Developing a novel microbial host and synthetic biology tools for valorizing waste polyethylene terephthalate and lignin-derived compounds

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Genetic Engineering of Crypthecodinium cohnii to Increase Growth and Lipid Accumulation

In this study, we evaluated suitable selected markers and optimized transformation protocols to develop a new genetic transformation methodology for DHA-producing Crypthecodinium cohnii. Additionally, ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), potentially involved in CO2 fixation under autotrophic conditions, was selected as the target for construction of a gene knockdown mutant. Our results show that the constructs were successfully inserted into the C. cohnii chromosome by homologous recombination. Comparative analysis showed that deletion of the RuBisCO gene promoted cell growth and increased the lipid content of C. cohnii under heterotrophic conditions compared with those of the wild-type. The liquid chromatography-mass spectrometry (LC-MS) based metabolomic analysis showed that the metabolites involved in energy metabolism were upregulated, suggesting that the deletion of the RuBisCO gene may contribute to the re-direction of more carbon or energy toward growth and lipid accumulation under heterotrophic conditions

Cellular engineering strategies toward sustainable omega-3 long chain polyunsaturated fatty acids production: State of the art and perspectives

Long-chain polyunsaturated fatty acids (LC-PUFAs) especially ω-3 fatty acids provide significant health benefits for human beings. However, ω-3 LC-PUFAs cannot be synthesized de novo in mammals. Traditionally, ω-3 LC-PUFAs are extracted from marine fish, and their production depends on sea fishing, which has not met ever-increasing global demand. To address the challenges, innovative cellular engineering strategies need to be developed. In nature, many fungi and microalgae are rich in ω-3 LC-PUFAs, representing promising sources of ω-3 LC-PUFAs. The latest progress in developing new cellular engineering strategies toward sustainable ω-3 LC-PUFAs production using fungi and microalga has demonstrated that they can to some extent address the supply shortage. In this review, we critically summarize the recent progress in enhancing the productivity in various ω-3 LC-PUFAs-producing organisms, as well as the latest efforts of biosynthesizing PUFAs in heterogenous biosystems. In addition, we also provide future perspectives in developing genetic toolkits for LC-PUFAs producing microbes so that cut-edging biotechnology such as gene stacking and genome editing can be further applied to increase the productivity of ω-3 LC-PUFAs

13C Metabolic Flux Analysis of Enhanced Lipid Accumulation Modulated by Ethanolamine in Crypthecodinium cohnii

The heterotrophic microalga Crypthecodinium cohnii has attracted considerable attention due to its capability of accumulating lipids with a high fraction of docosahexaenoic acid (DHA). In our previous study, ethanolamine (ETA) was identified as an effective chemical modulator for lipid accumulation in C. cohnii. In this study, to gain a better understanding of the lipid metabolism and mechanism for the positive effects of modulator ETA, metabolic flux analysis was performed using 13C-labeled glucose with and without 1 mM ETA modulator. The analysis of flux distribution showed that with the addition of ETA, flux in glycolysis pathway and citrate pyruvate cycle was strengthened while flux in pentose phosphate pathway was decreased. In addition, flux in TCA cycle was slightly decreased compared with the control without ETA. The enzyme activity of malic enzyme (ME) was significantly increased, suggesting that NADP+-dependent ME might be the major source of NADPH for lipid accumulation. The flux information obtained by this study could be valuable for the further efforts in improving lipid accumulation and DHA production in C. cohnii

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<p>In this study, we evaluated suitable selected markers and optimized transformation protocols to develop a new genetic transformation methodology for DHA-producing Crypthecodinium cohnii. Additionally, ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), potentially involved in CO₂ fixation under autotrophic conditions, was selected as the target for construction of a gene knockdown mutant. Our results show that the constructs were successfully inserted into the C. cohnii chromosome by homologous recombination. Comparative analysis showed that deletion of the RuBisCO gene promoted cell growth and increased the lipid content of C. cohnii under heterotrophic conditions compared with those of the wild-type. The liquid chromatography-mass spectrometry (LC-MS) based metabolomic analysis showed that the metabolites involved in energy metabolism were upregulated, suggesting that the deletion of the RuBisCO gene may contribute to the re-direction of more carbon or energy toward growth and lipid accumulation under heterotrophic conditions.</p

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<p>In this study, we evaluated suitable selected markers and optimized transformation protocols to develop a new genetic transformation methodology for DHA-producing Crypthecodinium cohnii. Additionally, ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), potentially involved in CO₂ fixation under autotrophic conditions, was selected as the target for construction of a gene knockdown mutant. Our results show that the constructs were successfully inserted into the C. cohnii chromosome by homologous recombination. Comparative analysis showed that deletion of the RuBisCO gene promoted cell growth and increased the lipid content of C. cohnii under heterotrophic conditions compared with those of the wild-type. The liquid chromatography-mass spectrometry (LC-MS) based metabolomic analysis showed that the metabolites involved in energy metabolism were upregulated, suggesting that the deletion of the RuBisCO gene may contribute to the re-direction of more carbon or energy toward growth and lipid accumulation under heterotrophic conditions.</p

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<p>In this study, we evaluated suitable selected markers and optimized transformation protocols to develop a new genetic transformation methodology for DHA-producing Crypthecodinium cohnii. Additionally, ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), potentially involved in CO₂ fixation under autotrophic conditions, was selected as the target for construction of a gene knockdown mutant. Our results show that the constructs were successfully inserted into the C. cohnii chromosome by homologous recombination. Comparative analysis showed that deletion of the RuBisCO gene promoted cell growth and increased the lipid content of C. cohnii under heterotrophic conditions compared with those of the wild-type. The liquid chromatography-mass spectrometry (LC-MS) based metabolomic analysis showed that the metabolites involved in energy metabolism were upregulated, suggesting that the deletion of the RuBisCO gene may contribute to the re-direction of more carbon or energy toward growth and lipid accumulation under heterotrophic conditions.</p

Image_6.JPEG

<p>In this study, we evaluated suitable selected markers and optimized transformation protocols to develop a new genetic transformation methodology for DHA-producing Crypthecodinium cohnii. Additionally, ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), potentially involved in CO₂ fixation under autotrophic conditions, was selected as the target for construction of a gene knockdown mutant. Our results show that the constructs were successfully inserted into the C. cohnii chromosome by homologous recombination. Comparative analysis showed that deletion of the RuBisCO gene promoted cell growth and increased the lipid content of C. cohnii under heterotrophic conditions compared with those of the wild-type. The liquid chromatography-mass spectrometry (LC-MS) based metabolomic analysis showed that the metabolites involved in energy metabolism were upregulated, suggesting that the deletion of the RuBisCO gene may contribute to the re-direction of more carbon or energy toward growth and lipid accumulation under heterotrophic conditions.</p

Image_3.JPEG

<p>In this study, we evaluated suitable selected markers and optimized transformation protocols to develop a new genetic transformation methodology for DHA-producing Crypthecodinium cohnii. Additionally, ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), potentially involved in CO₂ fixation under autotrophic conditions, was selected as the target for construction of a gene knockdown mutant. Our results show that the constructs were successfully inserted into the C. cohnii chromosome by homologous recombination. Comparative analysis showed that deletion of the RuBisCO gene promoted cell growth and increased the lipid content of C. cohnii under heterotrophic conditions compared with those of the wild-type. The liquid chromatography-mass spectrometry (LC-MS) based metabolomic analysis showed that the metabolites involved in energy metabolism were upregulated, suggesting that the deletion of the RuBisCO gene may contribute to the re-direction of more carbon or energy toward growth and lipid accumulation under heterotrophic conditions.</p