Liquid-like condensates that bind actin drive filament polymerization and bundling.

Liquid-like protein condensates perform diverse physiological functions. Previous work showed that VASP, a processive actin polymerase, forms condensates that polymerize and bundle actin. To minimize their curvature, filaments accumulated at the inner condensate surface, ultimately deforming the condensate into a rod-like shape, filled with a bundle of parallel filaments. Here we show that this behavior does not require proteins with specific polymerase activity. Specifically, we found that condensates composed of Lamellipodin, a protein that binds actin but is not an actin polymerase, were also capable of polymerizing and bundling actin filaments. To probe the minimum requirements for condensate-mediated actin bundling, we developed an agent-based computational model. Guided by its predictions, we hypothesized that any condensate-forming protein that binds actin could bundle filaments through multivalent crosslinking. To test this idea, we added an actin-binding motif to Eps15, a condensate-forming protein that does not normally bind actin. The resulting chimera formed condensates that drove efficient actin polymerization and bundling. Collectively, these findings broaden the family of proteins that could organize cytoskeletal filaments to include any actin-binding protein that participates in protein condensation.

Proteomes reveal metabolic capabilities of Yarrowia lipolytica for biological upcycling of polyethylene into high-value chemicals.

IMPORTANCE: Sustainable processes for biological upcycling of plastic wastes in a circular bioeconomy are needed to promote decarbonization and reduce environmental pollution due to increased plastic consumption, incineration, and landfill storage. Strain characterization and proteomic analysis revealed the robust metabolic capabilities of Yarrowia lipolytica to upcycle polyethylene into high-value chemicals. Significant proteome reallocation toward energy and lipid metabolisms was required for robust growth on hydrocarbons with n-hexadecane as the preferential substrate. However, an apparent over-investment in these same categories to utilize complex depolymerized plastic (DP) oil came at the expense of protein biosynthesis, limiting cell growth. Taken together, this study elucidates how Y. lipolytica activates its metabolism to utilize DP oil and establishes Y. lipolytica as a promising host for the upcycling of plastic wastes.

Gene Coexpression Connectivity Predicts Gene Targets Underlying High Ionic-Liquid Tolerance in Yarrowia lipolytica.

Microbial tolerance to organic solvents such as ionic liquids (ILs) is a robust phenotype beneficial for novel biotransformation. While most microbes become inhibited in 1% to 5% (vol/vol) IL (e.g., 1-ethyl-3-methylimidazolium acetate), we engineered a robust Yarrowia lipolytica strain (YlCW001) that tolerates a record high of 18% (vol/vol) IL via adaptive laboratory evolution. Yet, genotypes conferring high IL tolerance in YlCW001 remain to be discovered. In this study, we shed light on the underlying cellular processes that enable robust Y. lipolytica to thrive in inhibitory ILs. By using dynamic transcriptome sequencing (RNA-Seq) data, we introduced Gene Coexpression Connectivity (GeCCo) as a metric to discover genotypes conferring desirable phenotypes that might not be found by the conventional differential expression (DE) approaches. GeCCo selects genes based on their number of coexpressed genes in a subnetwork of upregulated genes by the target phenotype. We experimentally validated GeCCo by reverse engineering a high-IL-tolerance phenotype in wild-type Y. lipolytica. We found that gene targets selected by both DE and GeCCo exhibited the best statistical chance at increasing IL tolerance when individually overexpressed. Remarkably, the best combination of dual-overexpression genes was genes selected by GeCCo alone. This nonintuitive combination of genes, BRN1 and OYE2, is involved in guiding/regulating mitotic cell division, chromatin segregation/condensation, microtubule and cytoskeletal organization, and Golgi vesicle transport. IMPORTANCE Cellular robustness to cope with stressors is an important phenotype. Y. lipolytica is an industrial robust oleaginous yeast that has recently been discovered to tolerate record high concentrations of ILs, beneficial for novel biotransformation in organic solvents. However, genotypes that link to IL tolerance in Y. lipolytica are largely unknown. Due to the complex IL-tolerant phenotype, conventional gene discovery and validation based on differential gene expression approaches are time-consuming due to a large search space and might encounter a high false-discovery rate. Here, using the developed Gene Coexpression Connectivity (GeCCo) method, we identified and validated a subset of most promising gene targets conferring the IL-tolerant phenotypes and shed light on their potential mechanisms. We anticipate GeCCo being a useful method to discover the genotype-to-phenotype link.

Exploring Proteomes of Robust Yarrowia lipolytica Isolates Cultivated in Biomass Hydrolysate Reveals Key Processes Impacting Mixed Sugar Utilization, Lipid Accumulation, and Degradation.

