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Hopanoids

Hopanoids are a class of pentacyclic triterpenoid lipids primarily synthesized by bacteria, functioning as structural and functional analogs to sterols like cholesterol in eukaryotic cells.^[1] These rigid, planar molecules, featuring a fused ring system derived from squalene, integrate into bacterial membranes to enhance stability, modulate fluidity, and promote ordered lipid domains essential for cellular integrity.^[2] Produced by approximately 10% of known bacterial species, including diverse groups such as Proteobacteria, Cyanobacteria, and Acidobacteria, hopanoids are absent in eukaryotes but have been detected in some lichens and plants through bacterial associations.^[1] Structurally, hopanoids consist of a core scaffold with four six-membered rings (A–D) and one five-membered ring (E), totaling five fused rings in contrast to the four rings of sterols.^[1] They exist in two main forms: C30 hopanoids, such as diploptene and diplopterol, which lack extended side chains, and C35 bacteriohopanepolyols, like bacteriohopanetetrol, which feature polyhydroxylated side chains for greater polarity and membrane anchoring.^[2] Biosynthesis begins with the oxygen-independent cyclization of the linear precursor squalene by squalene-hopene cyclases (SHCs), encoded by genes like shc, followed by modifications via enzymes such as HpnP for methylation or HpnH for ribose addition to form extended variants.^[1] In some bacteria, such as Bradyrhizobium species, hopanoids covalently link to lipid A in the outer membrane, creating hybrid structures that span the bilayer.^[3] In bacterial membranes, hopanoids rigidify lipid packing by interacting with saturated phospholipids, promoting a liquid-ordered (L₀) phase akin to sterol-induced domains in eukaryotes, which inhibits gel-phase formation and enhances compartmentalization.^[2] They confer resistance to environmental stresses, including extreme pH, temperature fluctuations, osmotic pressure, and oxidative damage, as demonstrated in mutants lacking hopanoid synthesis that exhibit increased membrane permeability and growth defects.^[3] For instance, in Rhodopseudomonas palustris, hopanoids maintain pH homeostasis, while in Nostoc punctiforme, they bolster tolerance to osmotic and oxidative challenges.^[1] Beyond membrane function, hopanoids play pivotal roles in microbial ecology and symbiosis; in nitrogen-fixing bacteria like Bradyrhizobium diazoefficiens, they are essential for nodulation and efficient legume symbiosis, supporting plant growth through enhanced nitrogen fixation.^[3] Geologically, fossilized hopanoid derivatives, known as hopanes, serve as biomarkers tracing bacterial activity back at least 1.73 billion years,^[4] providing insights into ancient microbial ecosystems. Recent studies as of 2025 have re-established 2-methylhopanes as specific cyanobacterial biomarkers before 750 million years ago and highlighted horizontal gene transfer in the evolution of hopanoid biosynthesis.^[5]^[6] Their study also informs antibiotic development, as disrupting hopanoid pathways sensitizes bacteria to stressors, highlighting potential therapeutic targets.^[3]

Structure and properties

Core structure

Hopanoids are characterized by a pentacyclic triterpenoid core known as the hopane skeleton, which consists of four fused six-membered rings (designated A through D) and a terminal five-membered ring (E), forming a compact structure with 30 carbon atoms in its basic form.^[1] This rigid scaffold provides the foundational architecture for all hopanoids, enabling their role as membrane components in bacteria. In simple hopanoid variants, key functional groups distinguish basic forms such as hopanol and diploptene. Hopanol features a hydroxyl group at the C-3 position (3β-hydroxyhopane), contributing to its polarity and integration into lipid membranes.^[1] Diploptene, an unsaturated precursor, contains a double bond between C-22 and C-29 in the side chain (exocyclic methylene at C-22), which influences its biosynthetic pathway and membrane ordering properties.^[7] The hopane core exhibits specific stereochemistry at its eight chiral centers, defined as 5β,9β,10β,13β,14α,17α,18α,20R, which ensures the molecule's three-dimensional rigidity and proper orientation within bacterial membranes.^[1] This configuration is conserved across hopanoids and arises during the cyclization of the precursor squalene. The basic hopane formula is C₃₀H₅₂, reflecting its fully saturated hydrocarbon nature without side chain extensions.^[1] Structurally, hopanoids resemble eukaryotic sterols like cholesterol, sharing a similar planar ring system and side chain at C-17, but differ by incorporating an additional five-membered E ring and lacking an oxygen-containing functional group in the core side chain, making them pentacyclic rather than tetracyclic.^[1] Hopanoids exhibit rigidity due to their planar fused-ring system, which promotes ordered lipid packing in membranes, and vary in polarity based on functional groups, with hydroxylated forms enhancing amphiphilicity for bilayer integration.^[1]

