Metabolic Engineering Of Bacillus Licheniformis For Efficient Production Of 2-phenylethanol

Due to its rose-like odor,2-phenylethanol(2-PE)is widely used as a flavor and fragrance agent in the fields of food,cosmetics,and perfume cosmetics.Currently,2-PE is produced predominantly through chemical synthesis and extraction from plants.However,the increasing demand of“natural”2-PE for consumers cannot be met by these methods.Microbial production of 2-PE has emerged to be an appealing approach,due to its mild reaction condition,environmental friend process,sustainable,and natural product.However,the low titer and low tolerance in microbial fermentation process are main obstacles for its industrial-scale production.In this work,Bacillus licheniformis DW2,generally regarded as safe(GRAS)strain,was used as the initial host.Firstly,an integrated CRISPR interference(CRISPRi)system was constructed in B.licheniformis;an efficient biocatalysis system for the synthesis of 2-PE from L-phenylalanine(L-Phe)was developed,and an excellent recombinant strain for de novo production of 2-PE was achieved.The main results of this study were summarized as follows:1.Establishment of an efficient integrated CRISPR interference system in B.licheniformis.The dcas9 gene was integrated into B.licheniformis DW 2 genome,several endogenous genes,including yvm C,cyp X and essential gene rps C,were severed as the targets to test this CRISPRi system.This system could efficiently repression single gene,operon,and essential gene(from45.02%to 94.00%).Moreover,multiple genes were simultaneously repressed with high efficiency using the extended multiplexed CRISPRi system.As a case study,genes involved in by-products(acetoin,2,3-butanediol,acetate and lactate)synthetic and L-valine degradation pathways were selected as the silence targets.Repression of acetolactate decarboxylase(als D)and leucine dehydrogenase(bcd)led to 90.48%and 80.09%increases in L-valine titers,respectively.Compared with the control strain DW9i?leu A,the L-valine titers of combinatorial strain DW9i?leu A/p HYi-als D-bcd were increased by 1.27-fold and 2.89-fold,respectively,in flask and 3-L bioreactor.2.Screening of 2-PE stress-tolerant hosts,and construction of microbial cell factory for efficient production of 2-PE from L-Phe.Firstly,candidate hosts for 2-PE synthesis were screened by evaluating their tolerance to 2-PE.Compared to selected Escherichia coli,B.amyloliquefaciens and B.subtilis,B.licheniformis DW2 showed highest tolerance.Subsequently,phenylpyruvate decarboxylase,alcohol dehydrogenase,and aromatic amino acid transaminase from different hosts were screened to construct an efficient Ehrlich pathway for2-PE production.Based on our results,Kiv D from Lactococcus lactis and Yqh D from E.coli were the best phenylpyruvate decarboxylase and alcohol dehydrogenase,respectively.In combination with CRISPRi system,His C was identified as key transaminase for conversing phenylpyruvate to L-Phe.The best recombinant strain DE4(co-expression of Kiv D and Yqh D)produced 3.04 g/L 2-PE from 5.00 g/L L-Phe.To reduce the cost of fermentation,the fermentation process was further optimized using molasses as carbon resource,2-PE titer was increased to 4.41 g/L.In 3-L fed-batch fermentation,the maximum 2-PE titer reached 5.16 g/L with a yield of 0.65 g/g on L-Phe and productivity of 0.12 g/(L.h).3.2-phenylethanol dehydrogenase(s)in B.licheniformis were screened through genome mining and gene functional analysis.Through in silico analysis,19 alcohol dehydrogenases of B.licheniformis DW2 were classified into Group I,Group II and Group III.Subsequently,to screen the enzyme(s)responsible for conversion of phenylacetaldehyde to 2-PE,these genes was deleted individually in DWc9n derived from DW2 to obtain single gene knockout strains.We found that deletion of yug J gene significantly affected 2-PE synthesis,which led to 95%reduction in 2-PE titer.By reinstalling and overexpressing yug J gene,the mutants retained and enhanced the ability for conversion of phenylacetaldehyde to 2-PE.Enzyme kinetic study further revealed that YugJ was capable of converting phenylacetaldehyde to 2-PE with 3.69?M/min of V_max,6.49 m M of K_m,2.64 min^-1 of K_cat,and 0.41 min^-1m M^-1 of K_cat/K_m,respectively.4.Using a modular pathway engineering strategy and rational“push-pull-restrain”strategy,the Ehrlich pathway,central metabolic pathway,and phenylpyruvate pathway were engineered systematically to redirect carbon flux towards 2-PE in B.licheniformis DWc9n.(1)A hybrid Ehrlich pathway was constructed by co-expression of kiv D and yug J.The plasmid-expression of kiv D and integrated expression of yug J was the best combination for 2-PE production,and the attained strain PE4 produced 0.28 g/L of 2-PE.(2)Improvement of PEP supply by rewiring the central metabolic pathway.The effects of knockout of pyruvate kinase coding gene pyk,or(and)glucose PTS transport protein coding gene pts G,and overexpression of PEP synthase coding gene(pps1,pps2 and pps3)on 2-PE production were analyzed.Our results showed that deletion of pyk was the best approach for improvement of PEP supply and enhancement of 2-PE titer,the attained strain PE10 produced0.50 g/L of 2-PE.(3)To systematically evaluate the effects of key genes involved in phenylpyruvate pathway on 2-PE synthesis,aro A,Ecaro F,Ecaro G^fbr,phe A,Ecphe A^fbr,aro K,and aro D were overexpressed individually in strain PE10.Based on our results,overexpressing aro A,Ecaro G^fbr,phe A and Ecphe A^fbr significantly improved 2-PE titers,increased by 182%,99%,131%,and 261%,respectively,compared to PE10.Thus,through modular metabolic engineering strategy,the superior recombinant strain PE12(pyk?kiv D?yug J?phe A^fbr?)produced 1.81 g/L of 2-PE,which increased by 7.23-fold,compared to the initial strain PE1(0.22 g/L).(4)Elimination of the competing pathways to improve 2-PE production.The main byproducts of strain PE12 were acetoin(AC),2,3-butanediol(2,3-BD),shikimate(SA),3-dehydro-shikimate(DHS)and L-Phe.To eliminate the production of shikimate and 3-dehydro-shikimate,the shikimate catabolism was enhanced by substituting the native aro K promoter withstrongpromoterPbac A,andthecorrespondingstrain PE27(pyk?kiv D?yug J?phe A^fbrPbac A-aro K)produced 3.61 g/L of 2-PE.In combination with the overexpression of aro D and Ecaro G^fbr,the 2-PE production was increased to 4.25 g/L.Reducing L-Phe accumulation was attempted by deleting his C,and the attained strain PE33(pyk?his C?kiv D?yug J?phe A^fbr?aro D?aro G^fbr?Pbac A-aro K)produced 5.35 g/L of 2-PE,increased by 1.96-fold compared to PE12(1.81 g/L).The production of AC/2,3-BD,SA,DHS and L-Phe in PE33 were decreased by 19.37%?79.85%?85.70%and 61.32%,respectively,compared to PE12.(5)Optimization of fermentation process to further increase 2-PE titer.The fermentation process was further optimized,and the best performing strain PE33 was capable of producing5.38 g/L of 2-PE,increased by 23.45-fold compared to the initial strain PE1.The maximum 2-PE titer obtained in this study represents the highest titer for de novo production of 2-PE.