Yarrowia lipolytica is an oleaginous yeast exhibiting robust phenotypes beneficial for industrial biotechnology. The phenotypic diversity found within the undomesticated Y. lipolytica clade from various origins illuminates desirable phenotypic traits not found in the conventional laboratory strain CBS7504 (or W29), which include xylose utilization, lipid accumulation, and growth on undetoxified biomass hydrolysates. Currently, the related phenotypes of lipid accumulation and degradation when metabolizing nonpreferred sugars (e.g., xylose) associated with biomass hydrolysates are poorly understood, making it difficult to control and engineer in Y. lipolytica. To fill this knowledge gap, we analyzed the genetic diversity of five undomesticated Y. lipolytica strains and identified singleton genes and genes exclusively shared by strains exhibiting desirable phenotypes. Strain characterizations from controlled bioreactor cultures revealed that the undomesticated strain YB420 used xylose to support cell growth and maintained high lipid levels, while the conventional strain CBS7504 degraded cell biomass and lipids when xylose was the sole remaining carbon source. From proteomic analysis, we identified carbohydrate transporters, xylose metabolic enzymes, and pentose phosphate pathway proteins stimulated during the xylose uptake stage for both strains. Furthermore, we distinguished proteins involved in lipid metabolism (e.g., lipase, NADPH generation, lipid regulators, and ß-oxidation) activated by YB420 (lipid maintenance phenotype) or CBS7504 (lipid degradation phenotype) when xylose was the sole remaining carbon source. Overall, the results relate genetic diversity of undomesticated Y. lipolytica strains to complex phenotypes of superior growth, sugar utilization, lipid accumulation, and degradation in biomass hydrolysates. IMPORTANCE Yarrowia lipolytica is an important industrial oleaginous yeast due to its robust phenotypes for effective conversion of inhibitory lignocellulosic biomass hydrolysates into neutral lipids. While lipid accumulation has been well characterized in this organism, its interconnected lipid degradation phenotype is poorly understood during fermentation of biomass hydrolysates. Our investigation into the genetic diversity of undomesticated Y. lipolytica strains, coupled with detailed strain characterization and proteomic analysis, revealed metabolic processes and regulatory elements conferring desirable phenotypes for growth, sugar utilization, and lipid accumulation in undetoxified biomass hydrolysates by these natural variants. This study provides a better understanding of the robust metabolism of Y. lipolytica and suggests potential metabolic engineering strategies to enhance its performance.

The next generation of protein super-fibres: robust recombinant production and recovery of hagfish intermediate filament proteins with fibre spinning and mechanical-structural characterizations.

Native hagfish intermediate filament proteins have impressive mechanical properties. However, using these native fibres for any application is impractical, necessitating their recombinant production. In the only literature report on the proteins (denoted α and É£), heterologous expression levels, using E. coli, were low and no attempts were made to optimize expression, explore wet-spinning, or spin the two proteins individually into fibres. Reported here is the high production (~8 g l-1 of dry protein) of the hagfish intermediate filament proteins, with yields orders of magnitude higher (325-1000×) than previous reports. The proteins were spun into fibres individually and in their native-like 1:1 ratio. For all fibres, the hallmark α-helix to ß-sheet conversion occurred after draw-processing. The native-like 1:1 ratio fibres achieved the highest average tensile strength in this study at nearly 200 MPa with an elastic modulus of 5.7 GPa, representing the highest tensile strength reported for these proteins without chemical cross-linking. Interestingly, the recombinant α protein achieved nearly the same mechanical properties when spun as a homopolymeric fibre. These results suggest that varying the two protein ratios beyond the natural 1:1 ratio will allow a high degree of tunability. With robust heterologous expression and purification established, optimizing fibre spinning will be accelerated compared to difficult to produce proteins such as spider silks.

Differences in Endothelin B Receptor Isoforms Expression and Function in Breast Cancer Cells.

The endothelins and their receptors are best known for their regulation of the vascular system. Their widespread expression in epithelial cells and their overexpression in some tumors has prompted investigation into their ability to regulate cancer progression. In this study, we assessed the mRNA expression of the major endothelin B receptor gene (EDNRB) isoforms and found differences in both mRNA and protein expression in normal breast cells and breast cancer cell lines. Knocking down the EDNRB gene in breast cancer cells altered invasiveness toward endothelin 3 (ET3), and we observed EDNRB isoform-specific regulation of breast cancer cell invasion and cell signaling, as well as isoform- and subtype-specific differences in breast cancer patient survival. The results reported in this study emphasize the importance of the endothelin B receptor in breast cancer. To our knowledge, this study is the first to clarify the differential expression and roles of specific EDNRB isoforms in breast cancer.

Draft Genome Assemblies of Ionic Liquid-Resistant Yarrowia lipolytica PO1f and Its Superior Evolved Strain, YlCW001.

Adaptive laboratory evolution of Yarrowia lipolytica PO1f in the benchmark ionic liquid (IL; 1-ethyl-3-methylimidazolium acetate) produced a superior IL-tolerant microorganism, strain YlCW001. Here, we report the genome sequences of PO1f and YlCW001 to study the robustness of Y. lipolytica and its potential use as a microbial platform for producing fuels and chemicals.

Understanding and Eliminating the Detrimental Effect of Thiamine Deficiency on the Oleaginous Yeast Yarrowia lipolytica.

