Studies On The Key Enzymes For Aromatic And Carbohydrate Metabolism In Deep-sea Uncultivated Archaea

As a huge organic carbon pool,the ocean owns a broadly distributed biodegradable carbohydrates and refractory aromatics.Among them,the carbohydrates are the main component of the dissolved organic carbon(DOC),and the aromatics are more likely to accumulate in the sediments.Microbial degradation is an important driving force for marine organic carbon deposition and carbon cycle.Archaea may play an important role in the deep-sea organic matter conversion and carbon cycling.In the past,a lot of the genes and enzymes for the metabolism of carbohydrates and aromatics in bacteria were found,but only few in culturable thermophilic and halophilic archaea.In comparison,all the research on the metabolic pathways in the deep-sea uncultivated archaea are based on the metagenome-assembled genomes(MAGs)without direct genetic or biochemical evidences for carbohydrates and aromatics catabolism.Based on the distribution of aromatics in Guaymas hydrothermal vent,cold seep in the gulf of Cádiz and the content of DOC in Mariana trench as well as the microbial communities in these environments,we propose that Marine Benthic Group D(MBG-D),Anaerobic Methanotrophic archaea(ANME)and Marine Group II(MGII)may be involved in the biodegradation and utilization of aromatics or carbohydrates.The Guaymas Basin(Gulf of California)hydrothermal vent area contains petroleum aromatics.It is speculated that MBG-D in hydrothermal sediment may be involved in the aromatics`transformation in the carbon cycle.Reconstructed aromatic compounds degrading pathway based on MAGs of MBG-D originated from the hydrothermal sediment of Guaymas Basin showed that MBG-D might degrade aromatics via the Co A-ligation-mediated the phenylacetic acid(PAA)pathway,which was different from the way through introducing hydroxyl groups to activate the benzene ring.Transcriptome analysis showed that most of the genes involved in the PAA pathway were transcribed in situ.We then performed de novo DNA synthesis,heterogeneous expression,purification and enzymatic analysis,and finally confirmed two phenylacetyl-Co A ligases(PCL),PCL<sub>M8-3</sub> and PCL<sub>M10-15</sub>,were able to completely transform PAA to phenylacetyl-Co A(PA-Co A),with specific activities of 0.60±0.02and 0.55±0.01 U/mg respectively.In addition to the broad substrate spectra showing obvious activities against PAA and derivatives substituted by hydroxyl without preference,both PCLs showed strong acid tolerance(p H 5–7),high optimum temperatures(60°C and 80°C)and thermostability(stable at 60°C and 50°C).By alignment search,we found that the PCL genes were widely distributed among archaea,with presence in 75.9%MAGs of MBG-D.These results emphasize the essential role of MBG-D in aromatic degradation under an extreme and oligotrophic environment in the deep-sea sediments.Cadiz bay is an active area of mud volcano,where the refractory organic carbon was fixed by black carbon and petroleum aromatics in the sediment.It is speculated that ANME,which is abundant in cold seep environment,may be involved in the aromatics`transformation in the carbon cycle.Based on its MAGs from the gulf of Cádiz,the constructed aromatic degradation pathway suggested that ANME is capable of degrading aromatics via the ligation of Co A to PAA and benzoate(BA)before the reductive reaction,instead of introducing the hydroxyl group to the aromatic ring.Again,we performed the do novo DNA synthesis for three aromatic acid Co A ligases PCL₁₆₀,PCL₂₀₇₉ and BCL_ANME.Through heterogeneous expression,purification,and enzymatic analysis,we discovered PCL₁₆₀ exhibited specific activity of 0.19±0.01U/mg against PAA and BCL_ANME 9.49±0.23×10^-2 U/mg against BA.Time-course assay showed that PCL₁₆₀ was able to completely transform PAA to PA-Co A and so did BCL_ANMEagainst BA to benzoyl-Co A.For BCL_ANME,the optimum temperature was 30°C and the optimum p H was 9.It had a broad substrate spectrum,with obvious activity against 2-hydroxybenzoate and robust activities against BA,3-hydroxybenzoate and 4-hydroxybenzoate.Differently,the optimum temperature for PCL₁₆₀ was 60°C,but it retained stable below 37°C and the optimum p H is 10.The PCL₁₆₀ also had a broad substrate spectrum,with robust activities against PAA and derivatives substituted by hydroxyl without preference.And activities of both enzymes could be enhanced by adding divalent ions Mg²⁺or Mn²⁺.These results inferred the multi-function of ANME not only in methane anaerobic oxidation,but also the potential in aromatics metabolism and probabilities to be involved in the carbon cycle of deep-sea cold seep using different ways.There contains a high flux of organic carbon which is easy to degrade in the deeplayer of the Mariana trench where the heterotrophic archaeal group MGII abundant in seawater may be involved in the organic carbon transformation.In addition to the deep-sea sediments,there are also abundant archaeal groups in the seawater,among which MGII is one of the most abundant heterotrophic archaea.For the carbohydrate metabolism research in MGII,all the MAGs of MGII originated from Mariana Trench contained glycoside hydrolase(GH)-encoding genes but in different families.Five GH genes of the GH20 family in MGII from surface and deep-sea of Mariana Trench as well as Guaymas Basin hot spring and the Mediterranean Sea were synthesized de novo.After heterogeneous expression,purification,and enzymatic analysis,we found these members of GH20 family in MGII possess the?-galactosidase activity with the specific activities ranging from 4.40±0.16 to 46.12±1.14×10^-2 U/mg against o-Nitrophenyl-?-D-Galactopyranoside.In addition,the optimum temperature for all these five?-galactosidases was 50°C.These results further support the function of MGII in carbohydrate utilization and indicate an important role of MGII in carbon cycle for easily degradable organic carbon.Our future work will focus on identifying the initial aromatic substrates for MBG-D and ANME,searching for the missing enzymes in aromatics metabolic pathways,exploring the evolutionary relationship between aerobic and anaerobic pathways in microbial aromatics metabolism,and physiologically verifying the utilization of aromatics for uncultivated archaea using enriched sediments.Meanwhile,the enzymic investigation of the proteins and lipids utilization for MGII could also be involved.In general,these findings that MBG-D and ANME could degrade aromatics and MGII could hydrolyze carbohydrates respectively indicate the potential function of these archaea in the metabolism of refractory organic carbon in the deep-sea sediment or easily degradable organic carbon in the surface and deep-sea water.The characterization of the key enzymes involved in organic carbon metabolism in these archaea reveals that the marine archaea could carry out different metabolic strategies to take the advantage of the organic carbon source in the ocean,suggesting their pivotal driving role of these archaea in marine carbon cycle.