Cytosolic acetyl-CoA Research Articles

A modular pathway engineering strategy for the high-level production of \u03b2-ionone in Yarrowia lipolytica

BackgroundThe GRAS and oleaginous yeast Yarrowia lipolytica (Y. lipolytica) is an attractive cell factory for the production of chemicals and biofuels. The production of many natural products of commercial interest have been investigated in this cell factory by introducing heterologous biosynthetic pathways and by modifying the endogenous pathways. However, since natural products anabolism involves long pathways and complex regulation, re-channelling carbon into the product of target compounds is still a cumbersome work, and often resulting in low production performance.ResultsIn this work, the carotenogenic genes contained carB and bi-functional carRP from Mucor circinelloides and carotenoid cleavage dioxygenase 1 (CCD1) from Petunia hybrida were introduced to Y. lipolytica and led to the low production of β-ionone of 3.5 mg/L. To further improve the β-ionone synthesis, we implemented a modular engineering strategy for the construction and optimization of a biosynthetic pathway for the overproduction of β-ionone in Y. lipolytica. The strategy involved the enhancement of the cytosolic acetyl-CoA supply and the increase of MVA pathway flux, yielding a β-ionone titer of 358 mg/L in shake-flask fermentation and approximately 1 g/L (~ 280-fold higher than the baseline strain) in fed-batch fermentation.ConclusionsAn efficient β-ionone producing GRAS Y. lipolytica platform was constructed by combining integrated overexpressed of heterologous and native genes. A modular engineering strategy involved the optimization pathway and fermentation condition was investigated in the engineered strain and the highest β-ionone titer reported to date by a cell factory was achieved. This effective strategy can be adapted to enhance the biosynthesis of other terpenoids in Y. lipolytica.

Advanced Strategies for Production of Natural Products in Yeast.

Natural products account for more than 50% of all small-molecule pharmaceutical agents currently in clinical use. However, low availability often becomes problematic when a bioactive natural product is promising to become a pharmaceutical or leading compound. Advances in synthetic biology and metabolic engineering provide a feasible solution for sustainable supply of these compounds. In this review, we have summarized current progress in engineering yeast cell factories for production of natural products, including terpenoids, alkaloids, and phenylpropanoids. We then discuss advanced strategies in metabolic engineering at three different dimensions, including point, line, and plane (corresponding to the individual enzymes and cofactors, metabolic pathways, and the global cellular network). In particular, we comprehensively discuss how to engineer cofactor biosynthesis for enhancing the biosynthesis efficiency, other than the enzyme activity. Finally, current challenges and perspective are also discussed for future engineering direction.

Application of Stable Isotope Tracing to Elucidate Metabolic Dynamics During Yarrowia lipolytica α-Ionone Fermentation.

SummaryTargeted metabolite analysis in combination with 13C-tracing is a convenient strategy to determine pathway activity in biological systems; however, metabolite analysis is limited by challenges in separating and detecting pathway intermediates with current chromatographic methods. Here, a hydrophilic interaction chromatography tandem mass spectrometry approach was developed for improved metabolite separation, isotopologue analysis, and quantification. The physiological responses of a Yarrowia lipolytica strain engineered to produce ∼400 mg/L α-ionone and temporal changes in metabolism were quantified (e.g., mevalonate secretion, then uptake) indicating bottleneck shifts in the engineered pathway over the course of fermentation. Dynamic labeling results indicated limited tricarboxylic acid cycle label incorporation and, combined with a measurable ATP shortage during the high ionone production phase, suggested that electron transport and oxidative phosphorylation may limit energy supply and strain performance. The results provide insights into terpenoid pathway metabolic dynamics of non-model yeasts and offer guidelines for sensor development and modular engineering.

