Construction Of A N-acetylneuraminic Acidspecific Biosensor And Optimization Of Theproducer Strain

N-acetylneuraminic acid(NeuAc)and its derivatives take parts in many physiological and pathological processes,and have enormous potential in edible,pharmaceutical and health care applications.Due to the tedious process and low efficiency,extraction from natural materials is hard to meet the market demand.Nowadays,enzymatic synthesis and whole-cell biocatalysis are major tools for NeuAc production.However,both methods need supplementation of expensive precursors,pyruvate and GlcNAc,increasing the cost.Direct microbial fermentative NeuAc production from glucose or other unrelated carbon sources is an attractive alternative since it can synthesize NeuAc in one step without any direct precursors.Previously,a glucose initiated NeuAc biosynthetic pathway was constructed in E.coli(DN5/pNnsGM).During fermentation,substantial amount of GlcNAc and certain amount of N-acetylmannosamine(ManNAc)and pyruvate accumulated.Through exogenous pyruvate addition,NeuAc titer of DN5/pNnsGM improved by 104.6%,which suggested a shortage of pyruvate supply.We thus introduced the NeuAc synthetic pathway into a pyruvate producer strain,yet no NeuAc was detected.We also tried to increase pyruvate supply by overexpressing glk but failed to increase the NeuAc titer.Then,we used an irreversible NeuAc synthase neuB from E.coli K1 to replace nanA,trying to redirect metabolic flux into NeuAc production.DN5/pB produced 1.75 g/L NeuAc,1.55 times higher than DN5/pNnsGM.GlcNAc and ManNAc accumulation more than doubled,but pyruvate concentration decreased.To reduce acetate accumulation,the phosphate acetyltransferase(pta)of DN5 was inactivated,resulting in DN6.This modification decreased acetate titer from 4.62 g/L to 1.55 g/L.To increase intracellular PEP,glucose-specific PTS enzyme IIBC component(ptsG)of DN6 was inactivated,resulting in DN7.DN7/pB accumulated 1.48 g/L NeuAc in 48 h,slower than DN5/pB.ManNAc titer increased from 1.17 g/L to 2.63 g/L,and no pyruvate was detected.Subsequently,phosphoenolpyruvate carboxylase(ppc)was inactivated,resulting in DN8.DN8/pB accumulated 2 g/L NeuAc in 70 h,with a low glucose consumption rate of 0.26 g/L/h and a less biomass accumulation.This is probably due to the vital involvement of this gene in the C4 replenishment pathway which catalyzes the formation of oxaloacetic acid from PEP and CO2,replenishing the TCA cycle.Thus,knock out of this gene would slow down cell growth and glucose consumption.We performed fermentation optimization of DN8/pB on 1 L fermentor.Under 37?and high dissolved oxygen condition,4.15 g/L NeuAc was produced in 52 h.However,the strain grew poorly on 5 L fermentor and produced little NeuAc,which needs further optimization.The metabolic power of microorganisms to produce small molecules of biotechnological interest from simple substrates is truly amazing.However,due to the complicacy of microbial metabolism,rational design is always suboptimal for bioproduction purpose.In this view,random mutation and high-throughput screening strategy holds certain advantages.However,the characterization of mutants has become a new limiting factor,since most of the small molecules produced do not confer easily screened phenotypes.Biosensors solve this problem to a large extent.They sense the change of intracellular metabolite and report it with fluorescence,enzyme activity or coupled-cell growth,thus facilitating high-throughput screening.In 2013,Cho et al.reported a NeuAc aptamer with high specificity and affinity(?1.35 nM),which serves as an excellent candidate for constructing an in vivo active NeuAc biosensor.We constructed an intact NeuAc biosensor by fusing the aptazyme with a constitutive strong promoter PJ23106,a buffer sequence and a reporter gene.Upon binding of its ligand,a conformational change in the sensor part would lead to the self-cleavage of the ribozyme,exposing a 5' hydroxy that is recognized by the endogenous RNase J1,thus destabilizing the mRNA of downstream gene and decrease its expression.Using sfgfp as the reporter,12 mM exogenous NeuAc brought about a 11.8-fold