Efficient secretory expression of phospholipase D for the high-yield production of phosphatidylserine and phospholipid derivates from soybean lecithin

Phospholipase D(PLD)is an essential biocatalyst for the biological production of phosphatidylserine and phospholipid modification.However,the efficient heterologous expression of PLD is limited by its cell toxicity.In this study,a PLD was secretory expressed efficiently in Bacillus subtilis with an activity around 100 U/mL.A secretory expression system containing the signal peptide SPEstA and the dual-promoter PHpaII-SrfA was estab-lished,and the extracellular PLD activity further reached 119.22 U/mL through scale-up fermentation,191.30-fold higher than that of the control.Under optimum reaction conditions,a 61.61%conversion ratio and 21.07 g/L of phosphatidylserine production were achieved.Finally,the synthesis system of PL derivates was established,which could efficiently synthesis novel PL derivates.The results highlight that the secretory expression system constructed in this study provides a promising PLD producing strain in industrial application,and laid the foundation for the biosynthesis of phosphatidylserine and other PL derivates.As far as we know,this work re-ports the highest level of extracellular PLD expression to date and the enzymatic production of several PL der-ivates for the first time.

Characterization,heterologous expression and engineering of trehalase for biotechnological applications

Trehalose is a non-reducing disaccharide connected byα-1,1-glycosidic bonds;it is widely distributed in bacteria,fungi,yeast,insects,and plant tissues and plays various roles.It can be hydrolyzed by trehalase into two glucose molecules.Trehalases from different sources have been expressed in Escherichia coli,Pichia pastoris,Saccharomyces cerevisiae,baculovirus-silkworm,and other expression systems;however,it is most common in E.coli.The structural characteristics of different glycoside hydrolase(GH)family trehalases and the sources of trehalase have been analyzed.The catalytic mechanism of GH37 trehalase has also been elucidated in detail.Moreover,the molecular modification of trehalase has mainly focused on directed evolution for improving enzyme activity.We comprehensively reviewed the current application status and adaptable transformations was comprehensively overviewed in the context of industrial performance.We suggest that the level of recombinant production is far from meeting industrial requirements,and the catalytic performance of trehalase needs to be improved urgently.Finally,we discuss developmental prospects and future trends.

Enzymatic properties and inhibition tolerance analysis of key enzymes inβ-phenylethanol anabolic pathway of Saccharomyces cerevisiae HJ

Huangjiu is known for its unique aroma,primarily attributed to its high concentration ofβ-phenylethanol(ranging from 40 to 130 mg/L).Phenylalanine aminotransferase Aro9p and phenylpyruvate decarboxylase Aro10p are key enzymes in theβ-phenylethanol synthetic pathway of Saccharomyces cerevisiae^(HJ).This study examined the enzymatic properties of these two enzymes derived from S.cerevisiae^(HJ)and^(S288C).After substrate docking,Aro9p^(HJ)(-24.05 kJ/mol)and Aro10p^(HJ)(-14.33 kJ/mol)exhibited lower binding free energies compared to Aro9p^(S288C)(-21.93 kJ/mol)and Aro10p^(S288C)(-12.84 kJ/mol).ARO9 and ARO10 genes were heterologously expressed in E.coli BL21.Aro9p,which was purified via affinity chromatography,showed inhibition by L-phenylalanine(L-PHE),but the reaction rate Vmax(Aro9p^(HJ):23.89μmol⋅(min·g)^(-1)>Aro9p^(S288C):21.3μmol⋅(min·g)^(-1))and inhibition constant Ki values(Aro9p^(HJ):0.28 mol L^(-1)>Aro9p^(S288C)0.26 mol L^(-1))indicated that Aro9p from S.cerevisiae^(HJ)was more tolerant to substrate stress during Huangjiu fermentation.In the presence of the same substrate phenylpyruvate(PPY),Aro10p^(HJ)exhibited a stronger affinity than Aro10p^(S288C).Furthermore,Aro9p^(HJ)and Aro10p^(HJ)were slightly more tolerant to the final metabolitesβ-phenylethanol and ethanol,respectively,compared to those from^(S288C).The study suggests that the mutations in Aro9p^(HJ)and Aro10p^(HJ)may contribute to the increasedβ-phenylethanol concentration in Huangjiu.This is the first study investigating enzyme tolerance mechanisms in terms of substrate and product,providing a theoretical basis for the regulation of theβ-phenylethanol metabolic pathway.

Sequence and thermodynamic characteristics of terminators revealed by FlowSeq and the discrimination of terminators strength

The intrinsic terminator in prokaryotic forms secondary RNA structure and terminates the transcription.However,leaking transcription is common due to varied terminator strength.Besides of the representative hairpin and U-tract structure,detailed sequence and thermodynamic features of terminators were not completely clear,and the effect of terminator on the upstream gene expression was unclearly.Thus,it is still challenging to use terminator to control expression with higher precision.Here,in E.Coli,we firstly determined the effect of the 3′-end sequences including spacer sequences and terminator sequences on the expression of upstream and downstream genes.Secondly,terminator mutation library was constructed,and the thermodynamic and sequence features differing in the termination efficiency were analyzed using the FlowSeq technique.The result showed that under the regulation of terminators,a negative correlation was presented between the expression of upstream and downstream genes(r=0.60),and the terminators with lower free energy corelated with higher upstream gene expression.Meanwhile,the terminator with longer stem length,more compact loop and perfect U-tract structure was benefit to the transcription termination.Finally,a terminator strength classification model was established,and the verification experiment based on 20 synthetic terminators indicated that the model can distinguish strong and weak terminators to certain extent.The results help to elucidate the role of terminators in gene expression,and the key factors identified are crucial for rational design of terminators,and the model provided a method for terminator strength prediction.