Abstract C078: Substituted oxindoles as AMP-activated protein kinase (AMPK) inhibitors and their evaluation in models of leukemia

Abstract AMP-Activated protein kinase (AMPK) acts as a central metabolic sensor at the interface of metabolic and signaling networks. Activated AMPK promotes multiple catabolic processes to generate ATP, such as glucose uptake, glycolysis, fatty acid uptake and oxidation, and mitochondrial biogenesis. In addition, AMPK activation suppresses the cell cycle, and anabolic processes such as the mammalian Target of Rapamycin Complex 1 (mTORC1)-dependent protein synthesis and fatty acid biosynthesis via inactivating phosphorylation of cytosolic acetyl-CoA carboxylase 1 (ACC1) and mitochondrial ACC2. The activation of AMPK under conditions of energetic stress is modulated by an allosteric mechanism where the depletion of ATP promotes AMP binding at the γ subunit of AMPK with a subsequent conformational change in the catalytic α subunit. In the context of cancer AMPK has been reported to promote tumor survival and progression under hypoxia and the maintenance of cancer stem cells (CSCs). A major challenge to interrogating the therapeutic potential of targeting AMPK in cancer is the lack of potent and selective small molecule inhibitors. Compound C has been widely used as an AMPK inhibitor, but it lacks potency and has a poor selectivity profile. The multi-kinase inhibitor, sunitinib, has demonstrated potent nanomolar inhibition of AMPK activity, but has broad-spectrum activity across the kinome. Although sunitinib targets multiple kinases, its nanomolar potency for AMPK inhibition and large scope for chemical substitution on the core oxindole ring make it an attractive lead for AMPK inhibitor development. We have used a computational model of sunitinib docked into the ATP-binding site of the α subunit of AMPK to design and synthesize several series of oxindoles to examine structural modifications to improve AMPK inhibition and selectivity. These candidate inhibitors were evaluated against the activity of the α1 and α2 isoforms of AMPK using the TR-FRET assay and cell engagement was determined by ELISA, measuring p-ACC(Ser79), in the chronic myeloid leukemia K562 cell line. Our assays identified two novel, potent oxindole-based AMPK inhibitors that hadsignificantly reduced binding affinity when compared with sunitinib against members of the receptor tyrosine kinase (RTK) family, including VEGFR1, VEGFR2 and CSF1R, as well as BTK, a member of the B cell receptor (BCR) signaling pathway. Interestingly, cellular AMPK inhibition did not impact cell viability or result in cytotoxicity in K562 cells and may only sensitize these cells to chemotherapy. AMPK inhibition may be a more effective strategy to eliminate cancer cells in hypoxic microenvironments or against certain cell types, such as CSCs, that may be more reliant on AMPK for survival. Our current studies are designed to examine our AMPK inhibitors as single agents and in combination therapy in cancer cells grown under hypoxia and in CSC populations in order to eliminate these drug-resistant cancer cell populations with an aim to prevent cancer recurrence. In summary, we have developed two novel, potent AMPK inhibitors that were designed to interact with the DFG motif in the ATP-binding site of the α subunit of AMPK that are undergoing evaluation in advanced models of leukemia. Citation Format: Christopher J Matheson, Kimberly A Casalvieri, Donald S Backos, Craig T Jordan, Philip Reigan. Substituted oxindoles as AMP-activated protein kinase (AMPK) inhibitors and their evaluation in models of leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C078. doi:10.1158/1535-7163.TARG-19-C078

Engineering cytoplasmic acetyl-CoA synthesis decouples lipid production from nitrogen starvation in the oleaginous yeast Rhodosporidium azoricum

