Impact of heterologous production of carotenoids on <em>S. cerevisiae</em> metabolism

International audienceHeterologous production of high value chemicals like carotenoids has been performed in several yeasts species and recent advances in synthetic biology and biotechnology allowed spectacular yield improvements. Up to 18 mg / g dry cell weight beta-carotene have been recently reported in S. cerevisiae (1) and even 90 mg / g dry cell weight in the lipophilic yeast Y. lipolytica (2), thus rendering these organisms economically viable sources of carotenoids for the cosmetic or pharmaceutical industry. But even though product yields have been improved dramatically, still little is known about the impact of this pathway introduction on yeast overall metabolism. Intermediary metabolites of a designated pathway can have different fates in the cell e.g. accumulation (leading to a pathway bottleneck), degradation (leading to a final product yield decrease) or toxicity (leading to a cell growth defect). To better understand how S. cerevisiae is affected by the heterologous production of carotenoids we constructed several strains producing different levels of geranylgeranyl diphosphate and phytoene, respectively the precursor and the first product of the carotenoids synthesis pathway. By then performing a transcriptomic analysis on these strains, we were able to detect the host metabolic response to different levels of carotenoids production. From the analysis of up- and down-regulated genes, we highlight the central role of acetyl-coA for the cell, as genes responsible for its utilization or liberation are overrepresented. Acetyl-coA is situated at the crossroad of many metabolic routes like the glucose metabolism, the lipid biosynthesis or the cellular respiration and this is also the first brick leading to carotenoid production. To go more into detail, we show that the production of high amounts of phytoene has a main effect on lipid beta-oxidation, protein acylation and pyruvate decarboxylation, all biological processes which are involved in acetyl-coA production and utilization. We are now constructing mutant strains to better understand to which extent modifying these biological processes has an impact on the availability of acetyl-coA and on the carotenoids production yield. Even though this work is restricted to a very specific heterologous pathway in one yeast specie, we believe that this innovative approach can be successfully applied to other pathways and hosts, thus giving researchers new tools to improve heterologous compound production in microorganisms

Impact of heterologous production of carotenoids on <em>S. cerevisiae</em> metabolism

International audienceHeterologous production of high value chemicals like carotenoids has been performed in several yeasts species and recent advances in synthetic biology and biotechnology allowed spectacular yield improvements. Up to 18 mg / g dry cell weight beta-carotene have been recently reported in S. cerevisiae (1) and even 90 mg / g dry cell weight in the lipophilic yeast Y. lipolytica (2), thus rendering these organisms economically viable sources of carotenoids for the cosmetic or pharmaceutical industry. But even though product yields have been improved dramatically, still little is known about the impact of this pathway introduction on yeast overall metabolism. Intermediary metabolites of a designated pathway can have different fates in the cell e.g. accumulation (leading to a pathway bottleneck), degradation (leading to a final product yield decrease) or toxicity (leading to a cell growth defect). To better understand how S. cerevisiae is affected by the heterologous production of carotenoids we constructed several strains producing different levels of geranylgeranyl diphosphate and phytoene, respectively the precursor and the first product of the carotenoids synthesis pathway. By then performing a transcriptomic analysis on these strains, we were able to detect the host metabolic response to different levels of carotenoids production. From the analysis of up- and down-regulated genes, we highlight the central role of acetyl-coA for the cell, as genes responsible for its utilization or liberation are overrepresented. Acetyl-coA is situated at the crossroad of many metabolic routes like the glucose metabolism, the lipid biosynthesis or the cellular respiration and this is also the first brick leading to carotenoid production. To go more into detail, we show that the production of high amounts of phytoene has a main effect on lipid beta-oxidation, protein acylation and pyruvate decarboxylation, all biological processes which are involved in acetyl-coA production and utilization. We are now constructing mutant strains to better understand to which extent modifying these biological processes has an impact on the availability of acetyl-coA and on the carotenoids production yield. Even though this work is restricted to a very specific heterologous pathway in one yeast specie, we believe that this innovative approach can be successfully applied to other pathways and hosts, thus giving researchers new tools to improve heterologous compound production in microorganisms

