基因编辑益生菌在动物肠道健康上的研究及应用

doi:10.16431/j.cnki.1671-7236.2023.03.017

摘要/Abstract

摘要： 益生菌是一类有助于动物机体健康的微生物,在提高机体免疫力、改善肠道微生物平衡、促进养殖场环境等方面发挥重要作用。近年来,国内外学者对基因工程技术的探索已逐步深入,并在实际生产中加以应用。但是,目前为止绝大多数的研究将基因编辑技术着眼于真核生物。在此基础上,如何将基因编辑技术应用于益生菌,使益生菌发挥出更大的潜力是当前的研究热潮。因此通过基因工程技术将特定基因与益生菌进行基因编辑并表达,编辑后的益生菌可以表达特定的基因或者靶向对宿主发挥免疫调节作用。作者总结了基因编辑后的益生菌与宿主的相互作用,综述了CRISPR-Cas9技术在基因编辑乳酸菌及畜牧生产上的应用,同时经过分析比较不同基因编辑技术对乳酸菌进行基因编辑的方法,发现CRISPR-Cas9技术是目前针对乳酸菌和双歧杆菌等食品级益生菌相对灵活的基因编辑工具,能够实现益生菌在宿主体内发挥多重健康功效的目的,并对未来CRISPR-Cas9技术在基因编辑乳酸菌中的应用进行了展望,认为未来应将CRISPR-Cas9技术和其他基因编辑方法相结合,探索出更快更高效、简便的益生菌基因编辑技术。

关键词: 基因工程; 益生菌; 乳酸菌; CRISPR-Cas9技术; 免疫调节

Abstract: Probiotics are a kind of microorganisms that contribute to the health of animals.They play an important role in improving the immunity of animals,the balance of intestinal microorganisms and the environment of livestock farms.In recent years,scholars in domestic and foreign have gradually deepened their exploration of genetic engineering technology which applied it in practical production.However,the vast majority of research has focused on eukaryotes.On this basis,how to apply gene editing technology to probiotics and make probiotics play a greater potential is the current research upsurge.Therefore,through genetic engineering technology,specific genes and probiotics can be gene edited and expressed,and the edited probiotics can express specific genes or target to play an immunomodulatory role on the host.In this paper,the interaction between probiotics gene-edited and host have been summarized.The application of CRISPR-Cas9 technology in gene editing lactic acid bacteria and animal production is reviewed.Meanwhile,the methods of gene editing of lactic acid bacteria by different gene editing technologies are analyzed and compared.It is found that CRISPR-Cas9 technology is a relatively flexible gene editing tool for food-grade probiotics such as lactobacillus and bifidobacteria,so as to realize the purpose that probiotics can play multiple health effects in the host body,and the future application of CRISPR-Cas9 technology in gene editing lactobacillus is prospected.In the future,CRISPR-Cas9 technology will be combined with other gene editing methods to explore faster,more efficient and simple probiotic gene editing technology.

Key words: genetic engineering; probiotics; lactic acid bacteria; CRISPR-Cas9 technology; immune regulation

中图分类号:

Q815

陈少俊, 李刚, 奈子达, 刘娣, 姜新鹏. 基因编辑益生菌在动物肠道健康上的研究及应用[J]. 中国畜牧兽医, 2023, 50(3): 1016-1024.

CHEN Shaojun, LI Gang, NAI Zida, LIU Di, JIANG Xinpeng. Research and Application of Gene-edited Probiotics in Animal Intestinal Health[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1016-1024.