BackgroundOleaginous yeasts are able to accumulate very high levels of neutral lipids especially under condition of excess of carbon and nitrogen limitation (medium with high C/N ratio). This makes necessary the use of two-steps processes in order to achieve high level of biomass and lipid. To simplify the process, the decoupling of lipid synthesis from nitrogen starvation, by establishing a cytosolic acetyl-CoA formation pathway alternative to the one catalysed by ATP-citrate lyase, can be useful.ResultsIn this work, we introduced a new cytoplasmic route for acetyl-CoA (AcCoA) formation in Rhodosporidium azoricum by overexpressing genes encoding for homologous phosphoketolase (Xfpk) and heterologous phosphotransacetylase (Pta). The engineered strain PTAPK4 exhibits higher lipid content and produces higher lipid concentration than the wild type strain when it was cultivated in media containing different C/N ratios. In a bioreactor process performed on glucose/xylose mixture, to simulate an industrial process for lipid production from lignocellulosic materials, we obtained an increase of 89% in final lipid concentration by the engineered strain in comparison to the wild type. This indicates that the transformed strain can produce higher cellular biomass with a high lipid content than the wild type. The transformed strain furthermore evidenced the advantage over the wild type in performing this process, being the lipid yields 0.13 and 0.05, respectively.ConclusionOur results show that the overexpression of homologous Xfpk and heterologous Pta activities in R. azoricum creates a new cytosolic AcCoA supply that decouples lipid production from nitrogen starvation. This metabolic modification allows improving lipid production in cultural conditions that can be suitable for the development of industrial bioprocesses using lignocellulosic hydrolysates.

Metabolic engineering with ATP-citrate lyase and nitrogen source supplementation improves itaconic acid production in Aspergillus niger

BackgroundBio-based production of organic acids promises to be an attractive alternative for the chemicals industry to substitute petrochemicals as building-block chemicals. In recent years, itaconic acid (IA, methylenesuccinic acid) has been established as a sustainable building-block chemical for the manufacture of various products such as synthetic resins, coatings, and biofuels. The natural IA producer Aspergillus terreus is currently used for industrial IA production; however, the filamentous fungus Aspergillus niger has been suggested to be a more suitable host for this purpose. In our previous report, we communicated the overexpression of a putative cytosolic citrate synthase citB in an A. niger strain carrying the full IA biosynthesis gene cluster from A. terreus, which resulted in the highest final titer reported for A. niger (26.2 g/L IA). In this research, we have attempted to improve this pathway by increasing the cytosolic acetyl-CoA pool. Additionally, we have also performed fermentation optimization by varying the nitrogen source and concentration.ResultsTo increase the cytosolic acetyl-CoA pool, we have overexpressed genes acl1 and acl2 that together encode for ATP-citrate lyase (ACL). Metabolic engineering of ACL resulted in improved IA production through an apparent increase in glycolytic flux. Strains that overexpress acl12 show an increased yield, titer and productivity in comparison with parental strain CitB#99. Furthermore, IA fermentation conditions were improved by nitrogen supplementation, which resulted in alkalization of the medium and thereby reducing IA-induced weak-acid stress. In turn, the alkalizing effect of nitrogen supplementation enabled an elongated idiophase and allowed final titers up to 42.7 g/L to be reached at a productivity of 0.18 g/L/h and yield of 0.26 g/g in 10-L bioreactors.ConclusionUltimately, this study shows that metabolic engineering of ACL in our rewired IA biosynthesis pathway leads to improved IA production in A. niger due to an increase in glycolytic flux. Furthermore, IA fermentation conditions were improved by nitrogen supplementation that alleviates IA induced weak-acid stress and extends the idiophase.

Earlier Degraded Tapetum1 (EDT1) Encodes an ATP-Citrate Lyase Required for Tapetum Programmed Cell Death.

In flowering plants, the tapetum cells in anthers undergo programmed cell death (PCD) at the late meiotic stage, providing nutrients for further development of microspores, including the formation of the pollen wall. However, the molecular basis of tapetum PCD remains elusive. Here we report a tapetum PCD-related mutant in rice (Oryza sativa), earlier degraded tapetum 1 (edt1), that shows complete pollen abortion associated with earlier-than-programmed tapetum cell death. EDT1 encodes a subunit of ATP-citrate lyase (ACL), and is specifically expressed in the tapetum of anthers. EDT1 localized in both the nucleus and the cytoplasm as observed in rice protoplast transient assays. We demonstrated that the A and B subunits of ACL interacted with each other and might function as a heteromultimer in the cytoplasm. EDT1 catalyzes the critical steps in cytosolic acetyl-CoA synthesis. Our data indicated a decrease in ATP level, energy charge, and fatty acid content in mutant edt1 anthers. In addition, the genes encoding secretory proteases or lipid transporters, and the transcription factors known to regulate PCD, were downregulated. Our results demonstrate that the timing of tapetum PCD must be tightly regulated for successful pollen development, and that EDT1 is involved in the tapetum PCD process. This study furthers our understanding of the molecular basis of pollen fertility and fecundity in rice and may also be relevant to other flowering plants.

