| Fatty acid(FA)-derived chemicals with chain lengths of 6–10 carbons have unique value when used as biofuels,commodity and fine chemicals.Medium-chain alkanes/alkenes and fatty esters have lower melting temperature compared to their long-chain counterparts,which makes them more suitable to replace the fossil fuel such as gasoline.Medium-chain FAs have antibacterial and immunoregulatory effects,and their derivatives have been widely used in food and pharmaceutical production.For example,undecanone are used as flavors and floral fragrances,and octanoate lends aroma to wine.Recently,many advances have been made in microbial FA biosynthesis,but the content of medium-chain products remains very low.Thioesterase is a key enzyme in FA biosynthesis,and proved to determine the yield and compositions of FA products.However,the most frequently used bacterial thioesterase'Tes A lacking a signal peptide displays promiscuous activity for substrates with varying chain-length,and has enabled low production of medium-chain FAs.This study has applied semi-rational design strategy to engineer the substrate selectivity of'Tes A as follows:1.Semi-rational design improved the selectivity of'Tes A for short-and medium-chain substrates.E.coli thioesterase'Tes A can catalyze the hydrolysis of carboxylic esters and thioesters with varying chain-lengths,but it preferred long-chain substrates.Firstly,after analyzing the interaction between'Tes A and substrate,ten residues in the acyl binding pocket were selected and submitted to site-directed saturation mutagenesis(SSM).Secondly,based on the mutation effect acquired in the first-round mutagenesis,we conducted Combinatorial Active Site Test(CAST)to boost the selectivity.Finally,we recombined the beneficial mutations to yield the top-rank mutant.Three rounds of selectivity engineering yielded a series of mutants that preferred C2-p NP,C4-p NP,and C8-p NP substrates,respectively,indicating that we have successfully shifted the'Tes A selectivity for short-and medium-chain substrates.In order to investigate the selectivity of wild-type'Tes A and mutants for native substrates in FA biosynthesis,we synthesized three acyl-ACP substrates with varying chain-lengths(i.e.,C8-ACP,C12-ACP,and C16-ACP).The results showed that all of 10 tested mutants displayed enhanced selectivity for C8-ACP substrate.Among them,the top-ranking mutant RD-2 exhibited a 7.9-fold and 133-fold increase in C8-ACP activity and selectivity,respectively.2.'Tes A mutants greatly promoted the production of medium-chain FAs in E.coli.We constructed?tes A and?fad E-deleted MG1655(DE3)strain(“MG01”)to characterize the FA compositions of wild-type‘Tes A and mutants.After optimizing promoter and cultivation conditions,wild-type'Tes A synthesized 0.08 g/L of octanoic acid(C8)in flask fermentation,while the top-ranking mutant RD-2 enabled a 10-fold increase in C8titer.When this strain was fermented in a 5-L fed-batch bioreactor under optimized cultivation temperature and dissolved oxygen level,it reached highest cell density after36 h of fermentation and produced 2.7 g/L of free C8(45%,molar fraction),which is the highest-to-date free C8 titer reported using the E.coli type II fatty acid synthetic pathway.Furthermore,we also conducted adaptive laboratory evolution to strain MG01('Tes ARD-2)for improved C8 tolerance.It turned out that the evolved strain led to a 26%increase in C8 production titer and a 24%increase in total FAs titer,laying a foundation for enhancing the product tolerance of high-yield strains in the future.3.We revealed the mechanism underlying the changed selectivity observed in mutants.We resolved the crystal structures of'Tes A mutants CM-5(E142D/Y145G)and RD-1(M141L/E142D/Y145G)that exhibited obviously changed selectivity for acyl-ACP substrates.Through structural analysis and molecular dynamic simulation,the most obvious differences between these three structures occur for the flexible loops and the?F helix that interacts with the long-chain substrate of which the noticeably bent poses is unfavorable in the reshaped acyl-binding pocket of mutant structures;moreover,the continuous hydrophobic crevice in CM-5 and RD-1 exhibited increased affinity to medium-chain substrates.Pearson correlation analysis revealed a coefficient of 0.95between the specific activity of C8-ACP and the C8 titer values.Indeed,only mutants that increased or maintained C8-ACP activity have achieved higher C8 production in cells.Our study newly confirmed the essential role of the?F flexibility on substrate selectivity,which can support to identify promising targets for the selectivity engineering of thioesterases in SGNH family.Our study has been focused on the poor selectivity of key enzyme(i.e.,thioesterase'Tes A)in FA biosynthesis.We applied semi-rational design strategy to yield top-ranking mutant that led to a 133-fold increase in selectivity for medium-chain acyl-ACP substrate and much enhanced medium-chain FA production in cells,and revealed the mechanism that reshaping of acyl binding pocket along with increased hydrophobic crevice drives the changed selectivity in'Tes A mutants.These results demonstrate that reshaping the acyl binding pocket of a bacterial thioesterase enzyme by manipulating the hydrophobicity of multiple residues altered the substrate selectivity and therefore fatty acid product distributions in cells,which can guide future efforts to engineer thioesterases to achieve tailored product distributions. |
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