Recombinant Umbelopsis ramanniana Diacylglycerol O-acyltransferase 2B (DGAT2B)

What is Umbelopsis ramanniana DGAT2B and what is its primary function?

Umbelopsis ramanniana DGAT2B (Diacylglycerol O-acyltransferase 2B) is an essential enzyme that catalyzes the final and committed step in triacylglycerol (TG) biosynthesis. This enzyme transfers an acyl group from acyl-CoA to diacylglycerol (DAG), forming triacylglycerol, which is the main storage lipid in eukaryotic cells . The full-length protein consists of 349 amino acids and contains highly conserved motifs characteristic of the DGAT2 family . U. ramanniana DGAT2B is particularly important because it is part of the enzymatic machinery that enables this oleaginous fungus to accumulate significant amounts of storage lipids under specific conditions .

How does DGAT2B differ structurally from other DGAT enzymes?

DGAT2B belongs to the DGAT2 family, which differs significantly from the DGAT1 family in protein structure, evolutionary origin, and subcellular localization patterns. Specifically, U. ramanniana DGAT2B contains unique structural elements that distinguish it from other DGAT2 enzymes:

• The protein contains two transmembrane domains that are essential for proper membrane association and enzyme function

• The amino acid sequence (including MEQVQVTALLDHIPKVHWAPLRGIPLKRRLQTSAIVTWLALLPICLIIYLYLFTIPLLWPILIMYTIWLFFDKAPENGGRRISLVRKLPLWKHFANYFPVTLIKEGDLDPKGNYIMSYHPHGIISMAAFANFATEATGFSEQYPGIVPSLLTLASNFRLPLYRDFMMSLGMCSVSRHSCEAILRSGPGRSIVIVTGGASESLSARPGTNDLTLKKRLGFIRLAIRNGASLVPIFSFGENDIYEQYDNKKGSLIWRYQKWFQKITGFTVPLAHARGIFNYNAGFIPFRHPIVTVVGKPIAVPLLAEGETEPSEEQMHQVQAQYIESLQAIYDKYKDIYAKDRIKDMTMIA) contains specific regions that contribute to substrate specificity and catalytic efficiency

• When compared to DGAT2A from the same organism, DGAT2B shows distinct structural features that affect its catalytic properties and substrate preferences

What is known about the organism Umbelopsis ramanniana?

Umbelopsis ramanniana is a cosmopolitan oleaginous fungus with significant ecological and biotechnological importance:

• It was previously classified as Mortierella ramanniana but has been reclassified based on molecular phylogenetics

• Recent research has revealed that what was previously considered U. ramanniana actually consists of five cryptic species, suggesting greater biodiversity and potentially different enzymatic properties among these closely related fungi

• It is commonly found in soil, forest leaf litter, animal dung, and can grow on spore-producing bodies of ascomycete fungi

• U. ramanniana can function as an endophyte within xylem tissue of both healthy and declining conifers, though its exact effect on plant hosts remains unknown

• Taxonomically, it represents a unique group of zygomycete fungi that is distinct from the Mucoromycotina and Mortierellomycotina, forming an early diverging lineage within the Mucoralean fungi

• The fungus has notable biotechnological importance due to its oleaginous nature and tolerance to fungicides of the benomyl group

What expression systems are optimal for producing recombinant DGAT2B?

Based on the current research data, the following expression systems have been successfully employed for DGAT2B production:

• E. coli expression system: The commercial recombinant DGAT2B protein is successfully expressed in E. coli with an N-terminal His-tag . This system provides several advantages:

  • High yield of protein production

  • Relatively simple purification using affinity chromatography

  • Well-established protocols for optimization

• Heterologous yeast expression systems: For functional studies, DGAT2B has been successfully expressed in Saccharomyces cerevisiae, particularly in the quadruple mutant strain H1246 that is inherently defective in neutral lipid biosynthesis . This system offers:

  • A eukaryotic cellular environment more similar to the native conditions

  • The ability to perform functional complementation assays

  • A platform for studying enzyme kinetics in a cellular context

When designing expression strategies, researchers should consider the following methodological considerations:

• Codon optimization for the chosen expression system

• Selection of appropriate affinity tags (His-tag has been successfully used)

• Careful consideration of the purification strategy to maintain enzymatic activity

• Proper folding conditions to ensure the transmembrane domains adopt correct conformations

What are the recommended storage and handling conditions for recombinant DGAT2B?