Cytosolic Acetyl-CoA Generated by ATP-Citrate Lyase Is Essential for Acetylation of Cell Wall Polysaccharides.

Plant cell wall polysaccharides, including xylan, glucomannan, xyloglucan and pectin, are often acetylated. Although a number of acetyltransferases responsible for the acetylation of some of these polysaccharides have been biochemically characterized, little is known about the source of acetyl donors and how acetyl donors are translocated into the Golgi, where these polysaccharides are synthesized. In this report, we investigated roles of ATP-citrate lyase (ACL) that generates cytosolic acetyl-CoA in cell wall polysaccharide acetylation and effects of simultaneous mutations of four Reduced Wall Acetylation (RWA) genes on acetyl-CoA transport into the Golgi in Arabidopsis thaliana. Expression analyses of genes involved in the generation of acetyl-CoA in different subcellular compartments showed that the expression of several ACL genes responsible for cytosolic acetyl-CoA synthesis was elevated in interfascicular fiber cells and induced by secondary wall-associated transcriptional activators. Simultaneous downregulation of the expression of ACL genes was demonstrated to result in a substantial decrease in the degree of xylan acetylation and a severe alteration in secondary wall structure in xylem vessels. In addition, the degree of acetylation of other cell wall polysaccharides, including glucomannan, xyloglucan and pectin, was also reduced. Moreover, Golgi-enriched membrane vesicles isolated from the rwa1/2/3/4 quadruple mutant were found to exhibit a drastic reduction in acetyl-CoA transport activity compared with the wild type. These findings indicate that cytosolic acetyl-CoA generated by ACL is essential for cell wall polysaccharide acetylation and RWAs are required for its transport from the cytosol into the Golgi.

TSC1/mTOR-controlled metabolic-epigenetic cross talk underpins DC control of CD8+ T-cell homeostasis.

Dendritic cells (DCs) play pivotal roles in T-cell homeostasis and activation, and metabolic programing has been recently linked to DC development and function. However, the metabolic underpinnings corresponding to distinct DC functions remain largely unresolved. Here, we demonstrate a special metabolic–epigenetic coupling mechanism orchestrated by tuberous sclerosis complex subunit 1 (TSC1)-mechanistic target of rapamycin (mTOR) for homeostatic DC function. Specific ablation of Tsc1 in the DC compartment (Tsc1DC-KO) largely preserved DC development but led to pronounced reduction in naïve and memory–phenotype cluster of differentiation (CD)8+ T cells, a defect fully rescued by concomitant ablation of mTor or regulatory associated protein of MTOR, complex 1 (Rptor) in DCs. Moreover, Tsc1DC-KO mice were unable to launch efficient antigen-specific CD8+ T effector responses required for containing Listeria monocytogenes and B16 melanomas. Mechanistically, our data suggest that the steady-state DCs tend to tune down de novo fatty acid synthesis and divert acetyl-coenzyme A (acetyl-CoA) for histone acetylation, a process critically controlled by TSC1-mTOR. Correspondingly, TSC1 deficiency elevated acetyl-CoA carboxylase 1 (ACC1) expression and fatty acid synthesis, leading to impaired epigenetic imprinting on selective genes such as major histocompatibility complex (MHC)-I and interleukin (IL)-7. Remarkably, tempering ACC1 activity was able to divert cytosolic acetyl-CoA for histone acetylation and restore the gene expression program compromised by TSC1 deficiency. Taken together, our results uncover a crucial role for TSC1-mTOR in metabolic programing of the homeostatic DCs for T-cell homeostasis and implicate metabolic-coupled epigenetic imprinting as a paradigm for DC specification.

