Mining And Engineering Of Aldolases For Stereoselective Synthesis Of Aryl Vicinal Diols

Chiral aryl vicinal diols are important multifunctional compounds,which are often used as key chiral building blocks in industries of organic synthesis,pharmaceuticals and fine chemicals.Represented by aldolase,biological catalysts-mediated asymmetric synthesis of chiral aryl vicinal diols is a favored method due to its remarkable stereoselectivity,mild reaction conditions and high atomical utilization.However,the substrate spectrum of aldolases is generally quite narrow and the catalytic activity towards unnatural substrates of available aldolases is too low to satisfy the needs of industrialization.Therefore,enzyme library was constructed by gene mining and protein expression,followed by characterization of enzyme catalytic properties and protein engineering,aiming to develop novel biocatalysts with robust characteristics and desirable stereoselectivity for the synthesis of chiral aryl vicinal diols.Gene mining of aldolases was firstly conducted by searching the National Center for Biotechnology Information(NCBI)databank.Then conditions for the expression of recombinant proteins including inducing temperature,inducing time and chaperone coexpression were optimized.Finally,most of the recombinant proteins were heterologously expressed in E.coli BL21(DE3)with considerable solubility.At this point,an aldolase library containing 25 recombinant enzymes was constructed successfully.Subsequently,the constructed aldolase library was screened using a series of aldehydes as acceptors and three ketones(acetone,hydroxyacetone and dihydroxyacetone)as donors.As a result,two enzymes from Escherichia coli K12,namely D-fructose-6-phosphate aldolase A(FSAA)and D-fructose-6-phosphate aldolase B(FSAB),were found to possess the ability to catalyze the aldol addition of hydroxyacetone(HA)to cinnamaldehyde.After further optimization of conditions for the recombinant proteins expression,the soluble expression with high-efficiency for FSAA and FSAB was achieved.Subsequent characterization of enzyme catalytic properties showed that the optimal temperature for FSAA and FSAB towards cinnamaldehyde was 30?and 25 ?,respectively.Remarkably,FSAA was active in a wide temperature range(20?60?)and is more thermostable than FSAB.Considering that the thermostability is a crucial criterion in industrial application,it can be anticipated that FSAA will have a more promising application potential than FSAB.Therefore,the following studies were carried out with FSAA.When controlling the yield of spontaneous reaction below 1%,FSAA exhibited the best catalytic performance in citric acid-sodium citrate buffer(0.1 M,pH 6.5).Due to poor organic tolerance of FSAA,no cosolvent was added for later reactions.Considering the relatively low catalytic activity of FSAA in the target aldol additions,rational design was employed to optimize its catalytic properties.First,strategies involving decrease of steric hindrance and introduction of enzyme-substrate interactions were proposed to introduce mutations at key sites having direct interactions with the substrates.However,these strategies failed as judged by the lower activity of all constructed mutants when compared with the wild type.Subsequent analysis of the catalytic mechanism implied that a catalytic water molecule participates in the proton transfer during catalysis.As this catalytic water molecule forms hydrogen bonds with the active site residues Gln59,Thr109 and Tyr131,we attempted to mutate these water-interacting residues into amino acids with similar property and structure.As a result,four mutants were constructed(T109S,Y131F,Q59E and Q59N).Investigation of these four mutants revealed that only one mutant(Q59N)achieved improved activity,up to 3-fold higher than the wild type.whereas the other three mutants showed obviously decreased activities.Further site-directed mutagenesis of site 59 created two new mutants,Q59L and Q59T,with improved catalytic performance,showing 25-fold and 23-fold activity improvement over the wild type,respectively.Subsequent molecular dynamics(MD)simulations analysis revealed that the C-terminal a-helix from each monomer of the decameric FSAA runs across the active site of the neighbouring subunit forming interface interaction with this active site.The residue change in site 59 did have an effect on the orientation of this special C-terminal a-helix,thus influencing the activity of FSAA towards cinnamaldehyde.Although no direct interaction between site 59 and substrate was observed,this site affects the activity of FSAA through the residue interaction network.The results described above instructed us to expand the engineering targets in enzyme redesign to cover residues conferring possible network interactions rather than focusing only on the residues showing direct interactions with substrates.Considering that FSAA is featured by a special decametic structure with tightly packed subunits and distinctive interface interactions between adjacent subunits,a new comprehensive protein engineering strategy involving simultaneous modulation of both the intra-and inter-subunit interactions was proposed for modification of FSAA to efficiently accept thiophene-related heteroaromatic aldehydes as substrates in this research.Among these mutants created,the combinatorial variant FSAA I31T/Q59T/I195Q showed the best catalytic performance,with 27 to 278-fold activity improvement towards 2-thiophenecarboxaldehyde,3-thiophenecarboxaldehyde,benzothiophene-2-carboxaldehyde,2-furaldehyde and 2-pyridinecarboxaldehyde was created.In this triple mutant,site 6 was designed for modulation of intra-subunit interactions,while site 59 and site 195 were substituted for modulation of inter-subunit interactions.At the end,recombinant E.coli whole cells overexpressing this triple-site variant FSAA I31T/Q59T/I195Q was used to catalyze the aldol reactions of HA with heteroaromatic aldehydes described above at relative high substrate concentrations(350-500 mM)for the preparation of target chiral aryl vicinal diols.The conversion rates reached 70%-95%,demonstrating a wide application prospect.The dramatically improved catalytic performance of the combinatorial variant FSAA I31T/Q59T/I195Q unambiguously demonstrated the effectiveness of the synergistic modulation strategy exploited in the current research.For future tailoring of FSAA and other multimeric enzymes,synergistic modulation of both intra-and inter-subunit interactions would be a potent methodology.