Metabolite concentrations, fluxes, and free energies constitute the basis for understanding and controlling metabolism. Recently, using high-resolution mass spectrometry and multi-isotope tracing, improved flux quantitation led to determination of metabolic fates, pathway and nutrient contributions, and Gibbs free energy of reaction (ΔG) in central carbon metabolism using a relationship between reaction reversibility and thermodynamic driving force. We applied this quantitative analysis scheme towards the rational design of a pathway to assimilate formate in E. coli. Several synthetic pathways utilizing formate have been constructed in E. coli, indicative of its appeal as a raw material; however, these pathways either use formate as an electron donor rather than a carbon source or lose carbons as carbon dioxide. We have designed a pathway capable of incorporating formate into central carbon metabolism. Of the six engineered strains theoretically capable of operating this engineered metabplic pathway, we determined, through media sampling and tracing of the metabolic fate of 13C-formate, that the JXG2 strain incorporated formate into central carbon metabolism. Formate was responsible for 0.4% of carbons in hexose phosphate. Thus, we have engineered an E. coli strain that can serve as a platform for one-carbon utilization and sustainable biotechnology. At this point, further pathway optimization is needed, as flux through the engineered pathway is low; this may involve conducting targeted strain evolution on formate or knockout of competing pathways. When further optimized, our engineered metabolic pathway will be able to efficiently convert one-carbon compounds to advanced bioproducts.
The tumor microenvironment (TME) consists of cancer cells and other cell types including immune cells. Metabolism is a network of biochemical reactions that supports both T cells’ proliferation and anti-cancer activity. These reactions also promote cancer growth and progression by supplying energy and biochemical building blocks. While cancer cells can exploit the tumor microenvironment for additional nutrients, T cells must navigate these environments to recognize and kill cancers. Modifications to immune cell therapies are designed to overcome the metabolic adaptations that tumors use to outcompete healthy cells including T cells. One immune therapy uses chimeric antigen receptor (CAR)-T cells that are engineered to identify and kill cancers. To shed light on desirable metabolic features of CAR-T cells, we characterize metabolism across a panel of human T cells expressing the seven CARs such that non-signaling sequences are the only variable. Out of the seven CARs tested, T cells harboring anti-GD2 and rituximab anti-CD20 CARs display the most active metabolism with fast glucose and amino acid uptake while anti-CD19 and other anti-CD20 CAR T cells display minimal alteration in central carbon and nucleotide metabolism compared to control T cells. Interestingly, hyperactive CAR T cells use the increased nitrogen uptake to produce excess nucleotides, non-essential amino acids, and ammonia. In mice injected with lymphoma cells, modest waste secretion by CAR-T cells and high metabolic compatibility between cancer cells and CAR-T cells are recognized as features of efficacious CAR-T cell therapy.
We further explore how cancer cells are inelastic in the TME to meet the increased demands for energy and biosynthesis. Fast fermentation of cancer cells and poor vasculature and leads to accumulation of lactate in the TME. Despite thriving in the tumor microenvironment in vivo, cancer cells seldom grow in a glucose-depleted high-lactate medium. It is unclear how and why. Here, we find that a small window of glucose availability supports cancer cell proliferation regardless of tissue origin. In glucose-depleted environments, cells sustain growth for 24 hours using glutamine as a primary substrate for gluconeogenesis and nucleotide biosynthesis and lactate for the TCA cycle and aspartate and asparagine biosynthesis. We hypothesize that when glucose is supplied intermittently, cancer cells are displaying metabolic inelasticity, with the inability to adapt paradoxically helping them accelerate their growth. Without glucose, cancer cells operate glycolysis using glycogen. Upon glucose availability, cells restore glycogen to again operate glycolysis. Cancer cells are hardwired for glycolysis which allows them to proliferate without gluconeogenesis. Thus, intermittent glucose availability sustains cancer growth despite low average glucose levels in the tumor microenvironment.
Modern agriculture relies heavily on a small selection of widespread market-dominant herbicides, many of which share overlapping modes of action such as ALS inhibition from Chlorsulfuron and EPSP inhibition from Round-Up ready. This widespread use has caused a substantial increase in the number and types of herbicide resistant weeds throughout the world which underlies the near future need for herbicides with novel modes of action. Aspterric acid is a sesquiterpenoid herbicide which inhibits the DHAD enzyme of the branched chain amino acid pathway (BCAA); this enzyme is not targeted by any commercial herbicide on the market which makes aspterric acid an ideal candidate for a novel herbicide. Yarrowia lipolytica, an oleaginous yeast strain, stands out as a promising microbial platform for biosynthetic production of aspterric acid due to its high acetyl-CoA flux and a growing body of literature affirming Y. lipolytica as particularly suited for the over-production of terpenoids. The four ast genes (A,B,C,D) involved in the aspterric acid synthesis pathway were markerlessly integrated into the Y. lipolytica host genome, along with gene copies for HMGCR and FPPS which would overexpress commonly noted rate-limiting enzymes involved in terpenoid synthesis so as to further enhance carbon flux towards terpenoid production. However, the engineered strains of Y. lipolytica failed to produce aspterric acid and instead yielded an isomer, demonstrating the continued challenges that follow from attempts at heterologous pathway introduction into this microbe.
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