Variations and classification

Hopanoids exhibit structural diversity primarily through modifications to their side chain and functional groups attached to the pentacyclic hopane core, enabling their classification into several major categories based on carbon length, polarity, and chemical composition. The most prevalent group comprises bacteriohopanepolyols (BHPs), which are extended C35 compounds featuring a polyfunctionalized side chain derived from ribose, typically bearing four to six hydroxyl or amino groups for enhanced polarity and membrane anchoring. A key example is bacteriohopanetetrol (BHT), or bacteriohopane-32,33,34,35-tetrol, characterized by four hydroxyl groups on the C32–C35 side chain, making it one of the most abundant BHPs in bacterial membranes and environmental samples.^[1] Another subclass of BHPs includes amino-containing variants, such as aminotriol (35-aminobacteriohopane-32,33,34-triol), which incorporates an amino group at the C35 position alongside three hydroxyls, contributing to its distinct polarity and often associated with specific bacterial metabolisms like methanotrophy.^[1] In contrast, simpler C30 hopanoids lack the extended side chain and include diplopterol, a hopanoid alcohol with a hydroxyl group at C-22, serving as a biosynthetic precursor and found across diverse prokaryotes without the polyol complexity of BHPs.^[8] Geohopanoids represent diagenetic transformation products of these biohopanoids, primarily consisting of non-polar hopane hydrocarbons (e.g., C30–C35 hopanes) formed through defunctionalization and cyclization during sediment burial, preserving the core skeleton as geological biomarkers.^[1] Side chain variations further diversify hopanoids, with extensions from C31 to C35 achieved through direct alkylation or attachment of ribose-derived moieties, altering hydrophobicity and functionality. For instance, methylation at the 2-position (2-methyl) or 3-position (3-methyl) on the A-ring or side chain introduces steric bulk, as seen in 2-methyl-BHT linked to cyanobacterial producers or 3-methyl-BHT associated with methylotrophic bacteria.^[1] Specific ribosyl attachments yield compounds like ribosylhopane, a C35 intermediate featuring a ribofuranose-linked side chain, and adenosylhopane, a nucleoside analog with an adenosine moiety at C35, both exemplifying the transitional forms in BHP structural evolution and aiding in taxonomic classification of producing bacteria.^[1] Aromatic hopanoids arise as degradation derivatives, often through aromatization of rings during diagenesis, including tetra- and hexacyclic variants predominant in carbonate rocks and oils, which retain hopane-like skeletons but with fused aromatic systems for increased stability in geological contexts.^[9] These modifications, while not biosynthesized directly, reflect post-depositional alterations of biohopanoids like BHPs, contributing to the fossil record's diversity.

Biosynthesis

Squalene synthesis

Hopanoid biosynthesis begins with the formation of the linear triterpene precursor squalene, which is assembled from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) through sequential condensations. In the majority of bacteria, including those that produce hopanoids, IPP and DMAPP are generated via the 1-deoxy-D-xylulose 5-phosphate (DOXP) or methylerythritol 4-phosphate (MEP) pathway, a non-mevalonate route that starts from pyruvate and glyceraldehyde 3-phosphate. These C5 units are then elongated by farnesyl diphosphate synthase (encoded by ispA), first forming geranyl pyrophosphate (GPP) from DMAPP and IPP, followed by the addition of another IPP to yield farnesyl pyrophosphate (FPP). FPP serves as the immediate precursor for squalene synthesis.^[10] The conversion of two FPP molecules to squalene in hopanoid-producing bacteria proceeds via a distinctive three-enzyme pathway, differing from the single-enzyme squalene synthase used in eukaryotes. This process involves HpnD, which catalyzes the initial head-to-head condensation of two FPP units to form presqualene diphosphate (PSPP) and release one pyrophosphate (PPi); HpnC, which promotes the hydrolytic rearrangement of PSPP through a cyclopropylcarbinol intermediate to yield 10R-hydroxysqualene (HSQ) and a second PPi; and HpnE, an FAD-dependent short-chain dehydrogenase/reductase that reduces HSQ to squalene using NADPH (or NADH) as a cofactor. The genes encoding these enzymes (hpnD, hpnC, and hpnE) are typically clustered with other hopanoid biosynthesis genes, reflecting their coordinated role in the pathway. The overall reaction is:

$2 \text{ FPP} + \text{NADPH} \rightarrow \text{squalene} + 2 \text{PP}_\text{i} + \text{NADP}^+

This mechanism ensures efficient production of squalene as the substrate for hopanoid cyclization.^[11]^[12] While the DOXP/MEP pathway is predominant, certain bacteria, such as Zymomonas mobilis, utilize the alternative mevalonate pathway for IPP synthesis. This route begins with the condensation of three acetyl-CoA molecules to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), which is reduced to mevalonate; mevalonate is then sequentially phosphorylated, decarboxylated, and isomerized to IPP. Despite this variation in early steps, the downstream assembly of FPP and squalene remains analogous, highlighting the conservation of isoprenoid elongation mechanisms across bacterial lineages.^[13]

Squalene cyclization

The squalene-hopene cyclase (Shc), also known as hopene synthase, is a membrane-bound enzyme essential for hopanoid biosynthesis in bacteria. This monotopic protein, with a molecular mass of approximately 70-76 kDa, integrates partially into the cytoplasmic membrane through hydrophobic residues and positively charged amino acids flanking its transmembrane segments. Shc catalyzes the cyclization of the linear triterpene squalene into the pentacyclic hopene core in a single enzymatic step, without requiring an epoxide intermediate unlike eukaryotic oxidosqualene cyclases. The enzyme has been structurally characterized at 2.0 Å resolution from Alicyclobacillus acidocaldarius, revealing a barrel-shaped active site that accommodates the substrate.^[14] The cyclization mechanism begins with protonation of the terminal double bond at C-2/C-3 of squalene by an aspartate residue in the conserved DXDD motif (e.g., Asp376 in A. acidocaldarius Shc), generating an initial allylic carbocation. This initiates a cascade of electrophilic additions and ring closures, forming five fused rings (A-E) in a 6-6-6-6-5 configuration through stepwise cyclization via carbocation intermediates. Key intermediates include the A/B bicyclic cyclohexyl cation after the first two ring formations, followed by C-ring closure and subsequent D- and E-ring formations without viable five-to-six-membered ring expansions. The process concludes with deprotonation from the methyl group at C-24, yielding hopene (primarily diploptene) as the major product, with minor amounts of hopanol (diplopterol) under certain conditions. The overall reaction can be represented as:

