Engineering Aspergillus oryzae A-4 through the Chromosomal Insertion of Foreign Cellulase Expression Cassette to Improve Conversion of Cellulosic Biomass into Lipids

摘要

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.