversion efficiency. Furthermore, in a separate experimental set under the same VFA condition, co-expression of seACS with DGA1 further increased total lipid production from 0.632 g/L in the DGA1-overexpressing strain to 0.815 g/L, corresponding to a 1.29-fold increase relative to DGA1 alone. Collectively, these results demonstrate that stepwise metabolic engineering improved lipid production from mixed VFAs, with the highest lipid titer reaching 0.815 g/L.
[Consideration]The result confirm that enhancing TAG formation through DGA1 overexpression consistently improves lipid accumulation from mixed VFAs, In contrast, PEX10 deletion reduced growth and mixed-VFA consumption, highlighting the critical role of functional peroxisomes in the efficient utilization of mixed-acid substrates. The co-expression of seACS with DGA1 further suggests that increasing acetyl-CoA flux can promote lipid accumulation under mixed-VFA conditions.
[Conclusion]Stepwise metabolic engineering of Y. lipolytica demonstrated that improving TAG synthesis and acetyl-CoA supply can significantly increases lipid titers from waste-derived mixed VFA substrates, while peroxisome disruption hinders mixed-VFA utilization and are therefore unsuitable for this conversion context. These findings provide a viable strategy for valorizing mixed VFAs into microbial lipid feedstocks, supporting the development of waste-to-SAF and circular bioeconomy applications.
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