Cloning and Bioinformatics Analysis of Resveratrol Synthase Gene from Vitis vinifera

[Objectives] To obtain a resveratrol synthase gene of Vitis vinifera and make bioinformatics analysis. [Methods] Taking total RNA of V. vinifera as the template,by RT-PCR method,a complete c DNA sequence of resveratrol synthase gene was amplified from V. vinifera,and the resveratrol synthase gene was named as RS. The nucleic acid and protein sequences were analyzed using bioinformatics software.[Results]This sequence was 1179 bp in length,the similarity with reported resveratrol synthase gene reached 94%-99%,and the similarity with amino acid sequence reached 96%-99%; the RS gene encoded 392 amino acids,and amino acid sequence contained complete characteristic sequence GVLFGPGLT and active center sequence GCYAGGTVLR of stilbene synthase family; the predicted molecular weight was42. 78 k Da,the theoretical isoelectric point was 6. 57,the instability parameter was 35. 92,and it belonged to stable protein in the classification; the secondary structure was mainly α-helix,random coil and β-folding,α-helix content was 44. 13%,the random coil content was26. 53%,and β-folding content was 17. 66%. [Conclusions] The isolated RS gene is a resveratrol synthase gene from V. vinifera. This experiment is expected to lay a certain foundation for biosynthesis of resveratrol by the genetic engineering method.

体内连续进化技术的研究进展

实验室进化是遗传育种、提高微生物性能的重要方式。近几十年来,实验室进化的方法快速发展,应用也越来越广泛,但是常见的菌株进化策略以及针对特定蛋白的进化存在突变过程不连续,需要多轮操作、工作量大等缺点。微生物突变和筛选技术的进步促进了体内连续进化的发展,大大提高了实验室进化的效率。体内连续进化技术实现了体内突变,完美地把突变与筛选相结合,以最少的人工干预进化出特定表型。文中总结了近年来在微生物底盘中开发的基因组范围的体内连续进化技术,以及独立于基因组的针对特定蛋白的体内连续进化技术,主要对这些技术实现体内连续突变的原理及其相关应用进行了介绍。在此基础上,分析了现有技术的优缺点,并对体内连续进化技术的发展进行了展望。

Potential one-step strategy for PET degradation and PHB biosynthesis through co-cultivation of two engineered microorganisms

The management and recycling of plastic waste is a challenging global issue.Polyethylene terephthalate(PET),one of the most widely used synthetic plastics,can be hydrolyzed by a series of enzymes.However,upcycling the resulting monomers is also a problem.In this study,we designed a co-cultivation system,in which PET degradation was coupled with polyhydroxybutyrate(PHB)production.First,PETase from Ideonalla sakaiensis was expressed in Yarrowia lipolytica Po1f with a signal peptide from lipase.The engineered PETase-producing Y.lipolytica was confirmed to hydrolyze bis(2-hydroxyethyl)terephthalate(BHET)and PET powder into the monomers terephthalate(TPA)and ethylene glycol(EG).Simultaneously,a TPA-degrading Pseudomonas stutzeri strain isolated from PET waste was transformed with a recombinant plasmid containing the phb CAB operon from Ralstonia eutropha,which encodes enzymes for the biosynthesis of PHB.The two co-cultivated engineered microbes could directly hydrolyze BHET to produce the bioplastic PHB in one fermentation step.During this process,5.16 g/L BHET was hydrolyzed in 12 h,and 3.66 wt%PHB(3.54 g/L cell dry weight)accumulated in 54 h.A total of 0.31g/L TPA was produced from the hydrolyzation of PET in 228 h.Although PHB could not be synthesized directly from PET because of the low hydrolyzing efficiency of PETase,this study provides a new strategy for the biodegradation and upcycling of PET waste by artificial microflora.

Efficient production of 2′-fucosyllactose in unconventional yeast Yarrowia lipolytica

2′-Fucosyllactose(2′-FL)has great application value as a nutritional component and the whole cell biosynthesis of 2′-FL has become the focus of current research.Yarrowia lipolytica has great potential in oligosaccharide synthesis and large-scale fermentation.In this study,systematic engineering of Y.lipolytica for efficient 2′-FL production was performed.By fusing different protein tags,the synthesis of 2′-FL was optimized and the ubiquitin tag was demonstrated to be the best choice to increase the 2′-FL production.By iterative integration of the related genes,increasing the precursor supply,and promoting NADPH regeneration,the 2′-FL synthesis was further improved.The final 2′-FL titer,41.10 g/L,was obtained in the strain F5-1.Our work reports the highest 2′-FL production in Y.lipolytica,and demonstrates that Y.lipolytica is an efficient microbial chassis for the synthesis of oligosaccharides.