To maintain optimal activity of recombinant DGAT2B, the following storage and handling protocols are recommended:

Storage conditions:

• Store lyophilized powder at -20°C/-80°C upon receipt

• After reconstitution, store working aliquots at 4°C for up to one week

• For long-term storage, add glycerol to a final concentration of 5-50% (optimally 50%) and store at -20°C/-80°C

• Avoid repeated freeze-thaw cycles as these significantly reduce enzyme activity

Reconstitution protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 50% for long-term storage

• Aliquot to minimize freeze-thaw cycles

The protein is typically supplied in Tris/PBS-based buffer with 6% Trehalose at pH 8.0, which helps maintain stability during lyophilization and storage .

How does DGAT2B's enzymatic activity compare to other DGAT isoforms?

Comparative studies of DGAT isoforms have revealed important functional differences:

This comparative enzymatic efficiency data is particularly relevant when designing experiments to investigate lipid metabolism or when considering DGAT2B for biotechnological applications.

What domains and motifs are critical for DGAT2B catalytic activity?

Several structural elements are essential for DGAT2B function:

• Transmembrane domains: DGAT2B contains two transmembrane domains that are crucial for:

  • Proper localization to the endoplasmic reticulum (ER)

  • Membrane association necessary for accessing lipid substrates

  • Potentially creating a favorable environment for the catalytic reaction

• Conserved motifs: The protein contains three highly conserved motifs typical of DGAT2 family enzymes . Mutation studies in related DGAT2 enzymes indicate these are essential for catalytic activity.

• Unique structural elements: Studies with CtDGAT2 isozymes showed that the efficiency of triacylglycerol production is differentially affected by deletion, insertion, or replacement of amino acids in five regions exclusively present in these DGAT2 isozymes . By extension, similar regions in U. ramanniana DGAT2B likely play important roles in determining its catalytic properties.

Researchers investigating structure-function relationships should focus on these critical domains when designing site-directed mutagenesis experiments or truncation studies.

How can researchers effectively study DGAT2B's role in lipid droplet formation?

To investigate DGAT2B's contribution to lipid droplet biogenesis, researchers should consider these methodological approaches:

• Subcellular localization studies:

  • Immunofluorescence microscopy using tagged DGAT2B constructs to visualize its distribution during lipid droplet formation

  • Live-cell imaging with fluorescently tagged DGAT2B to track dynamic movement between the ER and forming lipid droplets

  • Subcellular fractionation to isolate lipid droplets and quantify associated DGAT2B

• Protein-protein interaction analysis:

  • Co-immunoprecipitation experiments to identify DGAT2B interaction partners

  • In situ proximity ligation assays to confirm interactions in cellular contexts

  • FRET/BRET approaches to study dynamic interactions during lipid droplet formation

• Functional assays:

  • Expression in lipid synthesis-deficient yeast strains (such as S. cerevisiae H1246)

  • Analysis of lipid droplet formation using fluorescent lipid dyes (Oil Red O, BODIPY, etc.)

  • Lipidomic analysis to determine the composition of triacylglycerols produced

• Gain and loss of function approaches:

  • Overexpression studies to assess enhanced lipid droplet formation

  • RNAi or CRISPR-based gene silencing/knockout to evaluate the necessity of DGAT2B

  • Rescue experiments with wild-type or mutant DGAT2B constructs

By combining these approaches, researchers can comprehensively characterize DGAT2B's specific contributions to lipid droplet biogenesis and lipid storage dynamics.

What experimental systems are most suitable for investigating DGAT2B interactions with other lipid metabolism enzymes?

DGAT2B functions within a complex network of lipid metabolism enzymes. To study these interactions, consider these experimental systems:

• Heterologous expression systems:

  • Saccharomyces cerevisiae quadruple mutant strain H1246 provides a neutral lipid biosynthesis-deficient background ideal for reconstitution experiments

  • Mammalian cell lines (HEK293T, COS-7) are suitable for co-expression studies of DGAT2B with potential partner proteins

• Protein interaction detection methods:

  • Co-immunoprecipitation experiments have successfully demonstrated interactions between DGAT2 and MGAT2

  • In situ proximity ligation assays provide evidence of protein interactions in their native cellular context

