Patchoulol Fermentation In Recombinant Escherichia Coli

As an important fragrance substance,patchoulol is the main component of patchouli oil which is widely used in the daily chemical industry.Because of its unique fragrance,it can be used to produce perfumes and insect repellents,and has potential medicinal value.Using metabolic engineering strategies to construct patchoulol synthesizing recombinant strains to realize the efficient patchoulol production will have greater economic value and environmental protection value.This study realized efficient synthesis of patchoulol in Escherichia coli through adding a short peptide tag to patchoulol synthase,engineering the key amino acid sites of patchoulol synthase,fusing patchoulol synthase to farnesyl pyrophosphate synthase,engineering the host chasis strain,and constructing a dynamic engineering system.The main results are as follows:（1）Adding fusion tags to patchoulol synthase to enhance the soluble expression.Five different peptide fusion tags were added to patchoulol synthase.The addition of T7A tag can effectively improve the soluble expression of patchoulol synthase in E.coli,and the titer of patchoulol in recombinant strain E.coli BL21（DE3）/p Mev+p ET-PTS2/T7A was 1.40-fold higher than the original strain in the shake flask experiment.（2）Fusion expression of patchoulol synthase to farnesyl pyrophosphate synthase.The encoding genes of farnesyl pyrophosphate synthase and patchoulol synthase were connected by five different linkers to shorten the distance between patchoulol synthase and substrate farnesyl pyrophosphate.Patchoulol titer in the recombinant strain E.coli BL21（DE3）/p Mev-△FPPs+p ET-FPPs-（PT）₄P-PTS2 was 1.94-fold higher than that of the original strain.（3）Molecular modification of patchoulol synthase.The amino acid residue which is highly conserved or positions close to the active center of patchoulol synthase were analyzed through amino acid sequence alignment and molecular structure modeling,and amino acid mutations were performed.Among them,the mutations of H454A,K458A,K458L and C415F increased the fermentation titer of patchoulol to 1.77-,1.65-,1.76-,and 1.96-fold compared to the original strain,respectively.The amino acid mutation was combined,and the C415F-H454A mutant can further increase the patchoulol titer to 2.88-fold higher than the original strain.（4）Engineering the host strain.Effects of deleting the central carbon metabolism pathway of the chasis strain and over-expressing transmembrane export protein and coenzyme recycling pathway on patchoulol fermentation were investigated in a fast-growing strain.The results showed that deleting by-product synthetic pathways of acetic acid,ethanol,succinic acid and lactic acid and over-expressing the efflux transporter Mac A and Msb A through substituting the chromosomal native promoter of the host strain with strong T7 promoter could improve patchoulol titer to 274.30 mg·L^-1.Further combining the above optimal strategies,patchoulol titer of the recombinant strain B0016-060H2/p Mev-△FPPs+p ET-FPPs-（PT）₄P-PTS2/C415F-H454A reached 338.64 mg·L^-1 in shake flask experiment.While cultivated in a 5-L bioreactor,the patchoulol titer reached 970.72 mg·L^-1 which was 2.87-fold higher than that obtained in the shake flask experiment.This was the highest titer reported for patchoulol fermentation with glucose as the sole carbon source.（5）The construction of dynamic regulation system.Several pyruvate dehydrogenase regulators（Pdh R）and their operator genes were used to construct pyruvate-resposive switches.The pyruvate-resposive switch was used to balance the relationship between cell growth and product synthesis,and the titer of patchoulol was further increase by 29.14%.These results provide materials for revealing the catalytic mechanism of patchoulol synthase,and help to promote patchoulol fermentation.