Bacteria can be genetically engineered to kill specific pathogens or inhibit their virulence. We previously developed a synthetic genetic system that allows a laboratory strain of Escherichia coli to ...sense and kill Pseudomonas aeruginosa in vitro. Here, we generate a modified version of the system, including a gene encoding an anti-biofilm enzyme, and use the probiotic strain Escherichia coli Nissle 1917 as host. The engineered probiotic shows in vivo prophylactic and therapeutic activity against P. aeruginosa during gut infection in two animal models (Caenorhabditis elegans and mice). These findings support the further development of engineered microorganisms with potential prophylactic and therapeutic activities against gut infections.
Environmental pH is a fundamental signal continuously directing the metabolism and behavior of living cells. Programming the precise cellular response toward environmental pH is, therefore, crucial ...for engineering cells for increasingly sophisticated functions. Herein, we engineer a set of riboswitch-based pH-sensing genetic devices to enable the control of gene expression according to differential environmental pH. We next develop a digital pH-sensing system to utilize the analogue-sensing behavior of these devices for high-resolution recording of host cell exposure to discrete external pH levels. The application of this digital pH-sensing system is demonstrated in a genetic program that autonomously regulated the evolutionary engineering of host cells for improved tolerance to a broad spectrum of organic acids, a valuable phenotype for metabolic engineering and bioremediation applications.Cells are exposed to shifts in environmental pH, which direct their metabolism and behavior. Here the authors design pH-sensing riboswitches to create a gene expression program, digitalize the system to respond to a narrow pH range and apply it to evolve host cells with improved tolerance to a variety of organic acids.
Abstract
Background
Amyrin is an important triterpenoid and precursor to a wide range of cosmetic, pharmaceutical and nutraceutical products. In this study, we metabolically engineered the oleaginous ...yeast,
Yarrowia lipolytica
to produce α- and β-amyrin on simple sugar and waste cooking oil.
Results
We first validated the in vivo enzymatic activity of a multi-functional amyrin synthase (CrMAS) from
Catharanthus roseus
, by expressing its codon-optimized gene in
Y. lipolytica
and assayed for amyrins. To increase yield, prevailing genes in the mevalonate pathway, namely
HMG1
,
ERG20
,
ERG9
and
ERG1
, were overexpressed singly and in combination to direct flux towards amyrin biosynthesis. By means of a semi-rational protein engineering approach, we augmented the catalytic activity of CrMAS and attained ~ 10-folds higher production level on glucose. When applied together, protein engineering with enhanced precursor supplies resulted in more than 20-folds increase in total amyrins. We also investigated the effects of different fermentation conditions in flask cultures, including temperature, volumetric oxygen mass transfer coefficient and carbon source types. The optimized fermentation condition attained titers of at least 100 mg/L α-amyrin and 20 mg/L β-amyrin.
Conclusions
The design workflow demonstrated herein is simple and remarkably effective in amplifying triterpenoid biosynthesis in the yeast
Y. lipolytica
.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Squalene is a highly sought-after triterpene compound in growing demand, and its production offers a promising avenue for circular economy practices. In this study, we applied metabolic engineering ...principles to enhance squalene production in the nonconventional yeast Yarrowia lipolytica, using waste cooking oil as a substrate. By overexpressing key enzymes in the mevalonate pathway — specifically ERG9 encoding squalene synthase, ERG20 encoding farnesyl diphosphate synthase, and HMGR encoding hydroxy-methyl-glutaryl-CoA reductase — we achieved a yield of 779.9 mg/L of squalene. Further co-overexpression of DGA1, encoding diacylglycerol acyltransferase, and CAT2, encoding carnitine acetyltransferase, in combination with prior metabolic enhancements, boosted squalene production to 1381.4 mg/L in the engineered strain Po1g17. To enhance the supply of the precursor acetyl-CoA and inhibit downstream squalene conversion, we supplemented with 6 g/L pyruvic acid and 0.7 mg/L terbinafine, resulting in an overall squalene titer of 2594.1 mg/L. These advancements underscore the potential for sustainable, large-scale squalene production using Y. lipolytica cell factories, contributing to circular economy initiatives by valorizing waste materials.
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•Up-cycling of waste cooking oil into squalene using engineered Y. lipolytica.•Overexpression of pathway genes directed metabolic flux towards squalene biosynthesis.•Co-overexpressing CAT2 and DGA1 boosted the squalene titers in Y. lipolytica.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
Itaconic acid (IA) is a high-value organic acid with a plethora of industrial applications. In this study, we seek to develop a microbial cell factory that could utilize waste cooking oil (WCO) as ...raw material for circular and cost-effective production of the abovementioned biochemical. Specifically, we expressed cis-aconitic acid decarboxylase (CAD) gene from
in either the cytosol or peroxisome of
and assayed for production of IA on WCO. To further improve production yield, the 10 genes involved in the production pathway of acetyl-CoA, an intermediate metabolite necessary for the synthesis of cis-aconitic acid, were individually overexpressed and investigated for their impact on IA production. To minimize off-target flux channeling, we had also knocked out genes related to competing pathways in the peroxisome. Impressively, IA titer up to 54.55 g/L was achieved in our engineered
in a 5 L bioreactor using WCO as the sole carbon source.
