Effects Of CofD Gene Knockout On Methanogenesis Of Methanobrevibacter Ruminantium

Methane emissions from ruminants is an important factor for increasing the greenhouse effect.In addition,the intake of 2-12%gross energy by ruminants always is translated into methane losses.Therefore,it is of great academic and practical significance to seek measures to reduce the methane emission from ruminant rumen.Methanobrevibacter ruminantium is the dominant methanogen in the rumen of ruminants.The main pathway of methane production is the reduction of CO₂ by H₂,and coenzyme F₄₂₀ is the key coenzyme in this pathway,and the CofD enzyme is the key enzyme in the process of coenzyme F₄₂₀ biosynthesis.In this research,the methanogen Methanobrevibacter ruminantium was isolatd from the rumen fluid,and the effects knockout of cofD gene on coenzyme F₄₂₀ production and methane production.in Mbr.ruminantium was studied..Trial 1.Isolation and identification of Mbr.ruminantiumThe Xuanhan cattle rumen fluid sample was multiplely concentrated and purified on optimized BRN?Bryant and Robison Nutrite?selective medium using separation technology with adopting serial dilution to pick a single colony in roll tubes invented by Hungate.The methanogens were isolated and screened successfully,and the isolated strain was identified.The results showed that:?1?the strain was short and smooth,which appeared about 0.7 ?m in diameter and about 0.8-1.8 ?m in length,single in existence or by 2-4 cells to form a short chain under the optical microscope,and blue green fluorescence under fluorescent microscope.?2?The isolated methanogens got the positive bands with the correct size of the amplified products,proven by using universal primers of 16S rDNA gene of methanogens?84F-1340R?to amplify the DNA.?3?The strain was sequenced,and the sequences similarity was up to 99%with NCBI Library of Methanobrevibacter ruminantium M1 16S rDNA sequence,which stated that it was Mbr.ruminantium.?4?The phylogenetic tree showed that the genetic relationship of isolated methanogens and Methanobrevibacter ruminantium Ml was 100%analyzed by UPGMA Mega software.?5?The Mbr.ruminantium was cultured,finding that the methane production gradually increased,and the hydrogen consumption also gradually increased with the proliferation of bacteria,indicating that the bacteria could effectively use H₂ to generate CH₄.Trial 2.Construction and validation of cotD knockout mutant Mbr.ruminantiumMethanobrevibacter rumin.antium isolated from trial 1 was used as the research object.The inserting inactivation method was used to knock out cofD gene to construct the cofD knockout vector.Finally,the constructed gene knockout plasmid pUC18-cofD-tet was transfered into the competent bacteria of Methanobrevibacter ruminantium,The results showed that the expression of cofD gene and the content of cofD protein in the mutant were lower than that of wild-type?P<0.05?.The cofD gene deleted strain of Mbr.ruminantium was successfully constructed.Trial 3.Comparison of coenzyme F₄₂₀ synthesis and methane production between cofD knockout and wild-type Mbr.ruminantiumThe growth curves of the mutant and wild-type Mbr.ruminantium were compared,and the methane yield and the content of coenzyme F₄₂₀ were compared after 12,24,36,48 and 96 h,respectively.The results showed that:?1?the logarithmic phase of mutant Mbr.ruminantium?16h?was lower than the wild-type?24h?,the proliferation of cells was significantly decreased.The maximum biomass and specific growth rate of wild-type Mbr.ruminantium was remarkable higher than the mutant?P<0.01?,and the maximum of biomass of the wild-type was 2 times than the mutant;?2?The content of coenzyme F₄₂₀ was significantly decreased after the cofD gene knockout?P<0.01?,and significant difference for the content of coenzyme F₄₂₀ was observed when cultured in the 36,48 and 96 h,and decreased by 29%,59%and 30%,respectively?P<0.01?;?3?The CH₄ production of the mutant were lower than the wild-type?P<0.05?.The maximum amount of H₂ consumed and the maximum amount of CH₄ produced were 14%and 2%of the wild-type Mbr.Ruminantium respectively.These results indicated that cofD knockout reduced the coenzyme F₄₂₀ synthesis and methane production in Mbr.ruminantium.In conclusion,this study successfully isolated and cultured Mbr.ruminantium originated from rumen fluid,and the cofD gene mutant strain of Mbr.ruminantium was successfully constructed.The growth rate and the reproductive ability Mbr.ruminantium.was reduced,and synthesis of coenzyme F₄₂₀ and the expression of CofD enzyme was decreased subsequently.Finally,its methanogenic activity to utilize hydrogen to reduce carbon oxide to form methane decreased.