Impact of heterologous production of carotenoids on S. cerevisiae metabolism

Heterologous production of high value chemicals like carotenoids has been performed in several yeasts species and recent advances in synthetic biology and biotechnology allowed spectacular yield improvements. Up to 18 mg / g dry cell weight beta-carotene have been recently reported in S. cerevisiae (1) and even 90 mg / g dry cell weight in the lipophilic yeast Y. lipolytica (2), thus rendering these organisms economically viable sources of carotenoids for the cosmetic or pharmaceutical industry. But even though product yields have been improved dramatically, still little is known about the impact of this pathway introduction on yeast overall metabolism. Intermediary metabolites of a designated pathway can have different fates in the cell e.g. accumulation (leading to a pathway bottleneck), degradation (leading to a final product yield decrease) or toxicity (leading to a cell growth defect). To better understand how S. cerevisiae is affected by the heterologous production of carotenoids we constructed several strains producing different levels of geranylgeranyl diphosphate and phytoene, respectively the precursor and the first product of the carotenoids synthesis pathway. By then performing a transcriptomic analysis on these strains, we were able to detect the host metabolic response to different levels of carotenoids production. From the analysis of up- and down-regulated genes, we highlight the central role of acetyl-coA for the cell, as genes responsible for its utilization or liberation are overrepresented. Acetyl-coA is situated at the crossroad of many metabolic routes like the glucose metabolism, the lipid biosynthesis or the cellular respiration and this is also the first brick leading to carotenoid production. To go more into detail, we show that the production of high amounts of phytoene has a main effect on lipid beta-oxidation, protein acylation and pyruvate decarboxylation, all biological processes which are involved in acetyl-coA production and utilization. We are now constructing mutant strains to better understand to which extent modifying these biological processes has an impact on the availability of acetyl-coA and on the carotenoids production yield. Even though this work is restricted to a very specific heterologous pathway in one yeast specie, we believe that this innovative approach can be successfully applied to other pathways and hosts, thus giving researchers new tools to improve heterologous compound production in microorganisms

Optogenetic control of beta-carotene bioproduction in yeast across multiple lab-scales

ABSTRACT Optogenetics arises as a valuable tool to precisely control genetic circuits in microbial cell factories. Light control holds the promise of optimizing bioproduction methods and maximize yields, but its implementation at different steps of the strain development process and at different culture scales remains challenging. In this study, we aim to control beta-carotene bioproduction using optogenetics in Saccharomyces cerevisiae and investigate how its performance translates across culture scales. We built four lab-scale illumination devices, each handling different culture volumes, and each having specific illumination characteristics and cultivating conditions. We evaluated optogenetic activation and beta-carotene production across devices and optimized them both independently. Then, we combined optogenetic induction and beta-carotene production to make a light-inducible beta-carotene producer strain. This was achieved by placing the transcription of the bifunctional lycopene cyclase / phytoene synthase CrtYB under the control of the pC120 optogenetic promoter regulated by the EL222-VP16 light-activated transcription factor, while other carotenogenic enzymes (CrtI, CrtE, tHMG) were expressed constitutively. We show that illumination, culture volume and shaking impact differently optogenetic activation and beta-carotene production across devices. This enabled us to determine the best culture conditions to maximize light-induced beta-carotene production in each of the devices, reaching a content of up to 880 μg/gCDW. Our study exemplifies the stakes of scaling up optogenetics in devices of different lab scales and sheds light on the interplays and potential conflicts between optogenetic control and metabolic pathway efficiency. As a general principle, we propose that it is important to first optimize both components of the system independently, before combining them into optogenetic producing strains to avoid extensive troubleshooting. We anticipate that our results can help designing both strains and devices that could eventually lead to larger scale systems in an effort to bring optogenetics to the industrial scale

Protein acetylation affects acetate metabolism, motility and acid stress response in Escherichia coli

Although protein acetylation is widely observed, it has been associated with few specific regulatory functions making it poorly understood. To interrogate its functionality, we analyzed the acetylome in Escherichia coli knockout mutants of cobB, the only known sirtuin-like deacetylase, and patZ, the best-known protein acetyltransferase. For four growth conditions, more than 2,000 unique acetylated peptides, belonging to 809 proteins, were identified and differentially quantified. Nearly 65%of these proteins are related to metabolism. The global activity of CobB contributes to the deacetylation of a large number of substrates and has a major impact on physiology. Apart from the regulation of acetyl-CoA synthetase, we found that CobB-controlled acetylation of isocitrate lyase contributes to the fine-tuning of the glyoxylate shunt. Acetylation of the transcription factor RcsB prevents DNA binding, activating flagella biosynthesis and motility, and increases acid stress susceptibility. Surprisingly, deletion of patZ increased acetylation in acetate cultures, which suggests that it regulates the levels of acetylating agents. The results presented offer new insights into functional roles of protein acetylation in metabolic fitness and global cell regulation