RESUMO
Sorting large libraries of cells for improved small molecule secretion is throughput limited. Here, we combine producer/secretor cell libraries with whole-cell biosensors using a microfluidic-based screening workflow. This approach enables a mix-and-match capability using off-the-shelf biosensors through either coencapsulation or pico-injection. We demonstrate the cell type and library agnostic nature of this workflow by utilizing single-guide RNA, transposon, and ethyl-methyl sulfonate mutagenesis libraries across three distinct microbes (Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica), biosensors from two organisms (E. coli and S. cerevisiae), and three products (triacetic acid lactone, naringenin, and L-DOPA) to identify targets improving production/secretion.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Microfluídica/métodos , Técnicas Biossensoriais , Escherichia coli/genética , Escherichia coli/metabolismo , Fluorescência , Levodopa/biossíntese , Mutagênese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Yarrowia/genética , Yarrowia/metabolismoRESUMO
Microbial fermentation offers a sustainable source of fuels, commodity chemicals, and pharmaceuticals, yet strain performance is influenced greatly by the growth media selected. Specifically, trace metals (e.g., iron, copper, manganese, zinc, and others) are critical for proper growth and enzymatic function within microorganisms yet are non-standardized across media formulation. In this work, the effect of trace metal supplementation on the lipid production profile of Yarrowia lipolytica was explored using tube scale fermentation followed by biomass and lipid characterization. Addition of iron (II) to the chemically defined Yeast Synthetic Complete (YSC) medium increased final optical density nearly twofold and lipid production threefold, while addition of copper (II) had no impact. Additionally, dose-responsive changes in lipid distribution were observed, with the percent of oleic acid increasing and stearic acid decreasing as initial iron concentration increased. These changes were reversible with subsequent iron-selective chelation. Use of rich Yeast Peptone Dextrose (YPD) medium enabled further increases in the production of two specialty oleochemicals ultimately reaching 63 and 47% of the lipid pool as α-linolenic acid and cyclopropane fatty acid, respectively, compared to YSC medium. Selective removal of iron (II) natively present in YPD medium decreased this oleochemical production, ultimately aligning the lipid profile with that of non-supplemented YSC medium. These results provide further insight into the proposed mechanisms for iron regulation in yeasts especially as these productions strains contain a mutant allele of the iron regulator, mga2. The work presented here also suggests a non-genetic method for control of the lipid profile in Y. lipolytica for use in diverse applications. KEY POINTS: ⢠Iron supplementation increases cell density and lipid titer in Yarrowia lipolytica. ⢠Iron addition reversibly alters lipid portfolio increasing linolenic acid. ⢠Removal of iron from YPD media provides a link to enhanced oleochemical production.
Assuntos
Yarrowia , Biomassa , Ácidos Graxos/química , Fermentação , Ferro , Yarrowia/genéticaRESUMO
Polyketides represent an extremely diverse class of secondary metabolites often explored for their bioactive traits. These molecules are also attractive building blocks for chemical catalysis and polymerization. However, the use of polyketides in larger scale chemistry applications is stymied by limited titers and yields from both microbial and chemical production. Here, we demonstrate that an oleaginous organism (specifically, Yarrowia lipolytica) can overcome such production limitations owing to a natural propensity for high flux through acetyl-CoA. By exploring three distinct metabolic engineering strategies for acetyl-CoA precursor formation, we demonstrate that a previously uncharacterized pyruvate bypass pathway supports increased production of the polyketide triacetic acid lactone (TAL). Ultimately, we establish a strain capable of producing over 35% of the theoretical conversion yield to TAL in an unoptimized tube culture. This strain also obtained an averaged maximum titer of 35.9 ± 3.9 g/L with an achieved maximum specific productivity of 0.21 ± 0.03 g/L/h in bioreactor fermentation. Additionally, we illustrate that a ß-oxidation-related overexpression (PEX10) can support high TAL production and is capable of achieving over 43% of the theoretical conversion yield under nitrogen starvation in a test tube. Next, through use of this bioproduct, we demonstrate the utility of polyketides like TAL to modify commodity materials such as poly(epichlorohydrin), resulting in an increased molecular weight and shift in glass transition temperature. Collectively, these findings establish an engineering strategy enabling unprecedented production from a type III polyketide synthase as well as establish a route through O-functionalization for converting polyketides into new materials.
Assuntos
Regulação da Expressão Gênica de Plantas/fisiologia , Engenharia Genética/métodos , Pironas/metabolismo , Yarrowia/metabolismo , Estrutura Molecular , Oxirredução , Pironas/química , Piruvatos/metabolismo , Yarrowia/genéticaRESUMO
Polyketides are a diverse class of molecules sought after for their valuable properties, including as potential pharmaceuticals. Previously, we demonstrated that the oleaginous yeast Yarrowia lipolytica is an optimal host for production of the simple polyketide, triacetic acid lactone (TAL). We here expand the capacities of this host by overcoming previous media challenges and enabling production of more complex polyketides. Specifically, we employ a ß-oxidation related strategy to improve polyketide production directly from defined media. Beyond TAL production, we establish biosynthesis of the 4-coumaroyl-CoA derived polyketides: naringenin, resveratrol, and bisdemethoxycurcumin, as well as the diketide intermediate, (E)-5-(4-hydroxyphenyl)-3-oxopent-4-enoic acid. In this background, we enable high-level de novo production of naringenin through import of both a heterologous pathway and a mutant Y. lipolytica allele. In doing so, we generated an averaged maximum titer of 898 mg/L naringenin, the highest titer reported to date in any host. These results demonstrate that Y. lipolytica is an ideal polyketide production host for more complex 4-coumaroyl-CoA derived products.
