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.

Enzymatic synthesis of nucleosides by nucleoside phosphorylase co-expressed in Escherichia coli

Nucleoside phosphorylase is an important enzyme involved in the biosynthesis of nucleosides.In this study,purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase were co-expressed in Escherichia coli and the intact cells were used as a catalyst for the biosynthesis of nucleosides.For protein induction,lactose was used in place of isopropylβ-D-1-thiogalactopyranoside(IPTG) .When the concentration of lactose was above 0.5 mmol/L,the ability to induce protein expression was similar to that of IPTG.We determined that the reaction conditions of four bacterial strains co-expressing these genes(TUD,TAD,DUD,and DAD) were similar for the biosyntheses of 2,6-diaminopurine nucleoside and 2,6-diaminopurine deoxynucleoside.When the substrate concentration was 30 mmol/L and 0.5%of the recombinant bacterial cell volume was used as the catalyst(pH 7.5) ,a greater than 90%conversion yield was reached after a 2-h incubation at 50°C.In addition,several other nucleosides and nucleoside derivatives were efficiently synthesized using bacterial strains co-expressing these recombinant enzymes.