\text{squalene} \xrightarrow{\text{Shc, H}^{+}} \text{hopene} + \text{H}^{+}

This protonation-deprotonation cycle ensures stereochemical fidelity, with free energy simulations indicating kinetic control favors the pentacyclic product (99% yield).^[15]^[16] Shc exhibits high stereospecificity, enforcing an all-chair conformation of squalene folded on its β-face to direct the cyclization cascade correctly. This results in trans fusions between all rings and the characteristic stereochemistry at chiral centers, preventing alternative folding modes that could lead to aberrant products. The enzyme's active site, lined with aromatic residues like tryptophan, stabilizes the carbocation intermediates through cation-π interactions, further enforcing this specificity.^[15] The genetic basis of Shc is encoded by the shc gene (also termed hpnF), often part of a biosynthetic cluster such as hpnABCDEF in bacteria. In Bradyrhizobium japonicum, the 1983 bp shc gene has been cloned and expressed in Escherichia coli, producing a soluble recombinant enzyme that cyclizes squalene to hopene and diplopterol in vitro. This gene shows 38-43% sequence similarity to eukaryotic oxidosqualene cyclases, highlighting evolutionary conservation. Similar shc homologs are found in diverse bacteria, including Zymomonas mobilis and Alicyclobacillus acidocaldarius, underscoring its role across prokaryotic hopanoid producers.^[17]

Functionalization and modifications

Following the cyclization of squalene to form the hopene core, such as diploptene, subsequent enzymatic modifications introduce functional groups to generate diverse hopanoids, including bacteriohopanepolyols (BHPs). These modifications primarily occur through oxidation, glycosylation, methylation, and other additions, enabling the lipids to integrate into bacterial membranes with enhanced properties. The extension to C-35 BHPs involves an ATP-dependent pathway where the radical S-adenosylmethionine (SAM) enzyme HpnH catalyzes the stereoselective addition of a 5'-deoxyadenosyl radical to diploptene, forming adenosylhopane as an intermediate. HpnG, a purine nucleoside phosphorylase, then cleaves the adenine to yield ribosylhopane, which serves as the scaffold for polyol chain assembly. Unidentified enzymes convert ribosylhopane to bacteriohopanetetrol (BHT), the most common BHP, through sequential addition and modification of the side chain to form the pentol structure at C-31 to C-35. Glycosyltransferases, such as HpnI, further modify BHT by attaching sugar moieties like N-acetylglucosamine, producing variants such as N-acetylglucosaminyl-BHT. Squalene-hopene cyclase variants (encoded by shc/hpnF) can influence the initial core structure, leading to stereoisomeric hopene products that affect downstream modifications.^[1]^[18]^[19] Additional modifications include methylation at C-2 or C-3 positions. The radical SAM methyltransferase HpnP catalyzes C-2 methylation on both C-30 and C-35 hopanoids, yielding 2-methyl derivatives like 2-methyl-BHT, which are prevalent in certain proteobacteria and cyanobacteria. Similarly, HpnR performs C-3 methylation, though less common. Other alterations encompass sulfation and amination; for instance, HpnO facilitates amination to form bacteriohopanepentol aminotriol by adding amino groups to the polyol chain, while sulfation occurs in select bacteria to produce sulfated BHPs, enhancing polarity. These steps are mediated by enzymes within the hpn biosynthetic gene cluster, which typically includes hpnB, hpnP, hpnH, hpnG, and glycosyltransferases like hpnI. The role of HpnB, predicted as a C-30 dehydrogenase, remains unconfirmed in BHP biosynthesis but may contribute to other hopanoid variants.^[20]^[1]^[21] Regulation of these modifications is governed by the hpn gene cluster, often organized as an operon adjacent to shc, ensuring coordinated expression. In many bacteria, such as alphaproteobacteria, hopanoid biosynthesis is oxygen-independent at the enzymatic level, but cluster expression can be oxygen-dependent, upregulated under aerobic conditions to support membrane adaptation during oxidative stress. This regulation links to environmental cues, with the cluster responding to growth phases or symbiosis signals in organisms like Bradyrhizobium.^[1]^[21]

