Recombinant strains engineered to express eGFP protein.

<p>(A) Construction of the recombinant expression vector for <i>A. oryzae</i> A-4; (B) Isolation of positive transformants by PCR checking; (C) Microscopic observations of mycelium samples exposed to ultraviolet (UV) and white light (WL), which collected from the transformant G-4 (A-4-GFP) and the wild-type A-4 (A-4-WT), respectively.</p

The lipid production (A, C) and cell growth (B) of A2-2 (black column), D1-B1 (gray column) and wild-type A-4 (dark gray column) in SmF experiments using glucose and maltose based media, respectively.

<p>The lipid production (A, C) and cell growth (B) of A2-2 (black column), D1-B1 (gray column) and wild-type A-4 (dark gray column) in SmF experiments using glucose and maltose based media, respectively.</p

Solid-state fermentation of wheat straw and bran mixture by D1-B1 (blank circle), A2-2 (closed circle) and wild-type A-4 (closed triangle).

<p>(A) Time courses of the filter paper activity (FPAase activity); (B) Time courses of the CMCase activity; (C) Time courses of the extracellular protein concentration; (D) Time courses of the loss in dry matter (LDM); (E) Time courses of the cell growth; (F) Time courses of the lipid yield.</p

The lipid production (A, C), cell growth (B), cellulase secretion (E, F) and extracellular protein secretion (D) of A2-2 (black column), D1-B1 (gray column) and wild-type A-4 (dark gray column) in SmF experiments using avicel and straw based media, respectively.

<p>The lipid production (A, C), cell growth (B), cellulase secretion (E, F) and extracellular protein secretion (D) of A2-2 (black column), D1-B1 (gray column) and wild-type A-4 (dark gray column) in SmF experiments using avicel and straw based media, respectively.</p

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

<div><p>A genetic modification scheme was designed for <i>Aspergillus oryzae</i> 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 <i>hlyA</i> 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 <i>celA</i> gene and with <i>celC</i> gene had significantly (<i>p</i><0.05) higher average FPAase activities than those strains integrated with <i>celB</i> gene and with <i>celD</i> gene. Through the assessment of cellulosic lipids accumulating abilities, <i>celA</i> transformant A2-2 and <i>celC</i> 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.</p></div

Preliminarily study of cellulase gene integration profiles in A2-2 and D1-B1, respectively.

<p>(A) Southern blot analysis. A part of <i>Amp</i>^r gene (0.7-kb) was amplified by PCR and used as the probe. (B) Primers designed for the insertion analysis of the target gene in A2-2 (<i>celA</i>) and D1-B1 (<i>celC</i>), and the theoretical results of PCR using the designed primers.</p

Cellulase activities, lipid production and cell growth of A2-E, A2-2, B11-C2, D1-B1 and D1-2(3).

<p>Incubation for cellulase activity measurement was conducted under submerged (SmF) conditions using wheat straw as substrate for 4 days. Lipid production and cell growth of transformants were determined under both submerged and solid-state conditions.</p>+<p>The control transformant introduced with the negative vector pPTRI without cellulase expression cassette.</p>++<p>The wild-type <i>A. oryzae</i> A-4.</p><p>*Submerged fermentation from wheat straw after 4 days.</p><p>**Solid state fermentation from wheat straw and bran mixture after 4 days.</p><p>***Loss in dry matter (LDM).</p><p>One-way ANOVA is used to test for differences: a, b means <i>p</i><0.05; A, B, C, D means <i>p</i><0.05; Values in brackets are standard errors.</p><p>Cellulase activities, lipid production and cell growth of A2-E, A2-2, B11-C2, D1-B1 and D1-2(3).</p

Lipid droplets accumulated in the mycelium of the reference strain A-4 (A) and the cell growth of strain A-4 grown on CD-plate without (-PT-h) and with 0.1 mg l⁻¹ PT-h (+PT-h) (B).

<p>(A) Strain A-4 was cultivated on wheat straw and bran mixture for 6 days. The Nile red stained fermented products were then microscopic observed under ultraviolet (UV) and white light (WL); (B) Inhibitory effects of PT-h on the cell growth of A-4. Incubation was performed at 30°C for 5 days.</p

Genes and plasmids used in this study.

a<p>DOGAN accession number (DOGAN, <a href="http://www.bio.nite.go.jp/dogan/" target="_blank">http://www.bio.nite.go.jp/dogan/</a>).</p><p>Genes and plasmids used in this study.</p