Overexpression of LAS21 in Cellulase-Displaying <i>Saccharomyces cerevisiae</i> for High-Yield Ethanol Production from Pretreated Sugarcane Bagasse

The valorization of lignocellulosic feedstocks into biofuels and biochemicals has received much attention due to its environmental friendliness and sustainability. However, engineering an ideal microorganism that can both produce sufficient cellulases and ferment ethanol is highly challenging. In this study, we have tested seven different genes that are involved in glycosylphosphatidylinositol (GPI) biosynthesis and remodeling for the improvement of cellulase activity tethered on the S. cerevisiae cell surface. It was found that the overexpression of LAS21 can improve β-glucosidase activity by 48.8% compared to the original strain. Then, the three cellulase genes (cellobiohydrolase, endoglucanase, and β-glucosidase) and the LAS21 gene were co-introduced into a diploid thermotolerant S. cerevisiae strain by a multiple-round transformation approach, resulting in the cellulolytic ECBLCCE5 strain. Further optimization of the bioprocess parameters was found to enhance the ethanol yield of the ECBLCCE5 strain. Scaling up the valorization of pretreated sugarcane bagasses in a 1 L bioreactor resulted in a maximum ethanol concentration of 28.0 g/L (86.5% of theoretical yield). Our study provides a promising way to improve the economic viability of second-generation ethanol production. Moreover, the engineering of genes involved in GPI biosynthesis and remodeling can be applied to other yeast cell surface display applications

Insights into the Prostanoid Pathway in the Ovary Development of the Penaeid Shrimp <i>Penaeus monodon</i>

<div><p>The prostanoid pathway converts polyunsaturated fatty acids (PUFAs) into bioactive lipid mediators, including prostaglandins, thromboxanes and prostacyclins, all of which play vital roles in the immune and reproductive systems in most animal phyla. In crustaceans, PUFAs and prostaglandins have been detected and often associated with female reproductive maturation. However, the presence of prostanoid biosynthesis genes remained in question in these species. In this study, we outlined the prostanoid pathway in the black tiger shrimp <i>Penaeus monodon</i> based on the amplification of nine prostanoid biosynthesis genes: <i>cytosolic phospholipase A2, hematopoietic prostaglandin D synthase, glutathione-dependent prostaglandin D synthase, prostaglandin E synthase 1, prostaglandin E synthase 2, prostaglandin E synthase 3, prostaglandin F synthase</i>, <i>thromboxane A synthase</i> and <i>cyclooxygenase</i>. TBLASTX analysis confirmed the identities of these genes with 51-99% sequence identities to their closest homologs. In addition, prostaglandin F_2α (PGF_2α), which is a product of the prostaglandin F synthase enzyme, was detected for the first time in <i>P. monodon</i> ovaries along with the previously identified PUFAs and prostaglandin E₂ (PGE₂) using RP-HPLC and mass-spectrometry. The prostaglandin synthase activity was also observed in shrimp ovary homogenates using <i>in vitro</i> activity assay. When prostaglandin biosynthesis was examined in different stages of shrimp ovaries, we found that the amounts of <i>prostaglandin F synthase</i> gene transcripts and PGF_2α decreased as the ovaries matured. These findings not only indicate the presence of a functional prostanoid pathway in penaeid shrimp, but also suggest a possible role of the PGF_2α biosynthesis in shrimp ovarian development. </p> </div

Mapping of essential residues in the predicted <i>P. monodon</i> PGES3 enzyme.

<p>Multiple sequence alignments of <i>P. monodon</i> PGES3 protein and their homologs reveal one conserved serine residue for the CKII phosphorylation site (black arrow), while the other phosphorylation site was not conserved (gray arrow). Genus and species used in this alignment are abbreviated as followed: <i>Penaeus</i> − <i>P. monodon</i>, Litopenaeus − <i>L. vannamei</i>, Danio − <i>Danio rerio</i>, <i>Pediculus</i> − <i>P. humanus corporis</i>, <i>Xenopus</i> − <i>Xenopus laevis</i>, <i>Caenorhabditis</i> − <i>Caenorhabditis elegans</i>, Drosophila − <i>Drosophila melanogaster</i>, Gallus − <i>Gallus gallus</i>, Bos − <i>B. taurus</i> and Homo − <i>H. sapiens</i>.</p

Relative expression levels of <i>PmPGES</i> and <i>PmPGFS</i> genes in each ovary stage.

