{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,29]],"date-time":"2026-04-29T22:28:55Z","timestamp":1777501735336,"version":"3.51.4"},"reference-count":32,"publisher":"Wiley","issue":"7","license":[{"start":{"date-parts":[[2025,4,18]],"date-time":"2025-04-18T00:00:00Z","timestamp":1744934400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Biotech &amp; Bioengineering"],"published-print":{"date-parts":[[2025,7]]},"abstract":"<jats:title>ABSTRACT<\/jats:title><jats:p><jats:italic>Aspergillus niger<\/jats:italic>, a filamentous fungus, is known as a cell factory due to its ability to produce large amounts of organic acids and industrial enzymes. Lipase B from <jats:italic>Candida antarctica<\/jats:italic> (CALB) is one of the most widely used lipases in industrial applications, including oil processing, papermaking, food, pharmaceuticals, and personal care products. In this study, the CRISPR\/Cas9 technique was employed to knock out the <jats:italic>pyrG<\/jats:italic> and <jats:italic>kusA<\/jats:italic> genes in <jats:italic>A. niger<\/jats:italic>. The CALB gene was integrated into the high\u2010production protein gene loci, such as <jats:italic>glaA<\/jats:italic> and <jats:italic>amyA<\/jats:italic>, to construct a multi\u2010copy CALB production engineered strain. Additionally, the <jats:italic>pepA<\/jats:italic>, <jats:italic>aglU<\/jats:italic>, and <jats:italic>bglA<\/jats:italic> genes were deleted, which minimized the background level of secreted proteins in <jats:italic>A. niger<\/jats:italic> and increased the production of CALB. After two rounds of gene editing, the <jats:italic>A. niger<\/jats:italic> with multi\u2010copy CALB was created, and the engineered <jats:italic>A. niger<\/jats:italic> CCTCC 206047.09 with high CALB yield was isolated. After 120\u2009h of liquid fermentation, the lipase activity reached 17.84\u2009U\/mL and the protein yield reached 10.21\u2009mg\/mL. In summary, an engineered <jats:italic>A. niger<\/jats:italic> strain with high lipase activity was successfully isolated by employing a CRISPR\/Cas9 system to integrate CALB into high\u2010expression loci, while simultaneously knocking out the host's highly expressed protein genes. These results provide an effective strategy for the high expression of both heterologous and homologous enzymes in <jats:italic>A. niger<\/jats:italic>.<\/jats:p>","DOI":"10.1002\/bit.29000","type":"journal-article","created":{"date-parts":[[2025,4,18]],"date-time":"2025-04-18T04:03:11Z","timestamp":1744948991000},"page":"1770-1779","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Heterologous Expression of <i>Candida antarctica<\/i> Lipase B in <i>Aspergillus niger<\/i> Using CRISPR\/Cas9\u2010mediated Multi\u2010Gene Editing"],"prefix":"10.1002","volume":"122","author":[{"given":"Hongmei","family":"Nie","sequence":"first","affiliation":[{"name":"Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou P. R. China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mengmeng","family":"Jin","sequence":"additional","affiliation":[{"name":"Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou P. R. China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zebin","family":"Wang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou P. R. China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9821-1062","authenticated-orcid":false,"given":"Jianyong","family":"Zheng","sequence":"additional","affiliation":[{"name":"Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou P. R. China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yinjun","family":"Zhang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou P. R. China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaojun","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Fundamental Medicine Xinyu University Xinyu P. R. China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2025,4,18]]},"reference":[{"key":"e_1_2_11_2_1","doi-asserted-by":"publisher","DOI":"10.1111\/febs.16891"},{"key":"e_1_2_11_3_1","doi-asserted-by":"publisher","DOI":"10.1080\/1040841X.2020.1828815"},{"key":"e_1_2_11_4_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12934-020-01428-8"},{"key":"e_1_2_11_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mimet.2019.105655"},{"key":"e_1_2_11_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.pep.2019.105482"},{"key":"e_1_2_11_7_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12934-018-0941-8"},{"key":"e_1_2_11_8_1","doi-asserted-by":"publisher","DOI":"10.3390\/jof8050467"},{"key":"e_1_2_11_9_1","doi-asserted-by":"publisher","DOI":"10.1186\/s40694-019-0076-7"},{"key":"e_1_2_11_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biotechadv.2020.107630"},{"key":"e_1_2_11_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biortech.2023.128962"},{"key":"e_1_2_11_12_1","doi-asserted-by":"publisher","DOI":"10.1186\/1475-2859-9-23"},{"key":"e_1_2_11_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbiotec.2006.12.021"},{"key":"e_1_2_11_14_1","doi-asserted-by":"publisher","DOI":"10.1186\/s40694-020-00095-z"},{"key":"e_1_2_11_15_1","doi-asserted-by":"publisher","DOI":"10.1007\/s11274-016-2168-3"},{"key":"e_1_2_11_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.fgb.2007.07.003"},{"key":"e_1_2_11_17_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0133085"},{"key":"e_1_2_11_18_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt1282"},{"key":"e_1_2_11_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0167-7799(02)01933-9"},{"key":"e_1_2_11_20_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13068-018-1139-3"},{"key":"e_1_2_11_21_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biortech.2012.01.013"},{"key":"e_1_2_11_22_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt0294-181"},{"key":"e_1_2_11_23_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0202868"},{"key":"e_1_2_11_24_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12934-024-02353-w"},{"key":"e_1_2_11_25_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jafc.3c04138"},{"key":"e_1_2_11_26_1","doi-asserted-by":"publisher","DOI":"10.1016\/0307-4412(83)90068-7"},{"key":"e_1_2_11_27_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10529-018-2540-5"},{"key":"e_1_2_11_28_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12934-021-01659-3"},{"key":"e_1_2_11_29_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.foodchem.2024.142274"},{"key":"e_1_2_11_30_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12934-020-01334-z"},{"key":"e_1_2_11_31_1","doi-asserted-by":"publisher","DOI":"10.13995\/j.cnki.11-1802\/ts.029413"},{"key":"e_1_2_11_32_1","doi-asserted-by":"publisher","DOI":"10.1021\/acssynbio.7b00456"},{"key":"e_1_2_11_33_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.procbio.2014.12.018"}],"container-title":["Biotechnology and Bioengineering"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/analyticalsciencejournals.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/bit.29000","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T20:00:57Z","timestamp":1760040057000},"score":1,"resource":{"primary":{"URL":"https:\/\/analyticalsciencejournals.onlinelibrary.wiley.com\/doi\/10.1002\/bit.29000"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,4,18]]},"references-count":32,"journal-issue":{"issue":"7","published-print":{"date-parts":[[2025,7]]}},"alternative-id":["10.1002\/bit.29000"],"URL":"https:\/\/doi.org\/10.1002\/bit.29000","archive":["Portico"],"relation":{},"ISSN":["0006-3592","1097-0290"],"issn-type":[{"value":"0006-3592","type":"print"},{"value":"1097-0290","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,4,18]]},"assertion":[{"value":"2024-11-24","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-04-05","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-04-18","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}]}}