Modular co-culture engineering of Yarrowia lipolytica for amorphadiene biosynthesis.

Amorphadiene is the precursor to synthesize the antimalarial drug artemisinin. The production of amorphadiene and artemisinin from metabolically engineered microbes may provide an alternate to plant secondary metabolite extraction. Microbial consortia can offer division of labor, and microbial co-culture system can be leveraged to achieve cost-efficient production of natural products. Using a co-culture system of Y. lipolytica Po1f and Po1g strains, subcellular localization of ADS gene (encoding amorphadiene synthase) into the endoplasmic reticulum, co-utilization of mixed carbon source, and enlargement of the endoplasmic reticulum (ER) surface area, we were able to significantly improve amorphadiene production in this work. Using Po1g/PPtM and Po1f/AaADSERx3/iGFMPDU strains and co-utilization of 5 µM sodium acetate with 20 g/L glucose in YPD media, amorphadiene titer were increased to 65.094 mg/L. The enlargement of the ER surface area caused by the deletion of the PAH1 gene provided more subcellular ER space for the action of the ADS-tagged gene. It further increased the amorphadiene production to 71.74 mg/L. The results demonstrated that the importance of the spatial localization of critical enzymes, and manipulating metabolic flux in the co-culture of Y. lipolytica can be efficient over a single culture for the bioproduction of isoprenoid-related secondary metabolites in a modular manner.

Rapid Ultrasensitive and High-Throughput Bioburden Detection: Microfluidics and Instrumentation.

Contamination detection often requires lengthy culturing steps to detect low-level bioburden. To increase the rate of detection and decrease the limit of detection (LOD), a system featuring microfluidics and a multichannel fluorometer has been developed. The eight-channel fluorometer enables parallel testing of multiple samples with the LOD as low as <1 cfu/mL. This low-cost system utilizes the slope of fluorescence intensity that serves as the criterion for bioburden detection. The redox indicator dye resazurin is used to monitor the presence of viable cells in this study and is reduced to resorufin with a high quantum yield at 585 nm. The sample under investigation is spiked with resazurin and loaded in a special-design microfluidic cassette, and the rate of change is observed via the fluorometer. The method was validated using primary Escherichia coli culture in comparison with a spectrophotometer which served as the gold standard. An optimized assay based on Luria-Bertani medium was developed. The impact on the assay sensitivity based on incubation and filtration steps was also explored. The assay is shown to pick up inadvertent contamination from test tubes and pipette tips showing its applicability in real-world settings. The data analysis demonstrated a comparable performance of the multichannel fluorometer vis-a-vis the conventional plate reader. The multichannel system is shown to detect bioburden presence in as low as 20 s for bacterial concentrations ≥5 cfu/mL after 6 h of incubation. Considering its portability, low cost, simplicity of operation, and relevant assay sensitivity, the system is well positioned to detect low-level bioburden in the laboratory, pharmaceutical, and field settings.

Synthetic biology, systems biology, and metabolic engineering of Yarrowia lipolytica toward a sustainable biorefinery platform.

Yarrowia lipolytica is an oleaginous yeast that has been substantially engineered for production of oleochemicals and drop-in transportation fuels. The unique acetyl-CoA/malonyl-CoA supply mode along with the versatile carbon-utilization pathways makes this yeast a superior host to upgrade low-value carbons into high-value secondary metabolites and fatty acid-based chemicals. The expanded synthetic biology toolkits enabled us to explore a large portfolio of specialized metabolism beyond fatty acids and lipid-based chemicals. In this review, we will summarize the recent advances in genetic, omics, and computational tool development that enables us to streamline the genetic or genomic modification for Y. lipolytica. We will also summarize various metabolic engineering strategies to harness the endogenous acetyl-CoA/malonyl-CoA/HMG-CoA pathway for production of complex oleochemicals, polyols, terpenes, polyketides, and commodity chemicals. We envision that Y. lipolytica will be an excellent microbial chassis to expand nature's biosynthetic capacity to produce plant secondary metabolites, industrially relevant oleochemicals, agrochemicals, commodity, and specialty chemicals and empower us to build a sustainable biorefinery platform that contributes to the prosperity of a bio-based economy in the future.

Genetically-encoded biosensors for analyzing and controlling cellular process in yeast.

Yeast has been a robust platform to manufacture a broad range of biofuels, commodity chemicals, natural products and pharmaceuticals. The membrane-bound organelles in yeast provide us the means to access the specialized metabolism for various biosynthetic applications. The separation and compartmentalization of genetic and metabolic events presents us the opportunity to precisely control and program gene expression for higher order biological functions. To further advance yeast synthetic biology platform, genetically encoded biosensors and actuators haven been engineered for in vivo monitoring and controlling cellular processes with spatiotemporal resolutions. The dynamic response, sensitivity and operational range of these genetically encoded sensors are determined by the regulatory architecture, dynamic assemly and interactions of the related proteins and genetic elements. This review provides an update of the basic design principles underlying the allosteric transcription factors, GPCR and optogenetics-based sensors, aiming to precisely analyze and control yeast cellular processes for various biotechnological applications.

