Enhancing lipase activity in Aspergillus niger through CRISPR/Cas9-mediated protease gene knockout and fermentation optimization.

IF 2.1 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Hongmei Nie, Zebin Wang, Zhenkai Lin, Yan Gao, Yinjun Zhang, Jianyong Zheng, Yong Cheng
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引用次数: 0

Abstract

The engineered Aspergillus niger strain AnCALB005 was selected as the research strain, which is a high-yield strain of Candida antarctica B lipase constructed in our laboratory. CRISPR/Cas9-mediated gene knockout was employed to construct the multiple protease-deficient strains targeting five genes (pepA, pepB, pepC, pepE and pepF) in the A. niger AnCALB005. Among the engineered variants, a triple-knockout strain lacking pepA, pepB, and pepF demonstrated 56% enhanced hydrolytic lipase activity relative to the parental strain. Fermentation culture conditions were initially screened through single-factor experiments. Building on these results, critical parameters were statistically determined via Plackett-Burman (PB) design. This was followed by a steepest ascent method combined with Box-Behnken (BB) response surface methodology. Key factors influencing lipase production (identified as maltose concentration, corn steep concentration, and shaking speed) were optimized. The final optimized fermentation conditions comprised: maltose (52 g/L), corn steep (52 g/L), K2HPO4 (5 g/L), soybean cake flour (30 g/L), initial pH 6.5, inoculation amount 10% (v/v), and shaking speed 220 rpm. Under the optimized fermentation conditions, Shake-flask validation of the engineered A. niger yielded a lipase activity of 46.66 U/mL, representing an increase of 92.01%. Scale-up fermentation in a 5 L bioreactor applying these optimized conditions over 120 h of cultivation achieved a lipase activity of 79.31 U/mL.

通过CRISPR/ cas9介导的蛋白酶基因敲除和发酵优化提高黑曲霉脂肪酶活性
本研究选用本实验室构建的高产南极念珠菌B脂肪酶黑曲霉工程菌株AnCALB005作为研究菌株。利用CRISPR/ cas9介导的基因敲除技术,在黑孢霉ancal005中构建了针对5个基因(pepA、pepB、pepC、pepE和pepF)的多重蛋白酶缺陷菌株。在工程变异株中,缺乏pepA、pepB和pepF的三敲除菌株的水解脂肪酶活性比亲本菌株提高了56%。通过单因素实验初步筛选发酵培养条件。在这些结果的基础上,通过Plackett-Burman (PB)设计统计确定关键参数。其次是最陡上升法结合Box-Behnken (BB)响应面法。优化了影响脂肪酶产量的关键因素(麦芽糖浓度、玉米浸泡浓度和摇动速度)。最终优化的发酵条件为:麦芽糖(52 g/L)、玉米浆(52 g/L)、K2HPO4 (5 g/L)、豆粕粉(30 g/L)、初始pH 6.5、接种量10% (v/v)、摇速220 rpm。在优化的发酵条件下,经摇瓶验证的黑曲霉脂肪酶活性为46.66 U/mL,提高了92.01%。在5l生物反应器中放大发酵120 h,脂肪酶活性达到79.31 U/mL。
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来源期刊
Biotechnology Letters
Biotechnology Letters 工程技术-生物工程与应用微生物
CiteScore
5.90
自引率
3.70%
发文量
108
审稿时长
1.2 months
期刊介绍: Biotechnology Letters is the world’s leading rapid-publication primary journal dedicated to biotechnology as a whole – that is to topics relating to actual or potential applications of biological reactions affected by microbial, plant or animal cells and biocatalysts derived from them. All relevant aspects of molecular biology, genetics and cell biochemistry, of process and reactor design, of pre- and post-treatment steps, and of manufacturing or service operations are therefore included. Contributions from industrial and academic laboratories are equally welcome. We also welcome contributions covering biotechnological aspects of regenerative medicine and biomaterials and also cancer biotechnology. Criteria for the acceptance of papers relate to our aim of publishing useful and informative results that will be of value to other workers in related fields. The emphasis is very much on novelty and immediacy in order to justify rapid publication of authors’ results. It should be noted, however, that we do not normally publish papers (but this is not absolute) that deal with unidentified consortia of microorganisms (e.g. as in activated sludge) as these results may not be easily reproducible in other laboratories. Papers describing the isolation and identification of microorganisms are not regarded as appropriate but such information can be appended as supporting information to a paper. Papers dealing with simple process development are usually considered to lack sufficient novelty or interest to warrant publication.
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