Guest Editorial: Enzymes in the Gut Microbiome As Contributors to Circulating Lipids

Microbial Enzymes: A Variable in Lipid Metabolism
It is well established that dietary input combined with genetic predisposition can substantially alter blood lipid profiles and susceptibility of humans to diseases such as metabolic syndrome and cardiovascular disease.^1,2 Less well understood is the role of gut microbiome enzymes mediating the association of dietary input and blood lipids important to human health, such as cholesterol, sphingolipids, and fatty acids. However, there has been significant progress in understanding how microbial enzymatic function can directly contribute to the overall circulating lipids and metabolite profiles.^3,4

Similar to the liver, the gut microbiome is a collection of microorganisms, including bacteria and archaea living in the digestive tract, and carrying enzymes which play a diverse role in digestion, biotransformation and detoxification of dietary components.⁵ Each human harbors a unique microbiome composition which is comprised of a taxonomically diverse set of bacteria primarily from the Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, and Verrucomicrobia phyla in the healthy state.⁶ In metabolic syndrome, microbial composition is altered and functionally contributes to aspects and outcomes of the disease.⁷ A more granular way to understand the role of the microbiome in lipid metabolism and its contribution to human health, is not through the study of the taxa of microbes associated, but the functional properties of the microbiome through their enzymes. This can be attained through metagenomic and meta-transcriptomic profiling of the human microbiome community. These technologies have developed to a point in the last 5-10 years where we are capable of utilizing ‘deep sequencing’ paired to profile the differences in the presence of microbiome enzymatic properties relevant to biotransformation and lipid production. Further, bioinformatic profiling of multi’omic data (metabolomics paired with metagenomics), along with the ability to genetically engineer bacteria to knockout genes of interest, have enabled us to determine which microbiome enzymes are able to synthesize or breakdown lipids of interest to human health and affect systemic lipid circulation in the blood.⁸ This is a new field with many unknown enzymes hypothetically contributing to the circulating lipid variability observed between different humans. While many microbial enzymes and functions could indirectly alter host metabolism and lipid homeostasis (such as production of pro- or anti-inflammatory small molecules), the focus here is on direct effect whereby a microbial species can bio-transform or directly synthesize bioactive lipids of importance to human health and disease. The narrative below highlights recent examples from the literature of particular microbial enzymes shown to directly impact lipid metabolism and circulating lipids.

Circulating Lipids Directly Altered by Enzymes in the Gut Microbiome
The presence, absence, and relative abundance of some lipid metabolites is directly controlled by functional output of a microbial enzyme. In some instances, these microbial enzymes are found in every human gut; in other cases, they are not. Examples presented are by no means exhaustive of the current literature but are meant to highlight recent keystone studies in this area of interest to human health and disease.

Cholesterol
Cholesterol is an essential building block of steroid hormones and cellular membranes in eukaryotic cells, and thus circulating serum cholesterol may be utilized as an important marker of human health.⁹ The notion that metabolism of cholesterol to coprostanol by the gut microbiome may lower serum cholesterol levels was proposed over 100 years ago. Yet relatively few studies have investigated this connection. A recent study from Kenny et al. was the first to do so for the human microbiome.¹⁰ In this study, the authors discovered the microbial enzyme IsmA which is found in a previously uncultured Firmicute species in the gut microbiome, and mediates the cholesterol to coprostanol conversion. The activity and presence of this enzyme in humans significantly impacts their total blood cholesterol levels as seen in 3 different human cohorts including the Framingham cohort.¹⁰ The impact of the enzyme is clinically relevant given it affects cholesterol concentrations in the blood with an odds ratio similar to that of Ezetimibe, an FDA-approved small molecule inhibitor of the intestinal cholesterol transporter and a clinically validated approach to lowering blood cholesterol.¹¹ The IsmA enzyme is present in a rare Firmicutes species and not present in every subject. Thus, some of the variability of cholesterol levels, cholesterol derivatives (such as bile acids and steroids) and blood lipid panels could be explained by whether the individual’s microbiome encodes this enzyme. Presence of this enzyme also increased the amount of high-density lipoprotein (HDL-c) and lowered low-density lipoprotein (LDL-c) in these 3 cohorts. Thus, there is interest in development of a potential probiotic to help correct those living with metabolic syndrome or have high cholesterol levels.¹²

Sphingolipids
Sphingolipids are both structural membrane and bioactive signaling molecules essential for the function of eukaryotic cells.¹³ Cells can both synthesize sphingolipids de novo and can also be salvaged from the diet. There are thousands of different sphingolipid metabolites each mediating key cellular functions such as apoptosis, cell proliferation, autophagy, inflammation, cell migration, cell cycles, and cell adhesion.^13,14 Defects in sphingolipid metabolism are causal in numerous human diseases. They have been shown to have important roles in the pathophysiology of cancer, metabolic syndrome (such as diabetes and obesity), autoimmune disease, and chronic inflammatory conditions such as inflammatory bowel disease.¹⁵ Circulating ceramide and sphingomyelin sphingolipids may be important disease biomarkers.¹⁵ Recently, it’s become apparent that there is a third source of sphingolipids that has been underappreciated to date: the gut microbiome.¹⁶ The serine palmitoyltransferase enzyme, which is the essential first committed step to sphingolipid synthesis, is also found in the Bacteroidetes species in the gut.¹⁷ These gut microbiome sphingolipids may be imported and enter host metabolic pathways changing the variability and abundance of ceramide and host sphingolipid species.¹⁸ The bacterial sphingolipids also are negatively correlated with inflammation and have immune functions.¹⁷ All humans harbor microorganisms with this enzyme and bacterial sphingolipids, with different levels of abundance. To what degree these microbial sphingolipids contribute to circulating lipids and disease phenotypes is unknown but represent an interesting area of exploration. The gut microbiome therefore is a previously unconsidered variable which mediates host sphingolipid metabolism and cellular function.