  • Cross-linking approaches can capture transient interactions

• Functional interaction analyses:

  • Enzyme activity assays with purified proteins to detect synergistic or inhibitory effects

  • Lipid analysis after co-expression of DGAT2B with other enzymes involved in TG synthesis

  • Rescue experiments in cells deficient in multiple lipid synthesis enzymes

• Membrane biology approaches:

  • Isolation of ER membranes to study how membrane composition affects DGAT2B interactions

  • Analysis of how fatty acid supplementation affects DGAT2B localization and its interactions with other proteins

Research has demonstrated that DGAT2 can utilize diacylglycerol generated by MGAT2 for TG synthesis , suggesting that DGAT2B may participate in similar enzyme complexes to enhance the efficiency of the TG synthesis pathway.

How can researchers analyze the impact of DGAT2B mutations on enzyme function and lipid metabolism?

To systematically study DGAT2B structure-function relationships, employ these methodological approaches:

• Site-directed mutagenesis strategy:

  • Target conserved motifs known to be essential for DGAT2 family function

  • Create mutations in the transmembrane domains to assess their role in localization and activity

  • Investigate the five regions unique to fungal DGAT2 isozymes through deletion, insertion, or replacement mutations

• Functional assessment of mutants:

  • In vitro enzyme activity assays using purified wild-type and mutant proteins

  • Complementation assays in S. cerevisiae H1246 strain to assess rescue of growth in fatty acid-containing media

  • Analysis of lipid accumulation in cells expressing mutant proteins using fluorescence microscopy and biochemical quantification

• Structural studies:

  • Protein modeling approaches to predict effects of mutations on structure

  • Limited proteolysis to analyze conformational changes induced by mutations

  • If feasible, X-ray crystallography or cryo-EM studies of the protein structure

• Comparative analysis:

  Mutation Type	Expected Effect on Activity	Methods to Assess
  Transmembrane domain mutations	Altered localization, reduced activity	Subcellular fractionation, immunofluorescence, activity assays
  Conserved motif mutations	Significant reduction in catalytic activity	In vitro enzyme assays, complementation studies
  Unique region modifications	Altered substrate specificity or catalytic efficiency	Lipidomic analysis of produced TGs, kinetic studies
  C-terminal truncations	Potential loss of regulatory interactions	Protein-protein interaction studies, activity assays

Studies with related DGAT2 enzymes have demonstrated that efficiency of triacylglycerol production is significantly affected by modifications in key structural regions, providing a foundation for similar investigations with DGAT2B .

How might DGAT2B be utilized in metabolic engineering for enhanced oil production?

DGAT2B offers significant potential for increasing lipid yields in engineered organisms:

The biotechnological value of DGAT2B stems from its demonstrated ability to rescue growth in transformed S. cerevisiae mutant cells by incorporating free fatty acids into triacylglycerol , suggesting it could be an effective tool for enhancing lipid production in various organisms.

What analytical methods are most appropriate for assessing DGAT2B activity in experimental systems?

Researchers can employ these analytical techniques to accurately evaluate DGAT2B function:

• In vitro enzyme assays:

  • Radiometric assays using [14C]-labeled acyl-CoA to measure incorporation into triacylglycerol

  • Spectrophotometric assays tracking either substrate consumption or product formation

  • HPLC or TLC-based methods to separate and quantify reaction products

• Cellular lipid analysis:

  • Fluorescent lipid dyes (BODIPY, Nile Red) for microscopy and flow cytometry-based quantification

  • Extraction and gravimetric determination of total lipids

  • Thin-layer chromatography for separation of neutral lipid classes

• Advanced analytical methods:

  • Lipidomics using LC-MS/MS to characterize the molecular species of triacylglycerols produced

  • Gas chromatography to analyze fatty acid composition of synthesized lipids

  • Stable isotope labeling to track flux through the triacylglycerol synthesis pathway

• Sensitivity and specificity considerations:

  Analytical Method	Sensitivity	Specificity	Best Used For
  Radiometric assays	Very high	High	Precise kinetic measurements
  Fluorescence methods	High	Moderate	High-throughput screening
  Mass spectrometry	High	Very high	Detailed product characterization
  Complementation assays	Moderate	High	Functional validation in vivo

When selecting methods, researchers should consider the specific research question, required sensitivity, available equipment, and whether quantitative or qualitative data is needed for their experimental objectives.