Short‑chain fatty acids(SCFAs)as potential resuscitation factors that promote the isolation and culture of uncultured bacteria in marine sediments

Many marine bacteria are difcult to culture because they are dormant,rare or found in low-abundances.Enrichment culturing has been widely tested as an important strategy to isolate rare or dormant microbes.However,many more mechanisms remain uncertain.Here,based on 16S rRNA gene high-throughput sequencing and metabolomics technology,it was found that the short-chain fatty acids(SCFAs)in metabolites were signifcantly correlated with uncultured bacterial groups during enrichment cultures.A pure culture analysis showed that the addition of SCFAs to media also resulted in high efciency for the isolation of uncultured strains from marine sediments.As a result,238 strains belonging to 10 phyla,26 families and 82 species were successfully isolated.Some uncultured rare taxa within Chlorobi and Kiritimatiellaeota were successfully cultured.Amongst the newly isolated uncultured microbes,most genomes,e.g.bacteria,possess SCFA oxidative degradation genes,and these features might aid these microbes in better adapting to the culture media.A further resuscitation analysis of a viable but non-culturable(VBNC)Marinilabiliales strain verifed that the addition of SCFAs could break the dormancy of Marinilabiliales in 5 days,and the growth curve test showed that the SCFAs could shorten the lag phase and increase the growth rate.Overall,this study provides new insights into SCFAs,which were frst studied as resuscitation factors in uncultured marine bacteria.Thus,this study can help improve the utilisation and excavation of marine microbial resources,especially for the most-wanted or key players.

Preparation and Magnetic Properties of Anisotropic SmCo5/Co Composite Particles

Anisotropic SmCo5/Co nanocomposite powders have been prepared by electroless Co deposition on commercial SmCo5 powders with hydrazine as reducer. The Co particles are mainly in the range of 8–27 nm and form dense/continuous soft magnetic coatings on the surface of SmCo5 powders. Exchange coupling happened between the coated Co soft magnetic particles and the SmCo5 hard phase. As a result, SmCo5/Co nanocomposite powders with remanence of73.58 emu/g and energy product of 13.74 MGOe were obtained in the optimum condition, as compared with those of70.52 emu/g and 13.40 MGOe for uncoated SmCo5 powders. The effects of Co adding amount on Co particle size, coating morphology, and magnetic properties of SmCo5/Co products were investigated.

Improving the biotransformation efficiency of soybean phytosterols in Mycolicibacterium neoaurum by the combined deletion of fbpC3 and embC in cell envelope synthesis

Biotransformation of soybean phytosterols into 9α-hydroxy-4-androstene-3,17-dione(9-OHAD)by mycobacteria is the core step in the synthesis of adrenocortical hormone.However,the low permeability of the dense cell envelope largely inhibits the overall conversion efficiency of phytosterols.The antigen 85(Ag85)complex encoded by fbpA,fbpB,and fbpC was proposed as the key factor in the combined catalysis of mycoloyl for producing mycolyl-arabinogalactan(m-AG)and trehalose dimycolate(TDM)in mycobacterial cell envelope.Herein,we confirmed that fbpC3 was essential for the biotransformation of trehalose monomycolate(TMM)to TDM in Mycolicibacterium neoaurum.The deficiency of this gene raised the cell permeability,thereby enhancing the steroid uptake and utilization.The 9-OHAD yield in the fbpC3-deficient 9-OHAD-producing strain was increased by 21.3%.Moreover,the combined deletion of fbpC3 and embC further increased the 9-OHAD yield compared to the single deletion of fbpC3.Finally,after 96 h of bioconversion in industrial resting cells,the 9-OHAD yield of 11.2 g/L was achieved from 20 g/L phytosterols and the productivity reached 0.116 g/L/h.In summary,this study suggested the critical role of the fbpC3 gene in the synthesis of TDM in M.neoaurum and verified the feasibility of improving the bioconversion efficiency of phytosterols through the cell envelope en-gineering strategy.

Multiplexed site-specific genome engineering in Mycolicibacterium neoaurum by Att/Int system

Genomic integration of genes and pathway-sized DNA cassettes is often an indispensable way to construct robust and productive microbial cell factories.For some uncommon microbial hosts,such as Mycolicibacterium and Mycobacterium species,however,it is a challenge.Here,we present a multiplexed integrase-assisted site-specific recombination(miSSR)method to precisely and iteratively integrate genes/pathways with controllable copies in the chromosomes of Mycolicibacteria for the purpose of developing cell factories.First,a single-step multi-copy integration method was established in M.neoaurum by a combination application of mycobacteriophage L5 integrase and two-step allelic exchange strategy,the efficiencies of which were~100%for no more than three-copy integration events and decreased sharply to~20%for five-copy integration events.Second,the R4,Bxb1 andΦC31 bacteriophage Att/Int systems were selected to extend the available integration toolbox for multiplexed gene integration events.Third,a reconstructed mycolicibacterial Xer recombinases(Xer-cise)system was employed to recycle the selection marker of gene recombination to facilitate the iterative gene manipulation.As a proof of concept,the biosynthetic pathway of ergothioneine(EGT)in Mycolicibacterium neoaurum ATCC 25795 was achieved by remodeling its metabolic pathway with a miSSR system.With six copies of the biosynthetic gene clusters(BGCs)of EGT and pentose phosphate isomerase(PRT),the titer of EGT in the resulting strain in a 30 mL shake flask within 5 days was enhanced to 66 mg/L,which was 3.77 times of that in the wild strain.The improvements indicated that the miSSR system was an effective,flexible,and convenient tool to engineer the genomes of Mycolicibacteria as well as other strains in the Mycobacteriaceae due to their proximate evolutionary relationships.