Plasmalogens
Plasmalogens are glycerophospholipids that contain a vinyl ether bond instead of an ester bond. Plasmalogens are indicated to have many important functions in mammalian cells including protection of cells from oxidative stress and are found at the highest concentrations in the neuronal and cardiovascular cell membranes.¹⁹ While the full extent of their function in humans is unknown, defects in plasmalogen synthesis and abundance is a factor in many neurological diseases including Alzheimer’s.²⁰ Some bacterial species from the gut microbiome are also capable of synthesizing and producing plasmalogens.²¹ Recently the enzymes responsible for plasmalogen synthesis were discovered in Clostridium species²², mapping to > 30% of all known microorganisms in the gut. However, whether these microbial plasmalogens enter host metabolic pathways and impact human disease remains to be seen and should be addressed by future studies.

Polyunsaturated Fatty Acids
Humans are capable of producing all the fatty acids needed except linoleic acids and alpha-linoleic acid.²³ These are the precursors to omega-6 and omega-3 polyunsaturated fatty acids (PUFAs), respectively, and cannot be synthesized by the human enzymatic machinery, and thus must be derived from our diets. The downstream metabolic products of both omega-6 and omegs-3 PUFAs are essential for membrane components and are important for regulating inflammation in the body.²⁴ The recent literature in this field shows the importance of maintaining a balanced ratio of omega-6: omega-3 fatty acid intake in order to protect against metabolic syndrome, cardiovascular disease, and cancer. Consumption of these fatty acids is unbalanced in the western diet, where humans consume significantly higher concentrations of omega-6 fatty acids.^25,26 Specifically the omega-3 fatty acids have anti-inflammatory effects^27,28 while omega-6 are pro-inflammatory, however both types of fatty acids (omega-3/6) can be oxidized easily and contribute to general oxidative stress of the body through lipid peroxidation, which is the oxidative breakdown of lipids into free radicals which can cause cellular damage.²⁹ As these PUFAs are derived from the diet, it has become clear there are enzymes in the microbiome which bio-transform these lipids before they enter host metabolic pathways, and thus modulating their effects on lipid metabolism. For example, Bifidobacterium and Lactobacillus species in the gut contain an enzyme which converts linoleic acid to conjugated linoleic acid and then to a molecule which is able to bind to G-protein coupled receptors. An anti-inflammatory signal is produced, and as a result the amount of linoleic acid converted to downstream products of omega-6 fatty acids, such as prostaglandins and eicosanoids, is reduced.³⁰ Dietary PUFAs can also be saturated by enzymes common in many microorganisms, limiting their oxidative potential through reducing their double bonds before they can be oxidized.³¹ Further, functionally different microbiome communities have been shown to significantly alter the inflammatory potential of dietary PUFAs.32 One study found that high concentrations of PUFAs in the gut may kill bacteria commonly associated with healthy state and promote the growth of bacteria with inflammatory disease associations such as Proteobacteria.³³ Further, common PUFA metabolites differ significantly in the inflamed gut microbiome.³⁴ These represent few examples; however this area is as yet understudied. There are likely many other examples of microbial enzymatic breakdown of PUFAs in bioactive metabolites which could modulate human health. Thus, how one metabolizes PUFAs may be dependent on dietary intake but also on the individual’s microbiome. A high abundance of omega-6 PUFAs in the stool and blood has been observed in patients with chronic inflammatory diseases and autoimmune diseases such as inflammatory bowel disease and rheumatoid arthritis.²⁸ Serum omega-6: omega-3 ratio is an evolving marker for cardiovascular health.²⁵ Thus modulation of PUFAs by the gut microbiome enzymes is an important factor in discordant responses seen in humans who may be more genetically susceptible to chronic inflammatory diseases, metabolic syndrome, and cardiovascular disease.

Conclusion: From Bench to Clinic
Our lipid profile is of paramount importance as a biomarker for many disease states such as cardiovascular disease and metabolic syndrome. There are many studies correlating genes and genetic SNPs to particular diseases in humans and their circulating lipid profiles. However, much less is known and appreciated about the variation in microbiome enzymatic function in individual humans, driving clinical outcomes and variability. Moving forward, there are potentially actionable steps to be taken which will both better understand and leveragethis phenomenon to treat and prevent disease in humans. Enzymes known to alter human health from the microbiome can either be inhibited or promoted in humans depending on the situation. Subjects could ultimately be screened using genetic techniques to understand the metabolic fingerprint of each individual microbiome. We can use these data for a more personalized medical approach to combating disease moving forward in the future. At a basic level, we should also be cognizant of the fact that many of the lipids circulating in our body are actually of microbial derivation and may contribute greatly to person-to-person susceptibility to disease.  

Disclosure statement: Dr. Brown has no financial disclosures to report.  
References are listed in the spring 2022 LipidSpin .pdf on www.lipid.org