inhibition of the fluorescence,demonstrating a successful coupling of NeuAc concentration and the expression of reporter.Then,reporter gene tetA was fused to the NeuAc aptazyme to develop a Ni2+-based selection system for growth-coupled selection.TetA encodes a tetracycline/H+ antiporter and can be used as both a positive and a negative selection marker.TetA confers the cells tetracycline resistance while renders them more sensitive to toxic metal salts such as NiCl2.The NeuAc biosensor would enable cells that accumulate more intracellular NeuAc to survive in the presence of NiCl2.To minimize interference,a modified M9 minimal medium previously used to produce GlcNAc was adopted.In a preliminary experiment,we determined the optimal selection concentration of Ni2+ in this medium to be 50 ?M,because under this concentration,growth inhibition caused by tetA expression was more obvious.On a concentration range of 0-86.4 ± 5.1 mg/gCDW intracellular NeuAc,the biosensor works soundly.We verified that the sensor was also active in Lactobacillus plantarumon a concentration range of 0-25.6 mM exogenous NeuAc,suggesting a similar working mechanism of ribozyme among different species and RNA-based biosensormay be more universal.To demonstrate the feasibility of the NeuAc biosensor for genetic selection,an enrichment assay using defined model libraries was performed as a proof of concept.A mixture of two species DN5/pSB and DN5/pSA at 1:100 ratio was cultured in M9B medium with 50 ?M Ni2+.After a single enrichment cycle,the high NeuAc producer DN5/pSB was enriched by more than 16-fold,indicating the feasibility of the designed NeuAc-biosensor in facilitating selection process during microbial evolution.We were encouraged to optimize the expression of glmS,GNA1,slr1975 and neuB via RBS engineering.We designed two sets of degenerate RBSs here,generating a possible library of 497,664 and 65,536 mutants respectively.Mutants from library 1 tend to accumulate more NeuAc than those from library 2 and thus Library 1 was used for further study.A three-cycle evolution process was performed.After each cycle,plasmids were extracted and re-transformed into fresh parental strain to prevent the adaptation of cells to nickel.Three plasmids p1e,p2e and p3e were enriched to a ratio more than 40%after cycle3.Predicted RBS strengths of neuB,slr1975 and GNA1 were higher in ple,p2e and p3e than those in pB,except for GNA1 in p2e,but the strength of glmS was less.The best producer DN5/p2e produced 2.12 g/L NeuAc(-34%improved com-pared with DN5/pB).Based on this,we chose the NeuAc synthase(NeuB)to increase its in vivo activity using biosensor-facilitated mutagenesis.After 4 cycles,mutants B1 and B3 were enriched to a ratio of 56.5%and 45%respectively.DN5/pB1 produced 2.24 g/L NeuAc,while DN5/pB3 produced 2.61 g/L(23%improved than DN5/p2e).Specific activity of B3 was 27%improved than the wild type and B1 was also slightly higher than the wild type(9.4%improved).To test the effect of single amino acid change and combinational sites mutation on enzymatic activity and NeuAc titer,NeuB mutants C1,C2 and C3 were constructed.Unfortunately,these eff orts did not improve the NeuAc titer further.To evaluate the NeuAc production potential of DN5/pB3,we performed two-stage fermentation in minimal medium.In this strategy,a first stage of biomass production is followed by a second stage of product fermentation.DN5/pB3 produced 8.31 g/L NeuAc after 44 h.Only small quantity of ManNAc,formate,acetate and ethanol were detected,but considerable amounts of pyruvate and GlcNAc accumulated.Through optimization of initial biomass,NeuAc titer was further improved to 12.52 g/L.In this work,we use the irreversible NeuAc synthase to catalyze the glucose initiated NeuAc synthesis,and modify the host strain to increase PEP supply.Through coupling of cell growth and NeuAc production,a high-throughput screening system was established and applied to optimize pathway and evolve key enzyme.This system can also be used to evolve other enzymes in the NeuAc pathway and should be suggestive for the development and utilization of other aptazyme-based biosensors.