Macrophage Atp-Citrate Lyase (Acly) Deficiency Affects Cellular Cholesterol Homeostasis And Stabilizes Atherosclerotic Lesions

Background and Aims: ATP-citrate lyase (Acly) is localized in the cytosol and converts citrate in to acetyl-CoA. Cytosolic acetyl-CoA synthesized by Acly serves several metabolic pathways, including the biosynthesis of cholesterol and fatty acids. Since expression of Acly is blunted in macrophage foam cells, which were found to be less inflammatory, we investigated the role of Acly in macrophage activation and atherosclerosis.

Abstract 792: Acetate production from glucose and coupling to mitochondrial metabolism in mammals

Abstract Background: In conditions of hyperactive cellular metabolism, excessive cellular nutrient uptake results in incomplete metabolism and excretion of intermediates. These intermediates may serve as unconventional fuel sources satisfy metabolic demands during nutrient scarcity. Interestingly, acetate metabolism provides a parallel pathway for acetyl-CoA production and allows for protein acetylation and lipogenesis independent of citrate conversion to acetyl-CoA. This pathway is important in tumorigenesis, immune alertness, neural plasticity, and other diverse contexts but the origin of acetate has been unclear. Thus, we have conducted a re-evaluation of endogenous acetate generation and the biological relevance. Method: Cancer cells were cultured in RPMI medium with 13C labelled nutrients in the presence of 18O2. Mouse models of soft tissue sarcoma were generated in a mixed 129/SVJae and C57BL/6 background. A jugular vein catheter was surgically implanted and exteriorized via a vascular access port, which allows infusion of [13C6]-glucose via the venous catheter. Acetate in medium and blood is quantified using [2H3] labelled acetate as the standard after 2-hydrazinoquinoline (HQ) derivatization, and other polar metabolites were directly analyzed after cold methanol extraction. All metabolites were measured using liquid chromatography coupled with high resolution mass spectrometer. Results: By employing multiple-isotope tracing technology, quantitative proteomics, and mouse genetics tools, we demonstrated that acetate is quantitatively generated from pyruvate, the end product of glycolysis and key node in central carbon metabolism in cancer cells and tumor. One reaction mechanism found to generate acetate occur through altered enzyme activity of thiamine-dependent keto acid dehydrogenases, which transforms their activity to keto acid decarboxylases. The other reaction mechanism to generate acetate occur by reaction with reactive oxygen species (ROS), a finding which potentially links this pathway to numerous physiological and pathophysiological processes. Thiamine starvation and the addition of exogeneous ROS greatly stimulated the ROS contribution to acetate production, which can be used to replenish intracellular acetyl groups. Thus, increased acetate production and release could potentially favor the neighboring cells deficient in cytosolic acetyl-CoA, as demonstrated by co-culturing ACLY KO cells with HCT116 cells. Conclusion: We have not only provided direct evidence that acetate arises from endogenous metabolism of glucose in mammalian cells, but also identified the regulatory mechanisms, which involve ROS and mitochondrial functions. Note: This abstract was not presented at the meeting. Citation Format: Xiaojing Liu, Daniel E. Cooper, Ahmad A. Cluntun, Marc O. Warmoes, Steven Zhao, Michael A. Reid, Juan Liu, Peder J. Lund, Mariana Lopes, Benjamin A. Garcia, Kathryn E. Wellen, David G. Kirsch, Jason W. Locasale. Acetate production from glucose and coupling to mitochondrial metabolism in mammals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 792.

Putting Stem Cells on a Low-Fat Diet Switches Their Pluripotent State.

Previous work demonstrating the importance of stem cell metabolism in regulating development ignited a debate on how intrinsic versus extrinsic conditions influence metabolic programming of epigenetics. In this issue of Cell Stem Cell, Cornacchia etal. show that exogenous lipids regulate an acetyl-CoA-dependent switch to influence the intrinsic pluripotent state of hPSCs.