参考文献

[1] THE INTEGRATIVE HMP(iHMP) RESEARCH NETWORK CONSORTIUM.The integrative human microbiome project:Dynamic analysis of microbiome-host omics profiles during periods of human health and disease[J].Cell Host Microbe,2014,16(3):276-289.
[2] KAILASAPATHY K,CHIN J.Survival and therapeutic potential of probiotic organisms with reference to Lactobacillus acidophilus and Bifidobacterium spp.[J].Immunology and Cell Biology,2000,78(1):80-88.
[3] WULLT M,JOHANSSON HAGSLATT M L,ODENHOLT I,et al.Lactobacillus plantarum 299V enhances the concentrations of fecal short-chain fatty acids in patients with recurrent clostridium difficile-associated diarrhea[J].Digestove Diseases and Sciences,2007,52(9):2082-2086.
[4] IZUMO T,MAEKAWA T,IDA M,et al.Effect of intranasal administration of Lactobacillus pentosus S-PT84 on Influenza virus infection in mice[J].International Immunopharmacology,2010,10(9):1101-1106.
[5] FERNANDEZ-DUARTE K P,OLAYA-GALAN N N,SALAS-CARDENAS S P,et al.Bifidobacterium adolescentis (DSM 20083) and Lactobacillus casei (Lafti L26-DSl):Probiotics able to block the in vitro adherence of Rotavirus in MA104 cells[J].Probiotics Antimicrob Proteins,2018,10(1):56-63.
[6] ERMOLENKO E I,FURAEVA V A,ISAKOV V A,et al.Inhibition of Herpes simplex virus type 1 reproduction by probiotic bacteria in vitro[J].Voprosy Virusologii,2010,55(4):25-28.
[7] ISABELLA V M,HA B N,CASTILLO M J,et al.Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria[J].Nature Biotechnology,2018,36(9):857-864.
[8] GANGAIAH D,RYAN V,VAN HOESEL D,et al.Recombinant Limosilactobacillus (Lactobacillus) delivering nanobodies against clostridium perfringens netb and alpha toxin confers potential protection from necrotic enteritis[J].Microbiologyopen,2022,11(2):293-299.
[9] HUANG Q,NIU T,ZOU B,et al.Lactobacillus plantarum surface-displayed ASFV (P14.5) can stimulate immune responses in mice[J].Vaccines (Basel),2022,10(3):335-345.
[10] ZHUANG Z,WU Z G,CHEN M,et al.Secretion of human interferon-beta 1B by recombinant Lactococcus lactis[J].Biotechnology Letters,2008,30(10):1819-1823.
[11] YIN S,ZHU H,SHEN M,et al.Surface display of heterologous beta-galactosidase in food-grade recombinant Lactococcus lactis[J].Current Microbiology,2018,75(10):1362-1371.
[12] KANG S,LIN Z,XU Y,et al.A recombinant bifidobacterium bifidum BGN4 strain expressing the streptococcal superoxide dismutase gene ameliorates inflammatory bowel disease[J].Microbial Cell Factories,2022,21(1):113-124.
[13] LE LINH H,THU N P A,DUNG T T X,et al.Yeast cell surface displaying VP28 antigen and its potential application for shrimp farming[J].Applied Microbiology Biotechnology,2021,105(16-17):6345-6354.
[14] ZHOU H,GAO Y,GAO G,et al.Oral administration of recombinant Lactococcus lactis expressing the cellulase gene increases digestibility of fiber in geese[J].Current Microbiology,2015,71(6):693-698.
[15] NIU H,XING J H,ZOU B S,et al.Immune evaluation of recombinant Lactobacillus plantarum with surface display of ha1-dcpep in mice[J].Frontiers in Immunology,2021,12:800965-800980.
[16] PAN N,LIU B,BAO X,et al.Oral delivery of novel recombinant Lactobacillus elicit high protection against Staphylococcus aureus pulmonary and skin infections[J].Vaccines (Basel),2021,9(9):984-998.
[17] HOU X L,YU L Y,LIU J,et al.Surface-displayed Porcine epidemic diarrhea viral (PEDV) antigens on lactic acid bacteria[J].Vaccine,2007,26(1):24-31.
[18] MA S,WANG L,HUANG X,et al.Oral recombinant Lactobacillus vaccine targeting the intestinal microfold cells and dendritic cells for delivering the core neutralizing epitope of Porcine epidemic diarrhea virus[J].Microbial Cell Factories,2018,17(1):20-32.
