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

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

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

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

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

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

Synthesis of a Water-Soluble Carboxylatobiphen[4]arene and Its Selective Complexation toward Acetylcholine

The first water-soluble biphen[4]­arene containing eight carboxylato moieties (carboxylatobiphen[4]­arene, CBP4) has been synthesized. Selective molecular recognition of acetylcholine (<b>ACh</b>) against choline (<b>Ch</b>) and betaine (<b>Bt</b>) and pH-responsive host–guest complexation in aqueous media are described

Synthesis of Pillar[5]arene Dimers and Their Cooperative Binding toward Some Neutral Guests

Three pillar[5]arene dimers, bridged by a flexible aliphatic chain (H1) or a relatively rigid phenylene unit (H2 and H3), were synthesized, with the possible synthetic strategies being discussed. The dimers could significantly enhance the binding affinities toward neutral model substrates in comparison with monomeric 1,4-dimethoxypillar[5]arene (H4) through the cooperative binding of two pillar[5]arene moieties. The molecular binding ability and selectivity are discussed from the viewpoints of the size/shape-fit concept and multiple recognition mechanism

Video2_Repair of fingertip defect with reverse digital artery island flap and repair of donor site with digital dorsal advancement flap.mp4

ObjectiveThe reverse digital artery island flap (RDAF) is widely used in repairing fingertip skin defects based on its good appearance and practicability. However, the donor area of the flap needs skin grafting, which can lead to complications. This retrospective study explored the clinical application of digital dorsal advance flap (DDAF) in repairing the donor site of the reverse digital artery island flap.MethodFrom June 2019 to February 2022, 17 patients with a soft tissue defect of the finger had been restored with the reverse digital artery island flap, and at the same time, the donor area was repaired with digital dorsal advance flap (DDAF). The sensitivity, the active range of motion (ROM) and patient satisfaction were assessed after the operation.ResultsAll flaps survived completely without skin grafting with only one linear scar. The sensory and motor functions of all patients recovered well. Assessment based on the Michigan Hand Outcomes Questionnaire (MHQ) showed satisfactory functional recovery for all patients.ConclusionsReconstruction using RDAF combined with DDAF represents an effective alternative for repairing fingertip skin defects.</p

Image1_Repair of fingertip defect with reverse digital artery island flap and repair of donor site with digital dorsal advancement flap.jpeg

ObjectiveThe reverse digital artery island flap (RDAF) is widely used in repairing fingertip skin defects based on its good appearance and practicability. However, the donor area of the flap needs skin grafting, which can lead to complications. This retrospective study explored the clinical application of digital dorsal advance flap (DDAF) in repairing the donor site of the reverse digital artery island flap.MethodFrom June 2019 to February 2022, 17 patients with a soft tissue defect of the finger had been restored with the reverse digital artery island flap, and at the same time, the donor area was repaired with digital dorsal advance flap (DDAF). The sensitivity, the active range of motion (ROM) and patient satisfaction were assessed after the operation.ResultsAll flaps survived completely without skin grafting with only one linear scar. The sensory and motor functions of all patients recovered well. Assessment based on the Michigan Hand Outcomes Questionnaire (MHQ) showed satisfactory functional recovery for all patients.ConclusionsReconstruction using RDAF combined with DDAF represents an effective alternative for repairing fingertip skin defects.</p