TSC1-mTOR-controlled metabolic-epigenetic crosstalk underlies DC control of CD8+ T cell homeostasis

Abstract Dendritic cells (DCs) play pivotal roles in T cell homeostasis and activation, and metabolic programing has been linked to DC development and function recently. However, the metabolic underpinnings corresponding to distinct DC functions remain largely unresolved. Here we demonstrate a special metabolic-epigenetic coupling mechanism orchestrated by TSC1-mTOR for the homeostatic DC function. Specific ablation of TSC1 in the DC compartment (TSC1DC-KO) largely preserved DC development, but led to pronounced reduction in naïve and memory-phenotype CD8+ T cells, a defect fully rescued by the simultaneous deletion of mTOR or Raptor in DCs. Moreover, TSC1DC-KO mice were unable to launch efficient antigen-specific CD8+ T effector responses required for containing Listeria monocytogenes and B16 melanomas. Mechanistically, our data suggest that steady-state DCs tune down de novo fatty acid synthesis through TSC1 and divert acetyl-CoA for histone acetylation. Correspondingly, TSC1-deficiency elevated ACC1 expression and fatty acid synthesis in the steady-state DCs, leading to impaired epigenetic imprinting on selective genes such as MHC-I and IL-7. Remarkably, tempering ACC1 activity was able to avert cytosolic acetyl-CoA for histone acetylation and restoration of the gene expression program compromised by TSC1-deficiency. Taken together, our results uncover a crucial role for TSC1-mTOR in metabolic programing the homeostatic DCs for T cell homeostasis, and implicate metabolic-coupled epigenetic imprinting as a paradigm for DC specification.

Mitochondrial Citrate Transporters CtpA and YhmA Are Required for Extracellular Citric Acid Accumulation and Contribute to Cytosolic Acetyl Coenzyme A Generation in Aspergillus luchuensis mut. kawachii.

Aspergillus luchuensis mut. kawachii (A. kawachii) produces a large amount of citric acid during the process of fermenting shochu, a traditional Japanese distilled spirit. In this study, we characterized A. kawachii CtpA and YhmA, which are homologous to the yeast Saccharomyces cerevisiae mitochondrial citrate transporters Ctp1 and Yhm2, respectively. CtpA and YhmA were purified from A. kawachii and reconstituted into liposomes. The proteoliposomes exhibited only counterexchange transport activity; CtpA transported citrate using countersubstrates, especially cis-aconitate and malate, whereas YhmA transported citrate using a wider variety of countersubstrates, including citrate, 2-oxoglutarate, malate, cis-aconitate, and succinate. Disruption of ctpA and yhmA caused deficient hyphal growth and conidium formation with reduced mycelial weight-normalized citrate production. Because we could not obtain a ΔctpA ΔyhmA strain, we constructed an S-tagged ctpA (ctpA-S) conditional expression strain in the ΔyhmA background using the Tet-On promoter system. Knockdown of ctpA-S in ΔyhmA resulted in a severe growth defect on minimal medium with significantly reduced acetyl coenzyme A (acetyl-CoA) and lysine levels, indicating that double disruption of ctpA and yhmA leads to synthetic lethality; however, we subsequently found that the severe growth defect was relieved by addition of acetate or lysine, which could remedy the acetyl-CoA level. Our results indicate that CtpA and YhmA are mitochondrial citrate transporters involved in citric acid production and that transport of citrate from mitochondria to the cytosol plays an important role in acetyl-CoA biogenesis in A. kawachiiIMPORTANCE Citrate transport is believed to play a significant role in citrate production by filamentous fungi; however, details of the process remain unclear. This study characterized two citrate transporters from Aspergillus luchuensis mut. kawachii Biochemical and gene disruption analyses showed that CtpA and YhmA are mitochondrial citrate transporters required for normal hyphal growth, conidium formation, cytosolic acetyl-CoA synthesis, and citric acid production. The characteristics of fungal citrate transporters elucidated in this study will help expand our understanding of the citrate production mechanism and facilitate the development and optimization of industrial organic acid fermentation processes.