Biological roles

Membrane stabilization

Hopanoids stabilize bacterial membranes primarily through their intercalation into lipid bilayers, where their rigid pentacyclic structure interacts with phospholipid acyl chains to promote the formation of liquid-ordered (Lₒ) phases and thereby reduce membrane permeability.^[22] This process modulates membrane fluidity by condensing the lipid packing and preventing excessive disorder, analogous to the role of cholesterol in eukaryotic plasma membranes.^[23] In model systems composed of bacterial lipids like lipid A, hopanoids such as diplopterol inhibit sharp gel-to-liquid crystalline phase transitions, effectively broadening the temperature range over which membranes maintain structural integrity.^[22] Like sterols, hopanoids enhance the order of saturated phospholipids, such as lipid A prevalent in bacterial outer membranes, by favoring interactions with saturated acyl chains that increase overall bilayer order.^[23] This ordering effect is particularly pronounced in saturated lipid environments, where hopanoids reduce the area per lipid molecule and limit passive diffusion across the membrane, contributing to barrier function. In contrast to cholesterol, which orders both saturated and certain unsaturated lipids more uniformly, hopanoids exhibit a stronger preference for saturated phospholipids, reflecting adaptations to the lipid composition of prokaryotic membranes.^[23] Experimental evidence from hopanoid-deficient mutants underscores their essential role in membrane stabilization; for instance, Δshc mutants in Rhodopseudomonas palustris display highly fluid outer membranes, increased permeability to detergents like SDS, and growth defects under environmental stresses that challenge membrane integrity.^[24] These mutants often exhibit slower growth rates and heightened sensitivity to antibiotics such as polymyxin B, attributable to disrupted lipid ordering and phase behavior.^[25] Complementation with exogenous hopanoids or sterols restores membrane order and rescues these phenotypes, confirming the functional equivalence in rigidity enhancement.^[23] In Gram-negative bacteria, hopanoids are predominantly enriched in the outer membrane, where they integrate with lipopolysaccharide (LPS) components like lipid A to maintain asymmetry and high lateral order, preventing phase separation and bolstering resistance to external perturbations.^[23] This distribution is critical for the structural robustness of the cell envelope, as evidenced by the severe outer membrane defects observed in hopanoid biosynthesis knockouts.^[1]

Stress response and adaptation

Hopanoids play a crucial role in bacterial adaptation to environmental stresses by reinforcing membrane integrity and modulating lipid dynamics, enabling survival under conditions such as elevated temperatures, low pH, and limited oxygen availability.^[1] In particular, extended hopanoids with a C35 side chain are essential for thermotolerance, allowing bacteria to maintain growth at temperatures such as 37°C, and for anaerobiosis, supporting microaerobic conditions with oxygen levels as low as 0.5%.^[26] Mutants lacking these C35 hopanoids, such as ΔhpnH strains in Bradyrhizobium diazoefficiens, exhibit heightened sensitivity to heat stress, failing to grow at 37°C, and to acidic environments, showing no growth at pH 6.^[27] These adaptive functions are evident in studies of hopanoid-deficient mutants, where loss of production impairs cellular resilience. For instance, in Rhodopseudomonas palustris, hopanoid deletion leads to disrupted lipid remodeling and reduced viability under microaerobic growth, highlighting the lipids' necessity for oxygen-limited environments.^[28] At the molecular level, hopanoids contribute to stress tolerance through specific interactions that preserve membrane function. They facilitate hydrogen bonding between lipid head groups, promoting ordered packing that resists phase transitions induced by stressors like ethanol or detergents.^[29] Additionally, hopanoids regulate proton and cation leakage across the membrane, maintaining pH homeostasis and preventing ion imbalances during acid exposure or thermal shifts.^[1] In Zymomonas mobilis, these inter-lipid hydrogen bonds and hydrophobic effects stabilize membranes against solvent penetration, with hopanoid composition directly influencing tolerance thresholds.^[29] The expression of hopanoid biosynthesis genes, such as hpnP in the hpn cluster, is upregulated under stress conditions to enhance adaptation. In Rhodopseudomonas palustris, the ECF sigma factor EcfG, part of the general stress response, induces transcription of the hpnP gene during heat shock, leading to increased hopanoid levels that bolster membrane stability.^[30] This regulatory mechanism ensures timely reinforcement of membrane properties in response to environmental challenges.

Symbiotic interactions

Hopanoids play a crucial role in the symbiotic interactions between rhizobial bacteria and legumes, particularly in facilitating nitrogen fixation within root nodules. In the Bradyrhizobium diazoefficiens-soybean symbiosis, hopanoids promote nodule formation and enhance nitrogenase activity, as demonstrated by studies showing that hopanoid-deficient mutants produce approximately 76% fewer nodules and exhibit 65% reduced nitrogen fixation compared to wild-type strains.^[31] These lipids are essential for the bacteria's transition from free-living to symbiotic states, enabling effective colonization and persistence in the host plant.^[31] The primary mechanism involves the stabilization of bacteroid membranes under microoxic conditions prevalent in nodules, where oxygen levels are low to protect oxygen-sensitive nitrogenase. Specific hopanoid classes, such as bacteriohopanetetrol (BHT), are vital for maintaining membrane rigidity and integrity during this process; BHT levels increase significantly under microaerobic conditions, aiding infection thread penetration and progression in host roots.^[26] In contrast, mutants lacking extended hopanoids like those produced by the hpnH gene show disrupted bacteroid envelopes, leading to nodule necrosis and impaired nitrogen fixation, particularly in symbioses with certain legumes.^[26] Evidence from hopanoid mutants underscores their indispensability for successful symbiosis, with complete depletion resulting in low bacteroid density, disorganized nodule structures, and symbiosis failure over extended periods.^[31] The 2015 analysis revealed differential impacts of hopanoid classes, where C35 hopanoids (including BHT) are critical for symbiotic performance under microoxic stress, while their effects are less pronounced in free-living states.^[26] This stress adaptation in symbiotic environments highlights hopanoids' targeted role beyond general membrane functions. Hopanoids are particularly prominent in rhizobial-legume interactions involving tropical crops, such as those with Aeschynomene species, where they enhance bacterial fitness in acidic, high-temperature soils conducive to these symbioses.^[31] Their production supports robust nitrogen-fixing partnerships essential for legume productivity in tropical agriculture.