Assuntos
Acil Coenzima A , Policetídeos/metabolismo , Pironas/metabolismo , Yarrowia , Acil Coenzima A/genética , Acil Coenzima A/metabolismo , Oxirredução , Yarrowia/genética , Yarrowia/metabolismoRESUMO
The nonconventional, oleaginous yeast, Yarrowia lipolytica is rapidly emerging as a valuable host for the production of a variety of both lipid and nonlipid chemical products. While the unique genetics of this organism pose some challenges, many new metabolic engineering tools have emerged to facilitate improved genetic manipulation in this host. This review establishes a case for Y. lipolytica as a premier metabolic engineering host based on innate metabolic capacity, emerging synthetic tools, and engineering examples. The metabolism underlying the lipid accumulation phenotype of this yeast as well as high flux through acyl-CoA precursors and the TCA cycle provide a favorable metabolic environment for expression of relevant heterologous pathways. These properties allow Y. lipolytica to be successfully engineered for the production of both native and nonnative lipid, organic acid, sugar and acetyl-CoA derived products. Finally, this host has unique metabolic pathways enabling growth on a wide range of carbon sources, including waste products. The expansion of carbon sources, together with the improvement of tools as highlighted here, have allowed this nonconventional organism to act as a cellular factory for valuable chemicals and fuels.
Assuntos
Engenharia Metabólica/métodos , Yarrowia/genética , Yarrowia/metabolismo , Acil Coenzima A/genética , Acil Coenzima A/metabolismo , Biocombustíveis , Ciclo do Ácido Cítrico/genética , Metabolismo dos Lipídeos/genética , Engenharia Metabólica/tendênciasRESUMO
A mutant excision+/integration- piggyBac transposase can be used to seamlessly excise a chromosomally integrated, piggyBac-compatible selection marker cassette from the Yarrowia lipolytica genome. This piggyBac transposase-based genome engineering process allows for both positive selection of targeted homologous recombination events and scarless or footprint-free genome modifications after precise marker recovery. Residual non-native sequences left in the genome after marker excision can be minimized (0-4 nucleotides) or customized (user-defined except for a TTAA tetranucleotide). Both of these options reduce the risk of unintended homologous recombination events in strains with multiple genomic edits. A suite of dual positive/negative selection marker pairs flanked by piggyBac inverted terminal repeats (ITRs) have been constructed and are available for precise genome engineering in Y. lipolytica using this method. This protocol specifically describes the split marker homologous recombination-based disruption of Y. lipolytica ADE2 with a piggyBac ITR-flanked URA3 cassette, followed by piggyBac transposase-mediated excision of the URA3 marker to leave a 50 nucleotide synthetic barcode at the ADE2 locus. The resulting ade2 strain is auxotrophic for adenine, which enables the use of ADE2 as a selectable marker for further strain engineering.
Assuntos
Elementos de DNA Transponíveis , Engenharia Genética/métodos , Transposases/metabolismo , Yarrowia/genética , Vetores Genéticos , Genoma Fúngico , Recombinação Homóloga , Fluxo de TrabalhoRESUMO
Enzymes are represented across a vast space of protein sequences and structural forms and have activities that far exceed the best chemical catalysts; however, engineering them to have novel or enhanced activity is limited by technologies for sensing product formation. Here, we describe a general and scalable approach for characterizing enzyme activity that uses the metabolism of the host cell as a biosensor by which to infer product formation. Since different products consume different molecules in their synthesis, they perturb host metabolism in unique ways that can be measured by mass spectrometry. This provides a general way by which to sense product formation, to discover unexpected products and map the effects of mutagenesis.
Assuntos
Técnicas Biossensoriais , Ensaios Enzimáticos/métodos , Engenharia Metabólica/métodos , Asteraceae/enzimologia , Asteraceae/genética , Biocatálise , Técnicas Analíticas Microfluídicas , Mutagênese , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Yarrowia/genética , Yarrowia/metabolismoRESUMO
Polyketides are a unique class of molecules with attractive bioactive and chemical properties. As a result, biorenewable production is being explored with these molecules as potential pharmaceutical, fuel, and material precursors. In particular, type III polyketide synthases enable access to a diverse class of chemicals using a relatively simple biochemical synthesis pathway. In this review, the recent advances in the engineering of microbial hosts for the production of type III PKS-derived polyketides are highlighted. In particular, the field has moved beyond simple proof-of-concept and has been exploring engineering efforts that have led to improved production scales. This review details engineering progress for the production of acetyl-CoA- and malonyl-CoA-derived polyketides including the products triacetic acid lactone and phloroglucinol as well as polyphenolic, phenylpropanoid-derived compounds including flavonoids, stilbenoids, and curcuminoids. Specifically, the authors focus on enumerating the metabolic engineering strategies employed and product titers achieved for these molecules. Finally, the authors highlight tools and strategies that can be leveraged to realize the potential of microbial production and diversification of these molecules.