Lin Hui, Wang Qun, Shen Qi, Ma Junwei, Fu Jianrong, Zhao Yuhua
Institute of Environment Resource and Soil Fertilizer, Zhejiang Academy of Agriculture Science, Hangzhou, China.
Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, China.
PLoS One. 2014 Sep 24;9(9):e108442. doi: 10.1371/journal.pone.0108442. eCollection 2014.
A genetic modification scheme was designed for Aspergillus oryzae A-4, a natural cellulosic lipids producer, to enhance its lipid production from biomass by putting the spotlight on improving cellulase secretion. Four cellulase genes were separately expressed in A-4 under the control of hlyA promoter, with the help of the successful development of a chromosomal genetic manipulation system. Comparison of cellulase activities of PCR-positive transformants showed that these transformants integrated with celA gene and with celC gene had significantly (p<0.05) higher average FPAase activities than those strains integrated with celB gene and with celD gene. Through the assessment of cellulosic lipids accumulating abilities, celA transformant A2-2 and celC transformant D1-B1 were isolated as promising candidates, which could yield 101%-133% and 35.22%-59.57% higher amount of lipids than the reference strain A-4 (WT) under submerged (SmF) conditions and solid-state (SSF) conditions, respectively. Variability in metabolism associated to the introduction of cellulase gene in A2-2 and D1-B1 was subsequently investigated. It was noted that cellulase expression repressed biomass formation but enhanced lipid accumulation; whereas the inhibitory effect on cell growth would be shielded during cellulosic lipids production owing to the essential role of cellulase in substrate utilization. Different metabolic profiles also existed between A2-2 and D1-B1, which could be attributed to not only different transgene but also biological impacts of different integration. Overall, both simultaneous saccharification and lipid accumulation were enhanced in A2-2 and D1-B1, resulting in efficient conversion of cellulose into lipids. A regulation of cellulase secretion in natural cellulosic lipids producers could be a possible strategy to enhance its lipid production from lignocellulosic biomass.
针对天然纤维素脂质生产菌米曲霉A-4设计了一种基因改造方案，通过聚焦提高纤维素酶分泌来增强其从生物质中生产脂质的能力。在染色体遗传操作系统成功开发的帮助下，四个纤维素酶基因在hlyA启动子的控制下分别在A-4中表达。PCR阳性转化体的纤维素酶活性比较表明，整合有celA基因和celC基因的转化体的平均滤纸酶活性显著（p<0.05）高于整合有celB基因和celD基因的菌株。通过评估纤维素脂质积累能力，分离出celA转化体A2-2和celC转化体D1-B1作为有潜力的候选菌株，在液体深层发酵（SmF）条件和固态发酵（SSF）条件下，它们产生的脂质量分别比参考菌株A-4（野生型）高101%-133%和35.22%-59.57%。随后研究了A2-2和D1-B1中与纤维素酶基因导入相关的代谢变化。结果表明，纤维素酶表达抑制了生物量形成，但增强了脂质积累；而由于纤维素酶在底物利用中的重要作用，在纤维素脂质生产过程中对细胞生长的抑制作用会被屏蔽。A2-2和D1-B1之间也存在不同的代谢谱，这不仅可归因于不同的转基因，还可归因于不同整合的生物学影响。总体而言,A2-2和D1-B1同时提高了糖化和脂质积累，实现了纤维素向脂质的高效转化。调节天然纤维素脂质生产菌中纤维素酶的分泌可能是提高其从木质纤维素生物质中生产脂质的一种可行策略。