Microbial Coculture for Flavonoid Synthesis.

Flavonoids are plant-derived natural products with human health-promoting benefits. The modularity and complexity of the flavonoid biosynthetic pathway allow us to leverage the metabolic characteristics of distinct microbial hosts and install structural functionalities beyond what monocultures can achieve. We discuss the promising future of applying microbial cocultures to improve the cost-efficiency and diversity of flavonoid biosynthesis.

Biotechnological Production of Flavonoids: An Update on Plant Metabolic Engineering, Microbial Host Selection, and Genetically Encoded Biosensors.

Flavonoids represent a diversified family of phenylpropanoid-derived plant secondary metabolites. They are widely found in fruits, vegetables, and medicinal herbs. There has been increasing interest on flavonoids because of their proven bioactivity associated with anti-obesity and anti-cancer, anti-inflammatory and anti-diabetic activity. Low bioavailability of flavonoids is a major challenge restricting their applications. Due to safety and economic issues, plant extraction or chemical synthesis could not provide a scalable route for large-scale production. Alternatively, reconstruction of biosynthetic gene clusters in plants and industrially relevant microbes offer significant promise for discovery and scalable synthesis of flavonoids. This review provides an update on biotechnological production of flavonoids. The recent advances on plant metabolic engineering, microbial host, and genetically encoded biosensors are summarized. Plant metabolic engineering holds the promise to improve the yield of specific flavonoids and expand the chemical space of novel flavonoids. The choice of microbial host provides the cellular chassis that could be tailored for various stereo- or regio-selective chemistries that are crucial for their bioactivities. When coupled with transcriptional biosensing, genetically encoded biosensors could be welded into cellular metabolism to achieve high throughput screening or dynamic carbon flux re-allocation to deliver efficient microbial workhorse. The convergence of these technologies will translate the vast majority of plant genetic resources into valuable flavonoids with pharmaceutical/nutraceutical values in the foreseeable future.

Debottlenecking mevalonate pathway for antimalarial drug precursor amorphadiene biosynthesis in Yarrowia lipolytica.

World Health Organization reports that half of the population in developing countries are at risk of malaria infection. Artemisinin, the most potent anti-malaria drug, is a sesquiterpene endoperoxide extracted from the plant Artemisia annua. Due to scalability and economics issues, plant extraction or chemical synthesis could not provide a sustainable route for large-scale manufacturing of artemisinin. The price of artemisinin has been fluctuating from 200$/Kg to 1100$/Kg, due to geopolitical and climate factors. Microbial fermentation was considered as a promising method to stabilize the artemisinin supply chain. Yarrowia lipolytica, is an oleaginous yeast with proven capacity to produce large quantity of lipids and oleochemicals. In this report, the lipogenic acetyl-CoA pathways and the endogenous mevalonate pathway of Y. lipolytica were harnessed for amorphadiene production. Gene overexpression indicate that HMG-CoA and acetyl-CoA supply are two limiting bottlenecks for amorphadiene production. We have identified the optimal HMG-CoA reductase and determined the optimal gene copy number for the precursor pathways. Amorphadiene production was improved further by either inhibiting fatty acids synthase or activating the fatty acid degradation pathway. With co-expression of mevalonate kinase (encoded by Erg12), a push-and-pull strategy enabled the engineered strain to produce 171.5 â€‹mg/L of amorphadiene in shake flasks. These results demonstrate that balancing carbon flux and manipulation of precursor competing pathways are key factors to improve amorphadiene biosynthesis in oleaginous yeast; and Y. lipolytica is a promising microbial host to expand nature's biosynthetic capacity, allowing us to quickly access antimalarial drug precursors.

Optimizing Oleaginous Yeast Cell Factories for Flavonoids and Hydroxylated Flavonoids Biosynthesis.