Thiamine is a vitamin that functions as a cofactor for key enzymes in carbon and energy metabolism in all living cells. While most plants, fungi, and bacteria can synthesize thiamine de novo, the oleaginous yeast Yarrowia lipolytica cannot. In this study, we used proteomics together with physiological characterization to elucidate key metabolic processes influenced and regulated by thiamine availability and to identify the genetic basis of thiamine auxotrophy in Y. lipolytica Specifically, we found that thiamine depletion results in decreased protein abundance for the lipid biosynthesis pathway and energy metabolism (i.e., ATP synthase), leading to the negligible growth and poor sugar assimilation observed in our study. Using comparative genomics, we identified the missing 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase (THI13) gene for the de novo thiamine biosynthesis in Y. lipolytica and discovered an exceptional promoter, P3, that exhibits strong activation and tight repression by low and high thiamine concentrations, respectively. Capitalizing on the strength of our thiamine-regulated promoter (P3) to express the missing gene from Saccharomyces cerevisiae (scTHI13), we engineered a thiamine-prototrophic Y. lipolytica strain. By comparing this engineered strain to the wild-type strain, we revealed the tight relationship between thiamine availability and lipid biosynthesis and demonstrated enhanced lipid production with thiamine supplementation in the engineered thiamine-prototrophic Y. lipolytica strain.IMPORTANCE Thiamine plays a crucial role as an essential cofactor for enzymes involved in carbon and energy metabolism in all living cells. Thiamine deficiency has detrimental consequences for cellular health. Yarrowia lipolytica, a nonconventional oleaginous yeast with broad biotechnological applications, is a native thiamine auxotroph whose affected cellular metabolism is not well understood. Therefore, Y. lipolytica is an ideal eukaryotic host for the study of thiamine metabolism, especially because mammalian cells are also thiamine auxotrophic and thiamine deficiency is implicated in several human diseases. This study elucidates the fundamental effects of thiamine deficiency on cellular metabolism in Y. lipolytica and identifies genes and novel thiamine-regulated elements that eliminate thiamine auxotrophy in Y. lipolytica Furthermore, the discovery of thiamine-regulated elements enables the development of thiamine biosensors with useful applications in synthetic biology and metabolic engineering.

Silkworms with Spider Silklike Fibers Using Synthetic Silkworm Chow Containing Calcium Lignosulfonate, Carbon Nanotubes, and Graphene.

Silkworm silk has become increasingly relevant for material applications. However, the industry as a whole is retracting because of problems with mass production. One of the key problems is the inconsistent properties of the silk. A means by which to improve the silk material properties is through enhanced sericulture techniques. One possible technique is altering the feed of the silkworms to include single-wall carbon nanotubes (SWNTs) or graphene (GR). Recently published results have demonstrated substantial improvement in fiber mechanical properties. However, the effect of the surfactant used to incorporate those materials into the feed on the fiber mechanical properties in comparison to normal silkworm silk has not been studied or reported. Thus, the total effect of feeding the SWNT and GR in the presence of surfactants on silkworms is not understood. Our study focuses on the surfactant [calcium lignosulfonate (LGS)] and demonstrates that it alone results in appreciable improvement of mechanical properties in comparison to nontreated silkworm silk. Furthermore, our study demonstrates that mixing the LGS, SWNT, and GR directly into the artificial diet of silkworms yields improved mechanical properties without decline below the control silk at high doses of SWNT or GR. Combined, we present evidence that mixing surfactants, in this case LGS, directly with the diet of silkworms creates a high-quality fiber product that can exceed 1 GPa in tensile strength. With the addition of nanocarbons, either SWNT or GR, the improvement is even greater and consistently surpasses control fibers. However, feeding LGS alone is a more economical and practical choice to consistently improve the mechanical properties of silkworm fiber.

Exceptional solvent tolerance in Yarrowia lipolytica is enhanced by sterols.

Green organic solvents such as ionic liquids (ILs) have versatile use but are inhibitory to microbes even at low concentrations of 0.5-1.0% (v/v) ILs. We discovered the oleaginous yeast Yarrowia lipolytica can grow in 10% (v/v) of 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]), which makes it more tolerant than most engineered microorganisms and naturally screened isolates. However, the underlying mechanism of IL tolerance in Y. lipolytica is not understood. Through adaptive laboratory evolution, in combination with physiological characterization and omics analysis, we shed light on the underlying mechanism of how Y. lipolytica restructures its membrane to tolerate different types of ILs at high levels up to 18% ILs. Specifically, we discovered that sterols play a key role for exceptional IL tolerance in Y. lipolytica.

Draft Genome Assemblies of Five Robust Yarrowia lipolytica Strains Exhibiting High Lipid Production, Pentose Sugar Utilization, and Sugar Alcohol Secretion from Undetoxified Lignocellulosic Biomass Hydrolysates.

Screening the genetic diversity of 45 Yarrowia lipolytica strains identified five candidates with unique metabolic capability and robustness in undetoxified switchgrass hydrolysates, including superior lipid production and efficient pentose sugar utilization. Here, we report the genome sequences of these strains to study their robustness and potential to produce fuels and chemicals.