Paleobiological significance

Geobiomarkers in sediments

Hopanoids serve as molecular fossils, known as geohopanoids or hopanes, that preserve evidence of ancient bacterial life in sedimentary rocks.^[32] These compounds undergo diagenetic transformations during burial, where biohopanoids lose functional groups through dehydration, forming hopenes that further stabilize via aromatization (double bond migration) and reduction (hydrogenation) into saturated hopanes.^[33] This process renders hopanes highly resistant to degradation, allowing their preservation in sediments for billions of years, with examples dating back to approximately 1.64 billion years ago in the Barney Creek Shale.^[34] Geohopanoids are important components of sedimentary organic matter and play a central role in petroleum geochemistry for correlating oils to source rocks.^[35] Their structural stability, stemming from the pentacyclic triterpenoid skeleton, facilitates long-term burial without significant alteration.^[33] Quantification of hopanoids in sediments typically employs gas chromatography-mass spectrometry (GC-MS), which separates and identifies hopane homologues based on mass-to-charge ratios.^[36] Recent protocols enable high-throughput extraction and analysis from complex matrices like soils, involving solvent extraction, saponification, and derivatization for polar hopanoids. In paleoenvironmental reconstruction, hopanes act as indicators of bacterial biomass and activity, reflecting the prevalence of prokaryotic communities in ancient settings.^[37] For instance, elevated hopane concentrations in Proterozoic sediments, such as those from approximately 1.32 billion years ago, signal dominant bacterial contributions to organic matter deposition and hydrocarbon generation.^[38]

2-Methylhopanoids

2-Methylhopanoids are a subclass of hopanoids characterized by a methyl group attached at the C-2 position of the hopane skeleton, distinguishing them from unsubstituted hopanoids.^[20] This methylation is catalyzed by the radical S-adenosylmethionine (SAM) enzyme HpnP, which is predominantly found in cyanobacteria and acts on bacteriohopanepolyol intermediates during hopanoid biosynthesis.^[20] The resulting 2-methylbacteriohopanepolyols serve as precursors to sedimentary 2-methylhopanes, which are diagenetically altered forms preserved in ancient rocks. In paleobiology, 2-methylhopanoids function as geobiomarkers for ancient cyanobacterial activity, particularly oxygenic photosynthesis. The 2-methylhopane index (2-MHI), calculated as the ratio of 2α-methylhopane to the sum of hopane and 2α-methylhopane concentrations, quantifies their relative abundance in sediments. Elevated 2-MHI values, often exceeding 1%, in 2.7 billion-year-old (Ga) shales from the Pilbara Craton in Western Australia indicate the presence of cyanobacteria and the advent of oxygenic photosynthesis by the late Archean. These findings, based on solvent-extracted bitumens analyzed via gas chromatography-mass spectrometry, suggest that cyanobacteria contributed significantly to primary production as early as 2.7 Ga. The initial interpretation positioned 2-methylhopanoids as exclusive biomarkers for cyanobacteria, stemming from observations that 2-methylbacteriohopanepolyols were abundant in cultured cyanobacteria and mats but rare or absent in most other bacteria. However, subsequent discoveries challenged this specificity, revealing 2-methylhopanoid production in diverse taxa. For instance, anoxygenic phototrophic bacteria such as Rhodopseudomonas palustris synthesize substantial quantities of 2-methylbacteriohopanepolyols via an orthologous HpnP enzyme, complicating their use as unambiguous proxies for oxygenic photosynthesis.^[39] Similarly, low levels of 2-methylhopanoids have been documented in methylotrophic bacteria, including pink-pigmented facultative methylotrophs related to Methylobacterium, further broadening potential biological sources. Recent advances, particularly genetic surveys and compound-specific isotope analyses, have refined the interpretive framework for 2-methylhopanoids. Phylogenetic analysis of hpnP genes indicates that C-2 methylation capability was present in the last common ancestor of crown-group cyanobacteria but was laterally transferred to Alphaproteobacteria only after approximately 750 million years ago (Ma).^[5] Consequently, elevated 2-MHI values in Precambrian sediments older than 750 Ma likely reflect primarily cyanobacterial sources, with carbon isotopic compositions (δ¹³C) of 2-methylhopanes often depleted relative to bulk organic matter, consistent with nitrogen-fixing cyanobacteria.^[5] In younger rocks, such as those from the mid-Proterozoic, revised 2-MHI calculations (typically <1%) account for non-cyanobacterial contributions, enhancing their utility in reconstructing ancient microbial ecosystems.^[5]