Plants possess myriads of secondary metabolites with a broad spectrum of health-promoting benefits. To date, plant extraction is still the primary route to produce high-value natural products which inherently suffers from economics and scalability issues. Heterologous expression of plant biosynthetic gene clusters in microbial host is considered as a feasible approach to overcoming these limitations. Oleaginous yeast produces a large amount of lipid bodies, the abundant membrane structure and the lipophilic environment provide the ideal environment for the regioselectivity and stereoselectivity of many plant-derived P450 enzymes. In this work, we used modular method to construct, characterize, and optimize the flavonoid pathways in Yarrowia lipolytica. We also evaluated various precursor biosynthetic routes and unleashed the metabolic potential of Y. lipolytica to produce flavonoids and hydroxylated flavonoids. Specifically, we have identified that chalcone synthase (CHS) and cytochrome P450 reductases (CPR) were the bottlenecks of hydroxylated flavonoid production. We determined the optimal gene copy number of CHS and CPR to be 5 and 2, respectively. We further removed precursor pathway limitations by expressing genes associated with chorismate and malonyl-CoA supply. With pH and carbon-nitrogen ratio (C/N) optimization, our engineered strain produced 252.4 mg/L naringenin, 134.2 mg/L eriodictyol, and 110.5 mg/L taxifolin from glucose in shake flasks. Flavonoid and its hydroxylated derivatives are most prominently known as antioxidant and antiaging agents. These findings demonstrate our ability to harness the oleaginous yeast as the microbial workhorse to expand nature's biosynthetic potential, enabling us to bridge the gap between drug discovery and natural product manufacturing.

Engineering acetyl-CoA metabolic shortcut for eco-friendly production of polyketides triacetic acid lactone in Yarrowia lipolytica.

Acetyl-CoA is the central metabolic node connecting glycolysis, Krebs cycle and fatty acids synthase. Plant-derived polyketides, are assembled from acetyl-CoA and malonyl-CoA, represent a large family of biological compounds with diversified bioactivity. Harnessing microbial bioconversion is considered as a feasible approach to large-scale production of polyketides from renewable feedstocks. Most of the current polyketide production platform relied on the lengthy glycolytic steps to provide acetyl-CoA, which inherently suffers from complex regulation with metabolically-costly cofactor/ATP requirements. Using the simplest polyketide triacetic acid lactone (TAL) as a testbed molecule, we demonstrate that acetate uptake pathway in oleaginous yeast (Yarrowia lipolytica) could function as an acetyl-CoA shortcut to achieve metabolic optimality in producing polyketides. We identified the metabolic bottlenecks to rewire acetate utilization for efficient TAL production in Y. lipolytica, including generation of the driving force for acetyl-CoA, malonyl-CoA and NADPH. The engineered strain, with the overexpression of endogenous acetyl-CoA carboxylase (ACC1), malic enzyme (MAE1) and a bacteria-derived cytosolic pyruvate dehydrogenase (PDH), affords robust TAL production with titer up to 4.76 g/L from industrial glacier acetic acid in shake flasks, representing 8.5-times improvement over the parental strain. The acetate-to-TAL conversion ratio (0.149 g/g) reaches 31.9% of the theoretical maximum yield. The carbon flux through this acetyl-CoA metabolic shortcut exceeds the carbon flux afforded by the native glycolytic pathways. Potentially, acetic acid could be manufactured in large-quantity at low-cost from Syngas fermentation or heterogenous catalysis (methanol carbonylation). This alternative carbon sources present a metabolic advantage over glucose to unleash intrinsic pathway limitations and achieve high carbon conversion efficiency and cost-efficiency. This work also highlights that low-cost acetic acid could be sustainably upgraded to high-value polyketides by oleaginous yeast species in an eco-friendly and cost-efficient manner.

Engineering metabolite-responsive transcriptional factors to sense small molecules in eukaryotes: current state and perspectives.

Nature has evolved exquisite sensing mechanisms to detect cellular and environmental signals surrounding living organisms. These biosensors have been widely used to sense small molecules, detect environmental cues and diagnose disease markers. Metabolic engineers and synthetic biologists have been able to exploit metabolites-responsive transcriptional factors (MRTFs) as basic tools to rewire cell metabolism, reprogram cellular activity as well as boost cell's productivity. This is commonly achieved by integrating sensor-actuator systems with biocatalytic functions and dynamically allocating cellular resources to drive carbon flux toward the target pathway. Up to date, most of identified MRTFs are derived from bacteria. As an endeavor to advance intelligent biomanufacturing in yeast cell factory, we will summarize the opportunities and challenges to transfer the bacteria-derived MRTFs to expand the small-molecule sensing capability in eukaryotic cells. We will discuss the design principles underlying MRTF-based biosensors in eukaryotic cells, including the choice of reliable reporters and the characterization tools to minimize background noise, strategies to tune the sensor dynamic range, sensitivity and specificity, as well as the criteria to engineer activator and repressor-based biosensors. Due to the physical separation of transcription and protein expression in eukaryotes, we argue that nuclear import/export mechanism of MRTFs across the nuclear membrane plays a critical role in regulating the MRTF sensor dynamics. Precisely-controlled MRTF response will allow us to repurpose the vast majority of transcriptional factors as molecular switches to achieve temporal or spatial gene expression in eukaryotes. Uncovering this knowledge will inform us fundamental design principles to deliver robust cell factories and enable the design of reprogrammable and predictable biological systems for intelligent biomanufacturing, smart therapeutics or precision medicine in the foreseeable future.

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, ß-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.