Engineering Aspergillus oryzae A-4 through the chromosomal insertion of foreign cellulase expression cassette to improve conversion of cellulosic biomass into lipids.

Hui Lin,Junwei Ma,Qi Shen,Yuhua Zhao,Jianrong Fu,Qun Wang

doi:10.1371/journal.pone.0108442

Hui Lin, Junwei Ma + Show 4 more

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https://doi.org/10.1371/journal.pone.0108442

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Abstract

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.

Highlights

Biodiesel is widely recognized as a type of green fuel, which has advantages of low sulfur content, being non-toxic and biodegradable, lack of aromatics, and excellent lubricity [1]
All DNA fragments for the construction of CelA, CelB, CelD and CelC expression vectors were cloned from A. oryzae ATCC 42149 genomic DNA
In 2002, Japanese researchers found that cell growth of a majority of detected filamentous fungi, including Aspergillus oryzae RIB138, Aspergillus niger IAM2561, Aspergillus nidulans FGSC89 and Trichoderma reesei IFO31326, could be suppressed when more than 0.1 mg l21 of pyrithiamine was added into the culture medium [17]

Summary

Introduction

Biodiesel is widely recognized as a type of green fuel, which has advantages of low sulfur content, being non-toxic and biodegradable, lack of aromatics, and excellent lubricity [1]. Testing strategies to establish more efficient, less-cost and sustainable technologies based on microbial fermentation for SCOs production from lignocellulosic biomass is under current investigation by various start-up biotechnology companies and research centers [2,3,4,5,6]. Among these researches and technologies, direct microbial conversion of lignocellulosic biomass into lipids, as an example of consolidated bioprocessing (CBP), is considered to be economically attractive for ‘‘third generation’’ biofuel production due to its simple feedstock processing and low energy inputs [7]. Hydrolysis of cellulose and hemicelluloses in biomass and

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