Presentation Information
[P04-518]Strategies for utilizing acetyl-CoA around peroxisomes in bioproduction
○Ryosuke Mitsui1, Genki Akai1, Tomokazu Shirai1 (1. RIKEN (Japan))
Keywords:
Saccharomyces cerevisiae,Metabolic engineering,Peroxisome,Carnitine shuttle,Acetyl-CoA
[Purpose]
Many studies have reported that in Saccharomyces cerevisiae, a eukaryotic cell, the production of target compounds increases when enzymes involved in the target pathway are localized within mitochondria and utilize the high concentration of acetyl-CoA there (Avalos et al., 2013; Yee et al., 2019; Hammer et al., 2020). However, constructing metabolic pathways within organelles can lead to the accumulation of non-natural compounds and, in some cases, inhibit cell proliferation (Zhu et al., 2021). Recently, it was demonstrated that expressing carnitine acetyltransferase on the peroxisomal membrane enables the supply of acetyl-CoA from the carnitine shuttle between peroxisomes and mitochondria in the cytosol of S. cerevisiae (Yocum et al., 2022). Here, we attempted several metabolic manipulations using this enzyme display method to increase the production of acetyl-CoA-derived compounds. Specifically, we selected (R)-citramalate as the target compound and co-expressed carnitine acetyltransferase YAT2 and (R)-citramalate synthase cimA3.7 on the peroxisomal membrane of yeast. Next, we evaluated the effects of several gene knockouts to increase acetyl-CoA concentration and final product yield.
[Method]
Gene knockout was performed by removing the gene ORF with the CRISPR-Cas system. Yeast cells were inoculated in 5 mL of SCD+20 μM L-carnitine medium with an OD600 of 0.1 and cultured at 30°C with 180 rpm using a test tube. Culture was collected every 24 hours and used to measure OD600, and the supernatant was used to analyze metabolites using HPLC and GC-MS.
[Results]
Co-displaying of YAT2 and cimA3.7 on the peroxisomal surface increased (R)-citramalate production per OD by 1.2-fold. By knocking out a total of four genes in this strain—the endogenous YAT2 and LEU1 genes, as well as the CIT2 and MLS1 genes encoding enzymes that catalyze citrate and malate synthesis, which consume acetyl-CoA in the glyoxylate cycle—the (R)-citramalate production per OD600 increased 2.7-fold. In contrast, the production of the byproduct malate decreased with each additional gene knockout, and in the quadruple knockout strain, malate production was 0.4 times that of the control strain.
[Consideration]
Displaying enzymes did indeed increase (R)-citramalate acid production per OD600. However, since the subsequent gene knockout had a greater impact on increasing citramalate production, this suggests that inhibiting competing acetyl-CoA-consuming pathways is crucial for our approach. Since malate is a metabolite of the TCA cycle as well as the glyoxylate cycle, small amounts of malate were likely produced even in the quadruple knockout strain.
[Conclusion]
Our research offers a general approach and insights for metabolic engineering in eukaryotic cells, with applications in bio-manufacturing. The method in this study might be even more effective in microbial cells with high fatty acid production capacity, such as oleaginous yeasts.
Many studies have reported that in Saccharomyces cerevisiae, a eukaryotic cell, the production of target compounds increases when enzymes involved in the target pathway are localized within mitochondria and utilize the high concentration of acetyl-CoA there (Avalos et al., 2013; Yee et al., 2019; Hammer et al., 2020). However, constructing metabolic pathways within organelles can lead to the accumulation of non-natural compounds and, in some cases, inhibit cell proliferation (Zhu et al., 2021). Recently, it was demonstrated that expressing carnitine acetyltransferase on the peroxisomal membrane enables the supply of acetyl-CoA from the carnitine shuttle between peroxisomes and mitochondria in the cytosol of S. cerevisiae (Yocum et al., 2022). Here, we attempted several metabolic manipulations using this enzyme display method to increase the production of acetyl-CoA-derived compounds. Specifically, we selected (R)-citramalate as the target compound and co-expressed carnitine acetyltransferase YAT2 and (R)-citramalate synthase cimA3.7 on the peroxisomal membrane of yeast. Next, we evaluated the effects of several gene knockouts to increase acetyl-CoA concentration and final product yield.
[Method]
Gene knockout was performed by removing the gene ORF with the CRISPR-Cas system. Yeast cells were inoculated in 5 mL of SCD+20 μM L-carnitine medium with an OD600 of 0.1 and cultured at 30°C with 180 rpm using a test tube. Culture was collected every 24 hours and used to measure OD600, and the supernatant was used to analyze metabolites using HPLC and GC-MS.
[Results]
Co-displaying of YAT2 and cimA3.7 on the peroxisomal surface increased (R)-citramalate production per OD by 1.2-fold. By knocking out a total of four genes in this strain—the endogenous YAT2 and LEU1 genes, as well as the CIT2 and MLS1 genes encoding enzymes that catalyze citrate and malate synthesis, which consume acetyl-CoA in the glyoxylate cycle—the (R)-citramalate production per OD600 increased 2.7-fold. In contrast, the production of the byproduct malate decreased with each additional gene knockout, and in the quadruple knockout strain, malate production was 0.4 times that of the control strain.
[Consideration]
Displaying enzymes did indeed increase (R)-citramalate acid production per OD600. However, since the subsequent gene knockout had a greater impact on increasing citramalate production, this suggests that inhibiting competing acetyl-CoA-consuming pathways is crucial for our approach. Since malate is a metabolite of the TCA cycle as well as the glyoxylate cycle, small amounts of malate were likely produced even in the quadruple knockout strain.
[Conclusion]
Our research offers a general approach and insights for metabolic engineering in eukaryotic cells, with applications in bio-manufacturing. The method in this study might be even more effective in microbial cells with high fatty acid production capacity, such as oleaginous yeasts.
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