3-Methylhopanoids

3-Methylhopanoids are a subclass of bacteriohopanepolyols characterized by a methyl group at the C-3 position of the hopane skeleton, a modification catalyzed by the radical S-adenosylmethionine (SAM) enzyme HpnR, which is predominantly found in aerobic methanotrophic and methylotrophic bacteria.^[40] This methylation occurs post-cyclization during hopanoid biosynthesis and is linked to organisms capable of utilizing one-carbon compounds like methane under oxic conditions.^[40] As geological biomarkers, 3-methylhopanoids, particularly their diagenetic products like 3-methylhopanes, are elevated in ancient sediments associated with methane oxidation environments, serving as indicators of early aerobic methanotrophy. For instance, in the ~2.5 billion-year-old (Ga) Hamersley Basin formations in Western Australia, high abundances of 3β-methylhopanes alongside other sterane and hopane markers suggest the presence of microaerophilic methanotrophic bacteria during the Archean, coinciding with the Great Oxidation Event and the onset of widespread aerobic methane consumption.^[41] This interpretation is supported by their co-occurrence with 13C-depleted organic carbon isotopes (δ¹³C values as low as -30‰), which reflect the incorporation of isotopically light methane-derived carbon into bacterial biomass and lipids.^[41]^[42] Modern evidence for their production comes from cultured aerobic methanotrophs, such as Methylococcus capsulatus, where HpnR-mediated synthesis yields 3-methylbacteriohopanepolyols that exhibit strong 13C depletion (up to -60‰ relative to substrate), mirroring ancient signatures and confirming their utility as proxies for methanotrophic activity.^[40]^[42] Primarily of bacterial origin, 3-methylhopanoids are rarely detected in eukaryotes, with their distribution in the geological record primarily tracing carbon cycling processes in paleoenvironments influenced by methane fluxes, such as ancient wetlands, marine seeps, and oxygenated ocean margins.^[40]^[41] This biomarker specificity has enabled reconstructions of microbial ecosystems and global biogeochemical dynamics over Earth's history, particularly in linking biological innovations to atmospheric oxygenation.^[41]

Applications and future directions

Agricultural uses

Hopanoids have emerged as key components in biofertilizers designed to enhance symbiotic nitrogen fixation between rhizobial bacteria and leguminous crops. Inoculation of soils with hopanoid-producing strains of Bradyrhizobium diazoefficiens, such as the commercial inoculant USDA110, promotes efficient nodulation and nitrogen fixation in soybeans (Glycine max), a major tropical legume.^[43] This approach leverages the bacteria's natural production of hopanoids to improve symbiosis under field conditions, potentially reducing reliance on synthetic nitrogen fertilizers.^[44] Experimental evidence demonstrates that hopanoid-producing Bradyrhizobium strains significantly outperform hopanoid-deficient mutants in symbiotic performance. In greenhouse studies, plants inoculated with wild-type B. diazoefficiens developed approximately 100 nodules per plant, compared to only about 24 nodules (a 76% reduction) with hopanoid mutants, alongside a 72% decrease in nodule dry mass.^[43] Nitrogen fixation rates, measured by acetylene reduction assay, were ~2 × 10⁵ nmol/hour/plant in wild-type symbioses versus ~0.65 × 10⁵ nmol/hour/plant (a 65% reduction) in mutants, correlating with stunted plant growth and chlorosis in the latter.^[43] Patented biofertilizer formulations incorporating hopanoid-producing rhizobia, including Bradyrhizobium species, have been developed to apply these bacteria via seed coating or soil drenching, enhancing nodulation efficiency and plant vigor in legumes like soybean.^[44] At the mechanistic level, hopanoids stabilize bacteroid membranes within root nodules, conferring tolerance to stresses such as fluctuating pH, temperature, and osmotic pressure that commonly disrupt symbiosis.^[43] This membrane reinforcement supports persistent bacteroid occupancy and sustained nitrogenase activity, as evidenced by higher infection zone densities (~50% in wild-type versus ~33% in mutants) and improved motility for nodule invasion.^[43] Such stabilization promotes long-term symbiotic interactions, aligning with broader roles in plant-microbe associations.^[43] Despite these promising results from controlled experiments, challenges remain in scaling hopanoid-based biofertilizers for widespread agricultural use. Field trials are essential to validate performance under variable soil and climatic conditions, where environmental factors could influence hopanoid efficacy and bacterial survival.^[43] Ongoing research focuses on engineering rhizobial strains for consistent hopanoid production to ensure reliable yield benefits in tropical cropping systems.^[44]

Industrial applications

Hopanoids play a key role in industrial microbiology by enhancing the membrane stability of fermentative bacteria used in food production. Analysis of hopanoid content in these bacteria reveals diverse profiles, including bacteriohopanetetrol and diploptene, which correlate with their tolerance to fermentation stresses.^[45] In biofuel production, hopanoids improve microbial robustness against ethanol toxicity, a critical factor for efficient fermentation. In Zymomonas mobilis, a key ethanol producer, specific hopanoid compositions, particularly extended forms, mediate growth and survival under high ethanol concentrations by stabilizing membrane integrity and reducing permeability.^[29] Knockdown studies demonstrate that altering hopanoid levels directly impacts ethanol tolerance, suggesting their manipulation could optimize yields in industrial bioreactors.^[29] Similarly, hopanoids' role in membrane stabilization under stress conditions supports potential applications in bioremediation, where robust bacteria degrade pollutants in harsh environments.^[1] Emerging research focuses on engineering hopanoid-overproducing strains to boost production of industrial enzymes and lipids. Synthetic biology approaches enable enhanced hopanoid production, potentially increasing cellular tolerance to multiple stresses and facilitating scalable biomanufacturing.^[1] However, natural hopanoid yields in most bacteria remain low, necessitating advanced synthetic biology tools to achieve commercially viable levels.^[1]