Fatty Acid Synthesis and Degradation Interplay to Regulate the Oxidative Stress in Cancer Cells.

Both cytosolic fatty acid synthesis (FAS) and mitochondrial fatty acid oxidation (FAO) have been shown to play a role in the survival and proliferation of cancer cells. This study aimed to confirm experimentally whether FAS and FAO coexist in breast cancer cells (BCC). By feeding cells with 13C-labeled glutamine and measuring labeling patterns of TCA intermediates, it was possible to show that part of the cytosolic acetyl-CoA used in lipid synthesis is also fed back into the mitochondrion via fatty acid degradation. This results in the transfer of reductive potential from the cytosol (in the form of NADPH) to the mitochondrion (in the form of NADH and FADH2). The hypothesized mechanism was further confirmed by blocking FAS and FAO with siRNAs. Exposure to staurosporine (which induces ROS production) resulted in the disruption of simultaneous FAS and FAO, which could be explained by NADPH depletion.

Enhanced β-Amyrin Synthesis in Saccharomyces cerevisiae by Coupling An Optimal Acetyl-CoA Supply Pathway.

β-Amyrin is a plant-derived triterpenoid skeleton with wide applications in food and medical industry. β-Amyrin biosynthesis in Saccharomyces cerevisiae is derived from the mevalonate pathway with cytosolic acetyl-CoA as a precursor. In this work, endogenous and several heterologous acetyl-CoA synthesis pathways were coupled to β-amyrin production and a combinational acetyl-CoA supply route was demonstrated to be optimal due to more balanced redox cofactors, much lower energy consumption, and glucose utilization as well as significantly enhanced β-amyrin production (a 200% increase compared to the original β-amyrin-producing strain). Further disruption of an acetyl-CoA competing pathway led to a 330% increase in β-amyrin production as compared to the original strain. Finally, the engineered strain harboring the optimal pathway configuration achieved a final β-amyrin production of 279.0 ± 13.0 mg/L in glucose fed-batch fermentation, which is the highest as ever reported. This work provides an efficient platform for triterpenoid biosynthesis in Saccharomyces cerevisiae.

Simultaneously down-regulation of multiplex branch pathways using CRISPRi and fermentation optimization for enhancing β-amyrin production in Saccharomyces cerevisiae

The production of β-amyrin in Saccharomyces cerevisiae is still low due to the inability of effectively regulating the endogenous metabolic pathway for competitive synthesis of β-amyrin precursors. In this study, we focused on two branches of β-amyrin synthetics pathway that consume β-amyrin precursors (2,3-oxidosqualene and cytosolic acetyl-CoA) and regulated related genes (ADH1, ADH4, ADH5, ADH6, CIT2, MLS2 and ERG7). We developed a CRISPRi method by constructing a multi-gRNA plasmid to down-regulate the seven genes simultaneously, which is reported for the first time in S. cerevisiae. The average transcription inhibition efficiency of the seven genes reached as high as 75.5%. Furthermore, by optimizing the fermentation condition (including pH, inoculum size, initial glucose concentration and feed of glucose or ethanol) and increasing extracellular transportation via supplying methyl-β-cyclodextrin, β-amyrin concentration of engineered strain SGibSdCg increased by 44.3% compared with the parent strain SGib, achieving 156.7 mg/L which was the highest concentration of β-amyrin reported in yeast. The one-step down-regulation of multiple genes using CRISPRi showed high efficiency and promising future in improving the yields of natural products.

Understanding lipogenesis by dynamically profiling transcriptional activity of lipogenic promoters in Yarrowia lipolytica.

