Yeast surface display of leech hyaluronidase for the industrial production of hyaluronic acid oligosaccharides

Leech hyaluronidase(LHyal)is a hyperactive hyaluronic acid(HA)hydrolase that belongs to the hyaluronoglu-curonidase family.Traditionally,LHyal is extracted from the heads of leeches,but the recent development of the Pichia pastoris recombinant LHyal expression method permitted the industrial production of size-specific HA oligosaccharides.However,at present LHyal expressed by recombinant yeast strains requires laborious protein purification steps.Moreover,the enzyme is deactivated and removed after single use.To solve this problem,we developed a recyclable LHyal biocatalyst using a yeast surface display(YSD)system.After screening and charac-terization,we found that the cell wall protein Sed1p displayed stronger anchoring to the P.pastoris cell wall than other cell wall proteins.By optimizing the type and length of the linkers between LHyal and Sed1p,we increased the activity of enzymes displayed on the P.pastoris cell wall by 50.34%in flask cultures.LHyal-(GGGS)6-Sed1p activity further increased to 3.58×105 U mL−1 in fed-batch cultivation in a 5 L bioreactor.Enzymatic prop-erty analysis results revealed that the displayed LHyal-(GGGS)6-Sed1p generated the same oligosaccharides but exhibited higher thermal stability than free LHyal enzyme.Moreover,displayed LHyal-(GGGS)6-Sed1p could be recovered easily from HA hydrolysis solutions via low-speed centrifugation and could be reused at least 5 times.YSD of LHyal not only increased the utilization efficiency of the enzyme but also simplified the purification pro-cess for HA oligosaccharides.Thus,this study provides an alternative approach for the industrial preparation of LHyal and HA oligosaccharides.

Design of artificial small regulatory trans-RNA for gene knockdown in Bacillus subtilis

Bacillus subtilis as the Gram-positive model bacterium has been widely used in synthetic biology and biotechnology while the regulatory RNA tools for B.subtilis are still not fully explored.Here,a bottom-up approach is proposed for designing artificial trans-acting sRNAs.By engineering the intrinsic sRNA SR6,a minimized core scaffold structure consisting of an 8 bp stem,a 4 nt loop,and a 9 nt polyU tail was generated and proven to be sufficient for constructing sRNAs with strong repression activity(83%).Moreover,we demonstrate this artificial sRNA system functions well in an hfq-independent manner and also achieves strong repression efficiency in Escherichia coli(above 80%).A structure-based sRNA design principle was further developed for the automatic generation of custom sRNAs with this core scaffold but various sequences,which facilitates the manipulation and avoids structure disruption when fusing any base-pairing sequence.By applying these auto-designed sRNAs,we rapidly modified the cell morphology and biofilm formation,and regulated metabolic flux toward acetoin biosynthesis.This sRNA system with cross-species regulatory activities not only enriched the gene regulation toolkit in synthetic biology for B.subtilis and E.coli but also enhanced our understanding of trans-acting sRNAs.

Engineering Escherichia coli for high-yield production of ectoine

Ectoine is a natural macromolecule protector and synthesized by some extremophiles.It provides protections against radiation-mediated oxidative damages and is widely used as a bioactive ingredient in pharmaceutics and cosmetics.To meet its growing commercial demands,we engineered Escherichia coli strains for the high-yield production of ectoine.The ectABC gene cluster from the native ectoine producer Halomonas elongata was intro-duced into different Escherichia coli(E.Coil)strains via plasmids and 0.8 g L^(-1)of ectoine was produced in flask cultures by engineered E.coli BL21(DE3).Subsequently,we designed the ribosome-binding sites of the gene cluster to fine-tune the expressions of genes ectA,ectB,and ectC,which increased the ectoine yield to 1.6 g L^(-1).After further combinatorial overexpression of Corynebacterium glutamicum aspartate kinase mutant(G1A,C932T)and the H.elongate aspartate-semialdehyde dehydrogenase to increase the supply of the precursor,the titer of ectoine reached to 5.5 g L^(-1)in flask cultures.Finally,the engineered strain produced 60.7 g L^(-1)ectoine in fed-batch cultures with a conversion rate of 0.25 g/g glucose.