Medical and biotechnological potential

Hopanoids have emerged as promising targets for novel antibiotics due to their essential role in maintaining bacterial membrane integrity, particularly in Gram-negative pathogens resistant to conventional treatments. Inhibiting hopanoid biosynthesis, such as through squalene-hopene cyclase (Shc) blockers, sensitizes bacteria to environmental stresses and potentiates existing antibiotics. For instance, fosmidomycin, which disrupts the isoprenoid precursor pathway for hopanoids, reduces membrane hopanoid levels in Burkholderia species and enhances the efficacy of polymyxin B by increasing outer membrane permeability.^[46] Similarly, mutants lacking Shc exhibit heightened sensitivity to antibiotics like polymyxin B and erythromycin, suggesting that Shc inhibitors could serve as adjuvants in combination therapies against acid-tolerant or stress-resistant bacteria.^[47] The covalent linkage between hopanoids and lipid A in certain bacteria further underscores their contribution to envelope robustness, offering a specific vulnerability for therapeutic intervention. In Bradyrhizobium symbionts, hopanoid-lipid A hybrids rigidify the outer membrane, conferring resistance to oxidative, acidic, and detergent stresses; disrupting this interaction could compromise Gram-negative barriers, facilitating antibiotic penetration.^[3] Early inhibitors like 2,3-azasqualene have demonstrated selective toxicity against hopanoid-producing bacteria by blocking squalene cyclization, highlighting the feasibility of narrow-spectrum agents that spare sterol-dependent eukaryotes.^[48] In biotechnology, synthetic hopanoids show potential as cholesterol mimics in liposomal formulations for improved drug delivery. Their ability to order phospholipid bilayers and reduce permeability parallels sterols, enabling stable liposomes that encapsulate therapeutics with enhanced rigidity and controlled release. Experiments with diplopterol-incorporated liposomes confirm that hopanoids condense membranes and modulate phase behavior similarly to cholesterol, potentially addressing limitations in sterol-based systems for vaccine or gene delivery.^[49] Recent insights into hopanoid transport pathways, including a novel ATP-binding cassette system in Proteobacteria (as of a July 2025 preprint), open avenues for engineering bacterial production of tailored hopanoids for biomedical applications.^[50] Despite these advances, challenges persist in translating hopanoid-targeted strategies to clinical use, including off-target toxicity from broad isoprenoid inhibition and the need for high specificity against pathogenic bacteria. Research remains in early stages, with most evidence from in vitro and model organism studies, necessitating further pharmacokinetic and safety evaluations.^[1]