Lipogenesis is a complicated process involving global transcriptional reprogramming of lipogenic pathways. It is commonly believed that nitrogen starvation triggers a metabolic shift that reroutes carbon flux from Krebs cycles to lipogenesis. In this study, we systematically surveyed and dynamically profiled the transcriptional activity of 22 lipogenic promoters aiming to delineate a picture how nitrogen starvation regulates lipogenesis in Y. lipolytica. These lipogenic promoters drive the expression of critical pathways that are responsible for the generation of reducing equivalents (NADPH), carbon backbones (acetyl-CoA, malonyl-CoA, DHAP, etc.), synthesis and degradation of fatty acids. Specifically, our investigated promoters span across an array of metabolic pathways, including glycolysis, Krebs cycle, pentose phosphate pathway, mannitol cycle, glutamine-GABA cycle, fatty acid and lipid synthesis, glyoxylate, β-oxidation, and POM (pyruvate-oxaloacetate-malate) cycle. Our work provides evidences that mannitol cycle, glutamine-GABA cycle and amino acid degradation, pyruvate oxidation, and acetate assimilation pathways are lipogenesis-related steps involved in generating cytosolic NADPH and acetyl-CoA precursors. This systematic investigation and dynamic profiling of lipogenic promoters may help us better understand lipogenesis, facilitate the formulation of structure-based kinetic models, as well as develop efficient cell factories for fuels and chemical production in oleaginous species.

Metabolic engineering of Saccharomyces cerevisiae by using the CRISPR-Cas9 system for enhanced fatty acid production

Fatty acids produced by the yeast, Saccharomyces cerevisiae, are not only industrially important products, but also highly affect fermentation efficiency. Despite that S. cerevisiae is the potent metabolic platform, traditional plasmid-based genetic manipulations still impose concerns about safety and environmental impact. In this study, using the Cas9 system, S. cerevisiae, was metabolically engineered for increased production of fatty acids by increasing cytosolic acetyl-CoA production via disrupting the isocitrate dehydrogenase gene of the TCA cycle and introducing the cytosolic ATP-citrate lyase gene from Yarrowia lipolytica. By expressing the ATP-citrate lyase gene from Y. lipolytica, total fatty acid production increased by 37.1% compared to that by the wild-type yeast. This total fatty acid production herein increased ∼2 times compared to that by an engineered S. cerevisiae in a previous report, and unsaturated fatty acid levels also increased. Our results suggest that the Cas9 system for S. cerevisiae could be applied to engineer the yeast for industrial production of bio-based products.

Enhanced squalene biosynthesis in Yarrowia lipolytica based on metabolically engineered acetyl-CoA metabolism

As a bioactive triterpenoid, squalene is widely used in the food industry, cosmetics, and pharmacology. Squalene’s major commercial sources are the liver oil of deep-sea sharks and plant oils. In this study, we focused on the enhancement of squalene biosynthesis in Yarrowia lipolytica, with particular attention to the engineering of acetyl-CoA metabolism based on genome-scale metabolic reaction network analysis. Although the overexpression of the rate-limiting endogenous ylHMG1 (3-hydroxy-3-methylglutaryl-CoA reductase gene) could improve squalene synthesis by 3.2-fold over that by the control strain, the availability of the key intracellular precursor, acetyl-CoA, was found to play a more significant role in elevating squalene production. Analysis of metabolic networks with the newly constructed genome-scale metabolic model of Y. lipolytica iYL_2.0 showed that the acetyl-CoA pool size could be increased by redirecting carbon flux of pyruvate dehydrogenation towards the ligation of acetate and CoA or the cleavage of citrate to form oxaloacetate and acetyl-CoA. The overexpression of either acetyl-CoA synthetase gene from Salmonella enterica (acs*) or the endogenous ATP citrate lyase gene (ylACL1) resulted in a more than 50% increase in the cytosolic acetyl-CoA level. Moreover, iterative chromosomal integration of the ylHMG1, asc*, and ylACL1 genes resulted in a significant improvement in squalene production (16.4-fold increase in squalene content over that in the control strain). We also found that supplementation with 10 mM citrate in a flask culture further enhanced squalene production to 10 mg/g DCW. The information obtained in this study demonstrates that rationally engineering acetyl-CoA metabolism to ensure the supply of this key metabolic precursor is an efficient strategy for the enhancement of squalene biosynthesis.