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.

Characterization of putative mannoprotein in Kluyveromyces lactis for lactase production

Lactase is a member of theβ-galactosidase family of enzymes that can hydrolyze lactose into galactose and glucose.However,extracellular lactase production was still restricted to the process of cell lysis.In this study,lactase-producing Kluyveromyces lactis JNXR-2101 was obtained using a rapid and sensitive method based on the fluorescent substrate 4-methylumbelliferyl-β-D-galactopyranoside.The purified enzyme was identified as a neutral lactase with an optimum pH of 9.To facilitate extracellular production of lactase,a putative mannoprotein KLLA0_E01057g of K.lactis was knocked out.It could effectively promote cell wall degradation and lactase production after lyticase treatment,which showed potential on other extracellular enzyme preparation.After optimizing the fermentation conditions,the lactase yield from mannoprotein-deficient K.lactis JNXR-2101ΔE01057g reached 159.62 U/mL in a 5-L fed-batch bioreactor.

Development of a monitoring system for Huangjiu storage based on electrical conductivity

In order to quickly detect the rancidification of Huangjiu in pottery jars,this study developed a fast detection method based on the principle of electrical conductivity changes caused by microbial contamination.The change in total acid in Huangjiu was positively correlated with the increase of electrical conductivity.This method was applied to an online monitoring system for Huangjiu storage in stainless steel tanks.When the electrical conductivity exceeds the normal fluctuation range(mean+3 standard deviations)of previous data,the monitoring system recognizes microbial contamination.By optimizing the conductivity-temperature compensation coefficient and conductivity statistical method,the standard deviation of the method was reduced and the sensitivity of microbial pollution monitoring was improved.The ranges of conductivity and compensation coefficient of common types of Huangjiu were estimated.Interference in conductivity measurements due to environmental factors was minimised through the synchronous comparison of conductivity data for multiple tanks of Huangjiu.The standard deviation,which indicates the fluctuation range of the system,decreased from 143 to 2μS/cm.The monitoring system was then applied in Huangjiu storage tanks with capacities of 60 t and 300 t.Through the comparison of conductivity data change,the abnormal signals caused by microbial contamination during the storage of Huangjiu were found over time.Meanwhile,through offline detection of total acid in Huangjiu,the effectiveness of microbial contamination online detection was verified.

Development of a novel platform for recombinant protein production in Corynebacterium glutamicum on ethanol

Corynebacterium glutamicum represents an emerging recombinant protein expression factory due to its ideal features for protein secretion,but its applicability is harmed by the lack of an autoinduction system with tight regulation and high yield.Here,we propose a new recombinant protein manufacturing platform that leverages ethanol as both a delayed carbon source and an inducer.First,we reanalysed the native inducible promoter PICL from the acetate uptake operon and found that its limited capacity is the result of the inadequate translation initial architecture.The two strategies of bicistronic design and ribozyme-based insulator can ensure the high activity of this promoter.Next,through transcriptional engineering that alters transcription factor binding sites(TFBSs)and the first transcribed sequence,the truncated promoter PA256 with a dramatically higher transcription level was generated.When producing the superfolder green fluorescent protein(sfGFP)under 1%ethanol conditions,PA256 exhibited substantially lower protein accumulation in prophase but an approximately 2.5-fold greater final yield than the strong promoter PH36.This superior expression mode was further validated using two secreted proteins,camelid antibody fragment(VHH)and endoxylanase(XynA).Furthermore,utilizing CRISPRi technology,ethanol utilization blocking strains were created,and PA256 was shown to be impaired in the phosphotransacetylase(PTA)knockdown strains,indicating that ethanol metabolism into the tricarboxylic acid cycle is required for PA256 upregulation.Finally,this platform was applied to produce the“de novo design”protein NEO-2/15,and by introducing the N-propeptide of CspB,NEO-2/15 was effectively secreted with the accumulation 281 mg/L obtained after 24 h of shake-flask fermentation.To the best of our knowledge,this is the first report of NEO-2/15 secretory overexpression.

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.

Characterization and implications of prokaryotic ribosome-binding sites across species

The ribosome-binding site(RBS)in the 5′untranslated region is recognized by 16S rRNA to start translation and is an essential element of the gene expression system.RBSs have been widely applied in regulating gene expression in various scenarios,including Gram-negative or Gram-positive bacteria.Here,we first rationally designed and constructed an RBS mutant library containing 66 RBSs.The strength of these RBSs in E.coli and C.glutamicum was characterized individually.The RBS strength spanned about 200 and 15 times in the two species,respectively.The strength of RBSs in C.glutamicum was generally lower than that of in E.coli.A total of 18 RBSs showed similar strength(within twofold differences)between the species in our study,and the correlation analysis of the strength of RBSs between E.coli and C.glutamicum(R^(2)=0.7483)revealed that these RBSs can be used across species.The sequence analysis revealed that the RBS region with two Ts stated was beneficial for RBS to function cross-species.The RBS characterized here can be used to precisely regulate gene expression in both hosts,and the characteristics of cross-species RBSs provide basic information for RBS rational design.