[19] INATOMI T,AMATATSU M,ROMERO-PEREZ G A,et al.Dietary probiotic compound improves reproductive performance of Porcine epidemic diarrhea virus-infected sows reared in a Japanese commercial swine farm under vaccine control condition[J].Frontiers in Immunology,2017,8:1877-1885.
[20] JIANG X,HOU X,TANG L,et al.A phase trial of the oral Lactobacillus casei vaccine polarizes TH2 cell immunity against Transmissible gastroenteritis coronavirus infection[J].Applied Microbiology Biotechnology,2016,100(17):7457-7469.
[21] HOU X,JIANG X,JIANG Y,et al.Oral immunization against PEDV with recombinant Lactobacillus casei expressing dendritic cell-targeting peptide fusing coe protein of PEDV in piglets[J].Viruses,2018,10(3):106-120.
[22] LEBEER S,BRON P A,MARCO M L,et al.Identification of probiotic effector molecules:Present state and future perspectives[J].Current Opinion in Biotechnology,2018,49:217-223.
[23] GOH Y J,AZCARATE-PERIL M A,O’FLAHERTY S,et al.Development and application of a upp-based counterselective gene replacement system for the study of the S-layer protein SlpX of Lactobacillus acidophilus NCFM[J].Applied and Environmental Microbiology,2009,75(10):3093-3105.
[24] O’FLAHERTY S J,KLAENHAMMER T R.Functional and phenotypic characterization of a protein from Lactobacillus acidophilus involved in cell morphology,stress tolerance and adherence to intestinal cells[J].Microbiology (Reading),2010,156(Pt 11):3360-3367.
[25] JOHNSON B R,KLAENHAMMER T R.ACMB is an S-layer-associated beta-N-acetylglucosaminidase and functional autolysin in Lactobacillus acidophilus NCFM[J].Applied and Environmental Microbiology,2016,82(18):5687-5697.
[26] HYMES J P,JOHNSON B R,BARRANGOU R,et al.Functional analysis of an S-layer-associated fibronectin-binding protein in Lactobacillus acidophilus NCFM[J].Applied and Environmental Microbiology,2016,82(9):2676-2685.
[27] JOHNSON B,SELLE K,O’FLAHERTY S,et al.Identification of extracellular surface-layer associated proteins in Lactobacillus acidophilus NCFM[J].Microbiology (Reading),2013,159(Pt 11):2269-2282.
[28] GOH Y J,KLAENHAMMER T R.Functional roles of aggregation-promoting-like factor in stress tolerance and adherence of Lactobacillus acidophilus NCFM[J].Applied and Environmental Microbiology,2010,76(15):5005-5012.
[29] OH J H,VAN PIJKEREN J P.CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri[J].Nucleic Acids Research,2014,42(17):e131-e142.
[30] OH J H,ALEXANDER L M,PAN M,et al.Dietary fructose and microbiota-derived short-chain fatty acids promote bacteriophage production in the gut symbiont Lactobacillus reuteri[J].Cell Host and Microbe,2019,25(2):273-284.
[31] ZENG Z,ZUO F,MARCOTTE H.Putative adhesion factors in vaginal Lactobacillus gasseri DSM14869:Functional characterization[J].Applied and Environmental Microbiology,2019,85(19):00800-00819.
[32] REMUS D M,BONGERS R S,MEIJERINK M,et al.Impact of Lactobacillus plantarum sortase on target protein sorting,gastrointestinal persistence,and host immune response modulation[J].Journal of Bacteriology,2013,195(3):502-509.
[33] KANESAKI Y,MASUTANI H,SAKANAKA M,et al.Complete genome sequence of Bifidobacterium longum 105-A,a strain with high transformation efficiency[J].Genome Announcements,2014,2(6):1311-1314.
[34] O’CALLAGHAN A,VAN SINDEREN D.Bifidobacteria and their role as members of the human gut microbiota[J].Frontiers in Microbiology,2016,7:925-948.
[35] SAKANAKA M,HANSEN M E,GOTOH A,et al.Evolutionary adaptation in fucosyllactose uptake systems supports bifidobacteria-infant symbiosis[J].Science Advances,2019,5(8):eaaw7696-7702.
[36] YAMADA C,GOTOH A,SAKANAKA M,et al.Molecular insight into evolution of symbiosis between breast-fed infants and a member of the human gut microbiome Bifidobacterium longum[J].Cell Chemical Biology,2017,24(4):515-524.