A comparative study on the genomes,transcriptomes,and metabolic properties of Escherichia coli strains Nissle 1917,BL21(DE3),and MG1655

Escherichia coli is the most well-studied model prokaryote and has become an indispensable host for the biotech-nological production of proteins and biochemicals.In particular,the probiotic status of one E.coli strain,E.coli Nissle 1917(EcN)has helped it become a new favorite amongst synthetic biologists.To broaden its potential applications,here we assemble a comparative study on the genomes,transcriptomes,and metabolic properties of E.coli strains EcN,BL21(DE3),and MG1655.Comparative genomics data suggests that EcN possesses 1404 unique CDSs.In particular,EcN has additional iron transport systems which endow EcN with a higher tolerance to iron scarcity when compared to two other E.coli strains.EcN transcriptome data demonstrates that E.coli strains EcN,BL21(DE3),and MG1655 all have comparable activities of the central metabolic pathway,however only EcN inherits the arginine deiminase pathway.Additionally,we found that EcN displayed a lower expres-sion of ribosomal proteins compared to BL21(DE3)and MG1655.This comparative study on E.coli strains EcN,BL21(DE3),and MG1655 aims to provide a reference for further engineering EcN as a biotechnological tool.

Gene knockdown by structure defined single-stem loop small non-coding RNAs with programmable regulatory activities

Gene regulation by trans-acting small RNAs(sRNAs)has considerable advantages over other gene regulation strategies.However,synthetic sRNAs mainly take natural sRNAs(MicC or SgrS)as backbones and comprise three functional elements folding into two or more stem-loop structures:an mRNA base pairing region,an Hfq-binding structure,and a rho-independent terminator.Due to limited numbers of natural sRNAs and complicated backbone structures,synthetic sRNAs suffer from low activity programmability and poor structural modularity.Moreover,natural sRNA backbone sequences may increase the possibility of unwanted recombination.Here,we present a bottom-up approach for creating structure defined single-stem loop small non-coding RNAs(ssl-sRNAs),which contain a standardized scaffold of a 7 bp-stem-4 nt-loop-polyU-tail and a 24 nt basing pairing region covering the first eight codons.Particularly,ssl-sRNA requires no independent Hfq-binding structure,as the polyU tail fulfills the roles of binding Hfq.A thermodynamic-based scoring model and a web server sslRNAD(http://www.kangzlab.cn/)were developed for automated design of ssl-sRNAs with well-defined structures and programmable activities.ssl-sRNAs displayed weak polar effects when regulating polycistronic mRNAs.The ssl-sRNA designed by sslRNAD showed regulatory activities in both Escherichia coli and Bacillus subtilis.A streamlined workflow was developed for the construction of customized ssl-sRNA and ssl-sRNA libraries.As examples,the E.coli cell morphology was easily modified and new target genes of ergothioneine biosynthesis were quickly identified with ssl-sRNAs.ssl-sRNA and its designer sslRNAD enable researchers to rapidly design sRNAs for knocking down target genes.

Enhanced catalytic efficiency and substrate specificity of Streptomyces griseus trypsin by evolution‑guided mutagenesis

Streptomyces griseus trypsin(SGT)is a bacteria-sourced trypsin that could be potentially applied to industrial insulin productions.However,SGT produced by microbial hosts displayed low catalytic efficiency and undesired preference to lysine residue.In this study,by engineering theαsignal peptide in Pichia pastoris,we increased the SGT amidase activity to 67.91 U mL^(−1)in shake flask cultures.Afterwards,we engineered SGT by evolution-guided mutagenesis and obtained three variants A45S,V177I and E180M with increased catalytic efficiencies.On this basis,we performed iterative combinatorial mutagenesis and constructed a mutant A45S/V177I/E180M which the amidase activity reached 98 U mL^(−1)in shake flasks and 2506 U mL^(−1)in 3-L fed-batch cultures.Moreover,single mutation T190 to S190 increased the substrate catalytic preference of R to K and the R/K value was improved to 7.5,which was 2 times better than the animal-sourced trypsin.