Improving acarbose production and eliminating the by-product component C with an efficient genetic manipulation system of Actinoplanes sp.SE50/110

Theα-glucosidase inhibitor acarbose is commercially produced by Actinoplanes sp.and used as a potent drug in the treatment of type-2 diabetes.In order to improve the yield of acarbose,an efficient genetic manipulation system for Actinoplanes sp.was established.The conjugation system between E.coli carryingØC31-derived integrative plasmids and the mycelia of Actinoplanes sp.SE50/110 was optimized by adjusting the parameters of incubation time of mixed culture(mycelia and E.coli),quantity of recipient cells,donor-to-recipient ratio and the concentration of MgCl2,which resulted in a high conjugation efficiency of 29.4%.Using this integrative system,a cloned acarbose biosynthetic gene cluster was introduced into SE50/110,resulting in a 35%increase of acarbose titer from 2.35 to 3.18 g/L.Alternatively,a pIJ101-derived replicating plasmid combined with the counter-selection system CodA(sm)was constructed for gene inactivation,which has a conjugation frequency as high as 0.52%.Meanwhile,almost all 5-flucytosine-resistant colonies were sensitive to apramycin,among which 75%harbored the successful deletion of targeted genes.Using this replicating vector,the maltooligosyltrehalose synthase gene treY responsible for the accumulation of component C was inactivated,and component C was eliminated as detected by LC-MS.Based on an efficient genetic manipulation system,improved acarbose production and the elimination of component C in our work paved a way for future rational engineering of the acarbose-producing strains.

Genetic manipulation and tools in myxobacteria for the exploitation of secondary metabolism

Myxobacteria are famous for their capacity for social behavior and natural product biosynthesis.The unique sociality of myxobacteria is not only an intriguing scientific topic but also the main limiting factor for their ma-nipulation.After more than half a century of research,a series of genetic techniques for myxobacteria have been developed,rendering these mysterious bacteria manipulable.Here,we review the advances in genetic manipu-lation of myxobacteria,with a particular focus on the exploitation of secondary metabolism.We emphasize the necessity and urgency of constructing the myxobacterial chassis for synthetic biology research and the exploita-tion of untapped secondary metabolism.

Apprehending the potential of BABY BOOM transcription factors to mitigate cotton regeneration and transformation

Since the advent of transgenic technology,the incorporation of gene(s)encoding traits of economic importance in cotton is being practiced worldwide.However,factors like recalcitrant nature of cotton cultivars,in vitro regeneration via tissue culture(especially via somatic embryogenesis),genotype dependency,long and toilsome protocols impede the pace of development of transgenic cotton.Besides that,types and age of explants,media composition,plant growth regulators and other environmental factors affect in vitro cotton regeneration significantly.The studies of genetic control of in vitro regeneration in plants have elucidated the role of certain transcription factor genes that are induced and expressed during somatic embryogenesis.Among these transcription factors,BABY BOOM(BBM)plays a very important role in signal transduction pathway,leading to cell differentiation and somatic embryos formation.The role of BBM has been established in plant cell proliferation,growth and development even without exogenous growth regulators.This review intends to provide an informative summary of regeneration and transformation problems in cotton and the latest developments in utilization of BBM transcription factors in cotton.We believe that the use of BBM will not only ease cotton genetic improvement but will also accelerate cotton breeding programmes.

Efficient genetic manipulation in the developing brain of tree shrew using in utero electroporation and virus infection

Tree shrews（also named banxrings）,the small mammals native to Southeast Asia,are featured by moderate size,easy breeding,high reproductivity and close genetic background to primates（Xu et al.,2012;Xiao et al.,2017）.Tiee shrews possess both conserved and unique features compared to primates,and thus will become a suitable animal model with modest cost-effciency（Yao,2017）.

Genetic technologies for extremely thermophilic microorganisms of Sulfolobus, the only genetically tractable genus of crenarchaea

Archaea represents the third domain of life, with the information-processing machineries more closely resembling those of eukaryotes than the machineries of the bacterial counterparts but sharing metabolic pathways with organisms of Bacteria, the sister prokaryotic phylum. Archaeal organisms also possess unique features as revealed by genomics and genome comparisons and by biochemical characterization of prominent enzymes. Nevertheless, diverse genetic tools are required for in vivo experiments to verify these interesting discoveries. Considerable efforts have been devoted to the development of genetic tools for archaea ever since their discovery, and great progress has been made in the creation of archaeal genetic tools in the past decade. Versatile genetic toolboxes are now available for several archaeal models, among which Sulfolobus microorganisms are the only genus representing Crenarchaeota because all the remaining genera are from Euryarchaeota. Nevertheless, genetic tools developed for Sulfolobus are probably the most versatile among all archaeal models, and these include viral and plasmid shuttle vectors, conventional and novel genetic manipulation methods, CRISPR-based gene deletion and mutagenesis, and gene silencing, among which CRISPR tools have been reported only for Sulfolobus thus far. In this review, we summarize recent developments in all these useful genetic tools and discuss their possible application to research into archaeal biology by means of Sulfolobus models.

Tailoring in fungi for next generation cellulase production with special reference to CRISPR/CAS system

Cellulose is the utmost plenteous source of biopolymer in our earth,and fungi are the most efcient and ubiquitous organism in degrading the cellulosic biomass by synthesizing cellulases.Tailoring through genetic manipulation has played a substantial role in constructing novel fungal strains towards improved cellulase production of desired traits.However,the traditional methods of genetic manipulation of fungi are time-consuming and tedious.With the availability of the full-genome sequences of several industrially relevant flamentous fungi,CRISPR-CAS(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein)technology has come into the focus for the profcient development of manipulated strains of flamentous fungi.This review summarizes the mode of action of cellulases,transcription level regulation for cellulase expression,various traditional strategies of genetic manipulation with CRISPR-CAS technology to develop modifed fungal strains for a preferred level of cellulase production,and the futuristic trend in this arena of research.

Advances in NK cell production

Immunotherapy based on natural killer(NK)cells is a promising approach for treating a variety of cancers.Unlike T cells,NK cells recognize target cells via a major histocompatibility complex(MHC)-independent mechanism and,without being sensitized,kill the cells directly.Several strategies for obtaining large quantities of NK cells with high purity and high cytotoxicity have been developed.These strategies include the use of cytokine−antibody fusions,feeder cells or membrane particles to stimulate the proliferation of NK cells and enhance their cytotoxicity.Various materials,including peripheral blood mononuclear cells(PBMCs),umbilical cord blood(UCB),induced pluripotent stem cells(iPSCs)and NK cell lines,have been used as sources to generate NK cells for immunotherapy.Moreover,genetic modification technologies to improve the proliferation of NK cells have also been developed to enhance the functions of NK cells.Here,we summarize the recent advances in expansion strategies with or without genetic manipulation of NK cells derived from various cellular sources.We also discuss the closed,automated and GMP-controlled large-scale expansion systems used for NK cells and possible future NK cell-based immunotherapy products.

Regulation of antibiotic biosynthesis in actinomycetes:Perspectives and challenges

Actinomycetes are the main sources of antibiotics.The onset and level of production of each antibiotic is subject to complex control by multi-level regulators.These regulators exert their functions at hierarchical levels.At the lower level,cluster-situated regulators(CSRs)directly control the transcription of neighboring genes within the gene cluster.Higher-level pleiotropic and global regulators exert their functions mainly through modulating the transcription of CSRs.Advances in understanding of the regulation of antibiotic biosynthesis in actinomycetes have inspired us to engineer these regulators for strain improvement and antibiotic discovery.