[37] PLAVEC T V,BERLEC A.Engineering of lactic acid bacteria for delivery of therapeutic proteins and peptides[J].Applied Microbiology and Biotechnology,2019,103(5):2053-2066.
[38] BROEKAERT I J,WALKER W A.Probiotics as flourishing benefactors for the human body[J].Gastroenterol Nursing,2006,29(1):26-34.
[39] ABRIOUEL H,HERRMANN A,STARKE J,et al.Cloning and heterologous expression of hematin-dependent catalase produced by Lactobacillus plantarum CNRZ1228[J].Applied and Environmental Microbiology,2004,70(1):603-606.
[40] HIDALGO-CANTABRANA C,SANCHEZ B,ALVAREZ-MARTIN P,et al.A single mutation in the gene responsible for the mucoid phenotype of bifidobacterium animalis sub sp.Lactis confers surface and functional characteristics[J].Applied and Environmental Microbiology,2015,81(23):7960-7968.
[41] VAUGHAN E E,VAN DEN BOGAARD P T,CATZEDDU P,et al.Activation of silent gal genes in the lac-gal regulon of Streptococcus thermophilus[J].Journal of Bacteriology,2001,183(4):1184-1194.
[42] HOLS P,FERAIN T,GARMYN D,et al.Use of homologous expression-secretion signals and vector-free stable chromosomal integration in engineering of Lactobacillus plantarum for alpha-amylase and levanase expression[J].Applied and Environmental Microbiology,1994,60(5):1401-1413.
[43] GASPAR P,CARVALHO A L,VINGA S,et al.From physiology to systems metabolic engineering for the production of biochemicals by lactic acid bacteria[J].Biotechnology Advances,2013,31(6):764-788.
[44] KYLA-NIKKILA K,HUJANEN M,LEISOLA M,et al.Metabolic engineering of Lactobacillus helveticus CNRZ32 for production of pure l-(+)-lactic acid[J].Applied and Environmental Microbiology,2000,66(9):3835-3841.
[45] ZUO F L,CHEN S W,MARCOTTE H.Engineer probiotic bifidobacteria for food and biomedical applications-current status and future prospective[J].Biotechnology Advances,2020,45:107654.
[46] MOUGIAKOS I,BOSMA E F,WEENINK K,et al.Efficient genome editing of a facultative thermophile using mesophilic spCas9[J].ACS Synthetic Biology,2017,6(5):849-861.
[47] GUO T,XIN Y,ZHANG Y,et al.A rapid and versatile tool for genomic engineering in Lactococcus lactis[J].Microbial Cell Factories,2019,18(1):22-34.
[48] VAN DER ELS S,JAMES J K,KLEEREBEZEM M,et al.Versatile Cas9-driven subpopulation selection toolbox for Lactococcus lactis[J].Applied and Environmental Microbiology,2018,84(8):2752-2778.
[49] HIDALGO-CANTABRANA C,GOH Y J,PAN M,et al.Genome editing using the endogenous type Ⅰ CRISPR-Cas system in Lactobacillus crispatus[J].Proceeding of the National Academy of Science of the United States of America,2019,116(32):15774-15783.
[50] HUANG H,SONG X,YANG S.Development of a RecE/T-assisted CRISPR-Cas9 toolbox for Lactobacillus[J].Biotechnology Journal,2019,14(7):e1800690-1800702.
[51] LEENAY R T,VENTO J M,SHAH M,et al.Genome editing with CRISPR-Cas9 in Lactobacillus plantarum revealed that editing outcomes can vary across strains and between methods[J].Biotechnology Journal,2019,14(3):1700583-1700608.
[52] JIANG Y,QIAN F,YANG J,et al.CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum[J].Nature Communications,2017,8:15179-15190.
[53] SONG X,HUANG H,XIONG Z,et al.CRISPR-Cas9(D10A) nickase-assisted genome editing in Lactobacillus casei[J].Applied and Environmental Microbiology,2017,83(22):e01259-e01272.
[54] ZHANG J,ZONG W,HONG W,et al.Exploiting endogenous CRISPR-Cas system for multiplex genome editing in Clostridium tyrobutyricum and engineer the strain for high-level butanol production[J].Metabolic Engineering,2018,47:49-59.