Biochemical characterization of a polyethylene terephthalate hydrolase and design of high-throughput screening for its directed evolution

Polyethylene terephthalate(PET),one of the most widely used plastics in the world,causes serious environmental pollution.Recently,researchers have focused their efforts on enzymatic degradation of PET,which is an attractive way of degrading and recycling PET.In this work,PET hydrolase Sb PETase from Schlegelella brevitalea sp.nov.was biochemically characterized,and rational design was performed based on its sequence similarity with the previ-ously reported Is PETase from Ideonella sakaiensis,resulting in a triple mutant with increased activity.Furthermore,using a sec-dependent signal peptide PeIB and colicin release protein Kil,we set up a high-efficiency secretion system of PETase in Escherichia coli BL21(DE3),enabling higher PETase secretion.Utilizing this secretion system,we established a high-throughput screening method named SecHTS(sec retion-based h igh-throughput s creening)and performed directed evolution of Is PETase and Sb PETase through DNA shuffling.Finally,we generated a mutant Is PETase S139T with increased activity from the mutant library.

The phage T4 DNA ligase mediates bacterial chromosome DSBs repair as single component non-homologous end joining

DNA double-strand breaks(DSBs)are one of the most lethal forms of DNA damage that is not efficiently repaired in prokaryotes.Certain microorganisms can handle chromosomal DSBs using the error-prone non-homologous end joining(NHEJ)system and ultimately cause genome mutagenesis.Here,we demonstrated that Enterobacteria phage T4 DNA ligase alone is capable of mediating in vivo chromosome DSBs repair in Escherichia coli.The ligation efficiency of DSBs with T4 DNA ligase is one order of magnitude higher than the NHEJ system from Mycobacterium tuberculosis.This process introduces chromosome DNA excision with different sizes,which can be manipulated by regulating the activity of host-exonuclease RecBCD.The DNA deletion length reduced either by inactivating recB or expressing the RecBCD inhibitor Gam protein fromλphage.Furthermore,we also found single nucleotide substitutions at the DNA junction,suggesting that T4 DNA ligase,as a single component non-homologous end joining system,has great potential in genome mutagenesis,genome reduction and genome editing.

The type IX secretion system: Insights into its function and connection to glycosylation in Cytophaga hutchinsonii

The recently discovered type IX secretion system(T9SS)is limited to the Bacteroidetes phylum.Cytophaga hutchin-sonii,a member of the Bacteroidetes phylum widely spread in soil,has complete orthologs of T9SS components and many T9SS substrates.C.hutchinsonii can efficiently degrade crystalline cellulose using a novel strategy,in which bacterial cells must be in direct contact with cellulose.It can rapidly glide over surfaces via unclear mech-anisms.Studies have shown that T9SS plays an important role in cellulose degradation,gliding motility,and ion assimilation in C.hutchinsonii.As reported recently,T9SS substrates are N-or O-glycosylated at their C-terminal domains(CTDs),with N-glycosylation being related to the translocation and outer membrane anchoring of these proteins.These findings have deepened our understanding of T9SS in C.hutchinsonii.In this review,we focused on the research progress on diverse substrates and functions of T9SS in C.hutchinsonii and the glycosylation of its substrates.A model of T9SS functions and the glycosylation of its substrates was proposed.

Lysine acetylation decreases enzyme activity and protein level of Escherichia coli lactate dehydrogenase

Lactate is an important bulk chemical with widespread applications and a major byproduct of other chemicals bioprocess in microbial fermentation.Lactate dehydrogenase A(LdhA)catalyzes the synthesis of lactate from pyruvate.Lysine acetylation is an evolutionarily conserved post-translational modification;however,the mech-anisms underlying the regulation of LdhA function by lysine acetylation in Escherichia coli remain poorly under-stood.Herein,we demonstrate acetylation of E.coli LdhA occurs via enzymatic and non-enzymatic mechanisms.Further,we show carbon source type and concentration affect the lysine acetylation status of LdhA via a non-enzymatic mechanism.Lysine acetylation significantly inhibits the enzymatic activity and protein level of LdhA.The results of the present study demonstrate lysine acetylation of E.coli LdhA is irreversible.Understanding of the effects of lysine acetylation on LdhA function may provide a new perspective for regulating lactate production in microbial synthesis.