Cell and gene therapies are an innovative solution to various severe diseases and unfulfilled needs. Adoptive cell therapy (ACT), a form of cellular immunotherapies, has been favored in recent years ...due to the approval of chimeric antigen receptor CAR-T products. Market research indicates that the industry’s value is predicted to reach USD 24.4 billion by 2030, with a compound annual growth rate (CAGR) of 21.5%. More importantly, ACT is recognized as the hope and future of effective, personalized cancer treatment for healthcare practitioners and patients worldwide. The significant global momentum of this therapeutic approach underscores the urgent need to establish it as a practical and standardized method. It is essential to understand how cell culture conditions affect the expansion and differentiation of T-cells. However, there are ongoing challenges in ensuring the robustness and reproducibility of the manufacturing process. The current study evaluated various adoptive T-cell culture platforms to achieve large-scale production of several billion cells and high-quality cellular output with minimal cell death. It examined factors such as bioreactor parameters, media, supplements and stimulation. This research addresses the fundamental challenges of scalability and reproducibility in manufacturing, which are essential for making adoptive T-cell therapy an accessible and powerful new class of cancer therapeutics.
Monoterpenoids are an important class of natural products that are derived from the condensation of two five‑carbon isoprene subunits. They are widely used for flavouring, fragrances, colourants, ...cosmetics, fuels, chemicals, and pharmaceuticals in various industries. They can also serve as precursors for the production of many industrially important products. Currently, monoterpenoids are produced predominantly through extraction from plant sources. However, the small quantity of monoterpenoids in nature renders this method of isolation non-economically viable. Similarly impractical is the chemical synthesis of these compounds as they suffer from high energy consumption and pollutant discharge. Microbial biosynthesis, however, exists as a potential solution to these hindrances, but the transformation of cells into efficient factories remains a major impediment. Here, we critically review the recent advances in engineering microbes for monoterpenoid production, with an emphasis on categorized strategies, and discuss the challenges and perspectives to offer guidance for future engineering.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
Bisabolene is a bioactive sesquiterpene with a wide range of applications in food, cosmetics, medicine, and aviation fuels. Microbial production offers a green, efficient, and sustainable ...alternative. In this study, we focused on improving the titers of α-bisabolene in Yarrowia lipolytica by applying two strategies, (i) optimizing the metabolic flux of α-bisabolene biosynthetic pathway and (ii) sequestering α-bisabolene in lipid droplet, thus alleviating its inherent toxicity to host cells. We showed that overexpression of DGA1 and OLE1 to increase lipid content and unsaturated fatty acid levels was essential for boosting the α-bisabolene synthesis when supplemented with auxiliary carbon sources. The final engineered strain Po1gαB10 produced 1954.3 mg/L α-bisabolene from the waste cooking oil under shake flask fermentation, which was 96-fold higher than the control strain Po1gαB0. At the time of writing, our study represents the highest reported α-bisabolene titer in the engineered Y. lipolytica cell factory. This work describes novel strategies to improve the bioproduction of α-bisabolene that potentially may be applicable for other high-value terpene products.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
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•Limonene titer was enhanced using combinatorial gene overexpression in Y. lipolytica.•Carbon sources were optimized to enhance the titers of D-limonene and L-limonene.•Waste cooking ...oil is the superior substrate for producing limonenes in Y. lipolytica.•The highest titer of limonenes till date was obtained at the shake-flasks level.
Limonene is a valuable monoterpene widely used in the food and pharmaceutical industries. Previously, we successfully engineered Yarrowia lipolytica to produce limonenes. In this study, we focused on improving the titers of limonenes in Y. lipolytica by optimizing the metabolic flux of the limonene biosynthetic pathway and the medium composition. First, we adopted a combinatorial gene (over)expression strategy to improve the production of limonenes, obtaining the highest titer production strains. Subsequently, the medium composition and fed-batch fermentation were optimized to improve limonene biosynthesis, and it was confirmed that waste cooking oil (WCO) is the superior substrate to produce limonenes in Y. lipolytica. Under optimal fermentation conditions, the titers of D-limonene and L-limonene were improved to 91.24 mg/L and 83.06 mg/L from WCO. These findings provide valuable insights into the engineering of Y. lipolytica for a higher-level production of limonene and its utilization in converting WCO into other industrial products.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
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