[55] 周秀清.丁酸梭菌CRISPR-Cas基因编辑工具的建立及应用[D].北京:中国农业科学院,2021. ZHOU X Q.Establishment and application of CRISPR-Cas based gene editing tools in Clostridium butyricum[D].Beijing:Chinese Academy of Agricultural Sciences,2021.(in Chinese)
[56] O’CONNELL M M,O’DRISCOLL J,FITZGERALD G F,et al.Overcoming the restriction barrier to plasmid transformation and targeted mutagenesis in Bifidobacterium breve UCC2003[J].Microbial Biotechnology,2009,2(3):321-332.
[57] HIRAYAMA Y,SAKANAKA M,FUKUMA H,et al.Development of a double-crossover markerless gene deletion system in Bifidobacterium longum:Functional analysis of the alpha-galactosidase gene for raffinose assimilation[J].Applied and Environmental Microbiology,2012,78(14):4984-4994.
[58] SAKAGUCHI K,HE J,TANI S,et al.A targeted gene knockout method using a newly constructed temperature-sensitive plasmid mediated homologous recombination in bifidobacterium longum[J].Applied Microbiology and Biotechnology,2012,95(2):499-509.
[59] BISWAS I,GRUSS A,EHRLICH S D,et al.High-efficiency gene inactivation and replacement system for gram-positive bacteria[J].Journal Bacteriology,1993,175(11):3628-3635.
[60] VAN PIJKEREN J P,BRITTON R A.High efficiency recombineering in lactic acid bacteria[J].Nucleic Acids Research,2012,40(10):e76.
[61] YANG P,WANG J,QI Q.Prophage recombinases-mediated genome engineering in Lactobacillus plantarum[J].Microbial Cell Factories,2015,14:154-165.
[62] XIN Y,GUO T,MU Y,et al.Identification and functional analysis of potential prophage-derived recombinases for genome editing in Lactobacillus casei[J].FEMS Microbiology Letters,2017,364(24):243-252.
[63] SAKAGUCHI K,FUNAOKA N,TANI S,et al.The PYRE gene as a bidirectional selection marker in Bifidobacterium longum 105-A[J].Bioscience of Microbiota Food and Health,2013,32(2):59-68.
[64] ROBERTS A,BARRANGOU R.Applications of CRISPR-Cas systems in lactic acid bacteria[J].FEMS Microbiology Reviews,2020,44(5):523-537.
[65] PAN M,NETHERY M A,HIDALGO-CANTABRANA C,et al.Comprehensive mining and characterization of CRISPR-Cas systems in Bifidobacterium[J].Microorganisms,2020,8(5):720-738.
[66] QIN Z,YANG Y,YU S,et al.Repurposing the endogenous type i-e CRISPR/Cas system for gene repression in gluconobacter oxydans wsh-003[J].ACS Synthetic Biology,2021,10(1):84-93
[67] VENTO J M,CROOK N,BEISEL C L.Barriers to genome editing with CRISPR in bacteria[J].Journal of Industrial Microbiology & Biotechnology,2019,46(9-10):1327-1341.
[68] ZUO F,ZENG Z,HAMMARSTROM L,et al.Inducible plasmid self-destruction (IPSD) assisted genome engineering in Lactobacilli and Bifidobacteria[J].ACS Synthetic Biology,2019,8(8):1723-1729.
[69] WANG C,CUI Y,QU X.Optimization of electrotransformation (ETF) conditions in lactic acid bacteria (lab)[J].Journal of Microbiological Methods,2020,174:105944-105957.
[70] PARK M J,PARK M S,JI G E.Improvement of electroporation-mediated transformation efficiency for a Bifidobacterium strain to a reproducibly high level[J].Journal of Microbiological Methods,2019,159:112-119.
[71] WELKER D L,CROWLEY B L,EVANS J B,et al.Transformation of Lactiplantibacillus plantarum and Apilactobacillus kunkeei is influenced by recipient cell growth temperature,vector replicon,and DNA methylation[J].Journal of Microbiological Methods,2020,175:105967-106012.
[72] YASUI K,KANO Y,TANAKA K,et al.Improvement of bacterial transformation efficiency using plasmid artificial modification[J].Nucleic Acids Research,2009,37(1):3-10.
[73] BOTTACINI F,MORRISSEY R,ROBERTS R J,et al.Comparative genome and methylome analysis reveals restriction/modification system diversity in the gut commensal bifidobacterium breve[J].Nucleic Acids Research,2018,46(4):1860-1877.
[74] DONG H,ZHANG Y,DAI Z,et al.Engineering clostridium strain to accept unmethylated DNA[J].PLoS One,2010,5(2):e9038.