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Isoprenoid biosynthesis in bacteria: a novel pathway for the early ...
A novel biosynthetic route for the early steps of isoprenoid biosynthesis. The C5 framework of isoprenic units results most probably (i) from the condensation ...
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Biosynthesis of Squalene from Farnesyl Diphosphate in Bacteria
Apr 20, 2015 · Squalene synthase (SQS) is a bifunctional enzyme that catalyzes the condensation of two mols. of farnesyl diphosphate (FPP) to give presqualene ...
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Cloning and characterization of a gene from Escherichia coli ... - PNAS
The only genes identified in E. coli related to isoprenoid biosynthesis are ispA and ispB, which encode farnesyl-diphosphate synthase (21) and octaprenyl- ...
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Aug 1, 1993 · In contrast, no intermediates of this reaction sequence could be detected using cell-free extracts of Zymomonas mobilis and Escherichia coli.
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https://pubs.acs.org/doi/10.1021/acscentsci.5b00115
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https://www.pnas.org/doi/10.1073/pnas.95.5.2105
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A Comprehensive Study of Biohopanoid Production in ...
Nov 4, 2025 · In our search, we focused on the presence of squalene‐hopene cyclase (Shc; also referred to as HpnF), catalyzing the C30 hopanoid synthesis by ...
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Identification of a methylase required for 2-methylhopanoid ... - PNAS
Apr 26, 2010 · In this study, we identified a radical SAM methylase encoded by hpnP that is required for C-2 methylation of bacterial hopanoids. At present, ...
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Functional convergence of hopanoids and sterols in membrane ...
We show that diplopterol, the simplest bacterial hopanoid, has similar properties and that hopanoids are bacterial “sterol surrogates” with the ability to order ...
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Hopanoids as functional analogues of cholesterol in bacterial ...
We find that hopanoids determine bacterial outer membrane order in a manner analogous to sterol ordering in the eukaryotic plasma membrane.
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Hopanoid Production Is Required for Low-pH Tolerance ... - NIH
Hopanoids are pentacyclic triterpenoids that are thought to be bacterial surrogates for eukaryotic sterols, such as cholesterol, acting to stabilize membranes ...
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Specific Hopanoid Classes Differentially Affect Free-Living and ...
Oct 20, 2015 · ... hopanoids such as bacteriohopanetetrol (BHT) and aminotriol. Penta- and hexa-acylated lipidA contain 5 and 6 fatty acyl chains, respectively.Missing: variations geohopanoids
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Specific Hopanoid Classes Differentially Affect Free-Living and ...
Oct 20, 2015 · Our results also show that C35 hopanoids are necessary for symbiosis only with the host Aeschynomene afraspera but not with soybean. This ...
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Lipid remodeling in Rhodopseudomonas palustris TIE-1 ... - PubMed
Apr 29, 2015 · Lipid remodeling in Rhodopseudomonas palustris TIE-1 upon loss of hopanoids and hopanoid methylation. Geobiology. 2015 Sep;13(5):443-53.Missing: study | Show results with:study
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Distinct functional roles for hopanoid composition in the chemical ...
Aug 29, 2019 · We found that the ethanol caused hpn mutants to show a significantly larger growth defect compared to the control (Fig. 4C). This effect was ...
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The General Stress Response Factor EcfG Regulates Expression of ...
Thus, all genes within the R. palustris GSR regulatory locus and one gene within the hopanoid biosynthesis cluster responded to heat stress by upregulating ...
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Hopanoid lipids promote soybean–Bradyrhizobium symbiosis | mBio
Mar 6, 2024 · Squalene-hopene cyclase encoded by shc cyclizes linear squalene (IV) into the C30 short hopanoids, diploptene (I), and/or diplopterol (II).
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Lipids - Wiley Online Library - AOCS Publications
Nov 15, 2008 · The first incorporations of 13C-labeled precursors into bacterial hopanoids were designed in order to determine the origin of the C5 side-chain ...Bacterial Biohopanoids · Conclusion: Further... · Mep Pathway Enzymes As...
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The diagenetic continuum of hopanoid hydrocarbon transformation ...
Sep 1, 2021 · The purpose of this study is to document the progressive changes in abundance of hopanoid hydrocarbon compound classes and between individual isomers with ...Missing: seminal | Show results with:seminal
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https://journals.asm.org/doi/10.1128/mbio.01251-15
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Origin and Geochemical Implications of Hopanoids in Saline ...
Nov 4, 2021 · Hopanoids are ubiquitous in sediments and crude oils and are an important indicator for bacteria. (1−3) Previous research studies have shown ...Missing: seminal | Show results with:seminal
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(PDF) Investigation of hopanoid biomarkers in lake sediments by GC ...
Aug 9, 2025 · Hopanoids are mainly derived from bacteriohopanpolyols that occur especially in bacteria, show the importance of bacterial lipid ...
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Origin and Geochemical Implications of Hopanoids in Saline ... - PMC
A homologous series of hexacyclic sulfides with a 17α, 21β(H) hopane carbon skeleton ranging from C30 to C35 are identified by Cyr et al. in heavy oils of ...
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Hydrocarbon generation from bacterial biomass in ca. 1320 million ...
Nov 27, 2020 · Abundant hopanes, 13α(n-alkyl)-tricyclic terpanes and other biomarkers of bacteria prove that bacterial biogenic organic matter can generate ...
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Biosynthesis of 2-methylbacteriohopanepolyols by an anoxygenic ...
Here, we report the identification of significant quantities of 2-MeBHP in two strains of the anoxygenic phototroph Rhodopseudomonas palustris.Missing: methylotrophs algae
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Genetics re-establish the utility of 2-methylhopanes as ... - Nature
Oct 26, 2023 · However, the discovery of hopanoid C-2 methyltransferase (HpnP) in Alphaproteobacteria led to the downfall of this molecular proxy. In the ...Missing: HpnB dehydrogenase
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Discovery, taxonomic distribution, and phenotypic ... - PNAS
Jul 23, 2012 · In this study, we identified and deleted a gene, hpnR, required for methylation of hopanoids at the C-3 position in the obligate methanotroph Methylococcus ...
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A reconstruction of Archean biological diversity based on molecular ...
Bitumens extracted from 2.7 to 2.5 billion-year-old (Ga) shales of the Fortescue and Hamersley Groups in the Pilbara Craton, Western Australia, ...
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Carbon isotopic fractionation in lipids from methanotrophic bacteria
Experiments with cultured aerobic methane oxidising bacteria confirm that their biomarker lipids will be significantly depleted in 13C compared to the substrate ...
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https://www.researchgate.net/publication/258377488_Investigation_of_hopanoid_biomarkers_in_lake_sediments_by_GC-MS_and_RP-HPLC-APCI-MS
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US10131585B2 - Hopanoids producing bacteria ... - Google Patents
The biofertilizer, biofertilizer compositions, nitrogen-fixing rhizobia, seeds and related methods and systems herein described allow in several embodiments ...
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Analysis of hopanoids in bacteria involved in food technology and ...
Hopanoids are pentacyclic triterpenoids which are believed to act as reinforcers of membranes in certain prokaryotic microorganisms. A rapid and sensitive ...Missing: industrial processing
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De novo cholesterol biosynthesis in bacteria | Nature Communications
May 22, 2023 · Production of cyclic triterpenoid lipids, including hopanoids in bacteria and sterols in fungi, have been suggested to increase the ...
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Production of cholesterol-like molecules impacts Escherichia coli ...
Engineering of the microbial cell membrane is one strategy that can increase robustness and thus alter this range. In this work, we targeted cellular ...
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Hopanoid Production Is Required for Low-pH Tolerance ...
Hopanoids are pentacyclic triterpenoids that are thought to be bacterial surrogates for eukaryotic sterols, such as cholesterol, acting to stabilize ...
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Growth inhibition of hopanoid synthesizing bacteria by squalene ...
2,3-Dihydro-2-azasqualene, its N-oxide and its N,N-diethyl analogue, as well as 2,3-dihydro-2,3-iminosqualene are potent inhibitors of the squalene to hopanoid ...Missing: antibiotics | Show results with:antibiotics<|control11|><|separator|>
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Hopanoids, like sterols, modulate dynamics, compaction, phase ...
Our results show that diplopterol induces phase segregation, increases lipid compaction, and decreases permeability on phospholipid membranes.
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Origin and novel transport pathways of bacterial hopanoids - bioRxiv
Jul 29, 2025 · Here we analyze conservation of hopanoid biosynthesis across bacterial genomes using modern taxonomic tools. We find that hopanoids most likely ...