Highly Efficient Biosynthesis Of Unnatural α-amino Acid Based On Rational Engineering Of Key Enzymes And Adaptive Laboratory Evolution In Gluconobacter Oxydans

Unnaturalα-amino acids are widely used and highly valuable chiral building blocks in chemical synthesis and modern medicinal chemistry,which are important intermediates for various antibiotics drugs.Currently,the preparation of unnaturalα-amino acids is mainly achieved by chemical synthesis,which not only needs harsh reaction conditions and complex purification processes but also involves toxic cyanide bringing a huge burden to environmental protection.The biocatalytic methods have attracted much attention due to their high stereoselectivities,mild reaction conditions,and few by-products.Among them,the multi-enzyme cascade catalytic methods do not require the separation of intermediate products and can achieve one-step synthesis from inexpensive substrates to complex products.It is one of the most important strategies for the biosynthesis of high-value chemicals.In this work,Gluconobacter oxydans was employed as a host cell to construct a multi-enzyme cascade for the biosynthesis of unnaturalα-amino acids from epoxides.Adaptive laboratory evolution strategy was introduced to improve the tolerance of intermediate tolerance.Protein engineering was implemented to improve Lb MDH activity toward different substrates,and by combining with crystal structure analyses,the molecular mechanisms of Lb MDH variants for improving the catalytic efficiency were revealed.Finally,recombinant strains were constructed with adapted multi-enzyme cascades and different unnaturalα-amino acids were efficiently biosynthesized.The main results of this work are summarized as follows:（1）Based on the strong incomplete oxidation ability of G.oxydans,a multi-enzyme biocatalytic cascade for producing L-α-amino acids from epoxides was designed and constructed.Through gene screening,SpEH,Lb MDH,and BcLDH as key enzymes were identified.G.oxydans,which has strong membrane-bound alcohol and aldehyde dehydrogenase systems that can directly oxidize 1,2-diols to D-α-hydroxyl acids,was employed as the host cell.In addition,Lb MDH and BcLDH generated a self-sufficient cofactor system and without the need for additional expression of coenzyme regeneration enzymes,greatly optimized and simplified the synthetic route.The yields of L-5a and L-5b synthesized from rac-1a and rac-1b were 8.4 m M and 7.5 m M,respectively,which were much lower than the theoretical values.The low mandelic acid tolerance of G.oxydans led to the accumulation of intermediates,which was the limited step of this system.The ability of Lb MDH to catalyze D-3b was only 24.7%of D-3a,further limiting the application potential of the catalytic system.（2）Enhancing the D-3a tolerance of G.oxydans by the adaptive laboratory evolution strategy to further improve the ability to produce D-α-hydroxyl acids and the molecular mechanisms of increased tolerance were revealed by membrane phenotype analysis and transcriptome analysis.The G.oxydans cells were continuously passage cultured in fresh medium with gradually increased D-3a concentration to enhance adaptability.Finally,an adapted strain that can tolerate 6 g/L D-3a was isolated（named G.oxydans STA）.The conversion rate of D-3a from 1a was increased from 0.37 g/（L·h）to 0.70 g/（L·h）by the recombinant STA compared to wild-type strain and the production of D-3b-d also were improved.The membrane phenotype analysis revealed that the membrane integrity and hydrophobicity of STA were significantly improved compared to the wild-type stain.By transcriptome analysis and functional verification of key genes,six genes including Mar R,FUSC,glt B,glt D,acr A and Ara C were identified associating with D-3a tolerance.In addition,the RND efflux pump subunit protein Acr A also played an important role in cell tolerance of osmotic pressure and low p H environments.（3）Based on rational and semi-rational engineering of the crystal structure of Lb MDH,variants with improved enzymatic activities of aromatic and aliphatic substrates were obtained,and their molecular mechanisms were explored by crystallization.The crystal structure of Lb MDH was resolved at 1.75?,and through sequence alignment and structural analysis,the active center and substrate channel of Lb MDH were rationally modified.The variant L243W was proved to have improved enzymatic activity to D-3b,the K_m was reduced by 3.9 times compared to the wild-type,and the k_cat/K_m was increased by 2.3 times.By analyzing the conservation of 8 loop regions around the active center,5 non-conservative residues were identified.After site-saturation mutagenesis,the positive mutations were combined and A103G/T143G was identified with k_cat/K_m of 512.3 S^-1·m M^-1,which is 2.9times that of WT.Subsequently,the crystal structures of A103G/T143G（2.55?）and L243W（2.00?）were resolved.Based on crystal structure alignments,hydrogen bond interaction analysis of active centers and substrate channel visualization analyses,the reduced substrate channel of L243W variant and the expanded catalytic caver of A103G/T143G variant were revealed,which led to the diametrically opposite enzymatic activities towards aliphatic and aromatic substrates,respectively.Molecular dynamics simulation results revealed that A103G/T143G had higher catalytic stability compared to the wild-type.（4）Recombinant strains for synthesizing unnaturalα-amino acids were constructed with adapted multi-enzyme cascade of SpEH,BcLDH,and Lb MDH wild-type and its variants by the screened novel constitutive promoters.Seven endogenous strong promoters were screened from G.oxydans transcriptome data,among which,P₁₂₇₈₀ was the strongest for SpEH and the expression level improved 3.8 times compared to P_lac,and 1a-d were transformed into D-3a-d by using this recombinant strain with 40.6-94.8%conversion.P₀₄₇₅₀ was the strongest promoter for BcLDH expression with 2.4 times improvement,co-expression BcLDH with Lb MDH wild-type and its variants in G.oxydans and L-5a-d were successfully biosynthesized from 2a-d with 40.1-83.4%conversion and 99%e.e.Co-expressing SpEH with Lb MDH and BcLDH in G.oxydans to whole-cell bio-transforming 1a-e to L-5a-e,the conversion were 39.4-84.3%and the e.e.value also up to 99%.