<p>Wild broodstock from the Andaman Sea (N=27) were captured and dissected to obtain ovary samples used in the real-time PCR analysis. Each graph represents the average copy number of prostaglandin biosynthesis gene transcripts normalized against <i>EF1α</i> in each ovary stage. (A) <i>PmPGES1</i>, (B) <i>PmPGES2</i>, (C) <i>PmPGES3</i> and (D) <i>PmPGFS</i>. Error bars show standard deviations and asterisks indicate significant changes between stages (p < 0.05).</p

Mapping of essential residues in the predicted <i>P. monodon</i> PGES1 enzyme.

<p>Multiple sequence alignments of <i>P. monodon</i> PGES1 and their homologs showed a conserved residue for the MAPEG family (white star), catalytic residues that interact with PGH₂ (white arrow head), essential residues for H-bonding to GSH (black arrows) and consensus sequence required for oxygenation product (underline). Genus and species used in this alignment are abbreviated as followed: <i>Penaeus</i> − <i>P. monodon</i>, Litopenaeus − <i>L. vannamei</i>, <i>Crassostrea</i> − <i>Crassostrea virginica</i>, Homarus − <i>H. americanus</i>, <i>Pediculus</i> − <i>Pediculus humanus corporis</i>, <i>Culex</i> − <i>Culex quinquefasciatus</i>, <i>Tribolium</i> − <i>Tribolium castaneum</i>, Equus − <i>Equus caballus</i>, Bos − <i>Bos taurus</i> and Homo − <i>Homo sapiens</i>.</p

Mapping of essential residues in the predicted <i>P. monodon</i> PGES2 enzyme.

<p>Multiple sequence alignments of <i>P. monodon</i> PGES2 protein and their homologs were performed, revealing conserved Cys residues at the active site (black arrows) and the N-terminal sequence of the mature enzyme (underline). Genus and species used in this alignment are abbreviated as followed: <i>Penaeus</i> − <i>P. monodon</i>, <i>Pediculus</i> − <i>P. humanus corporis</i>, Caligus − <i>Caligus rogercresseyi</i>, Mus − <i>Mus musculus</i> and Homo − <i>H. sapiens</i>.</p

The proposed prostanoid biosynthesis pathway in <i>P. monodon</i>.

<p>PUFAs, prostaglandins and full-length prostanoid biosynthesis genes identified in this study (black) were used to outline the <i>P. monodon</i> prostanoid pathway based on the previously published pathway in mammals. Prostanoids that have yet to be identified in <i>P. monodon</i> are shown in gray.</p

Differential distribution of eicosanoids and polyunsaturated fatty acids in the Penaeus monodon male reproductive tract and their effects on total sperm counts

Eicosanoids, which are oxygenated derivatives of polyunsaturated fatty acids (PUFAs), serve as signaling molecules that regulate spermatogenesis in mammals. However, their roles in crustacean sperm development remain unknown. In this study, the testis and vas deferens of the black tiger shrimp Penaeus monodon were analyzed using ultra-high performance liquid chromatography coupled with Orbitrap high resolution mass spectrometry. This led to the identification of three PUFAs and ten eicosanoids, including 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) and (±)15-hydroxyeicosapentaenoic acid ((±)15-HEPE), both of which have not previously been reported in crustaceans. The comparison between wild-caught and domesticated shrimp revealed that wild-caught shrimp had higher sperm counts, higher levels of (±)8-HEPE in testes, and higher levels of prostaglandin E(2) (PGE(2)) and prostaglandin F(2α) in vas deferens than domesticated shrimp. In contrast, domesticated shrimp contained higher levels of (±)12-HEPE, (±)18-HEPE, and eicosapentaenoic acid (EPA) in testes and higher levels of 15d-PGJ(2), (±)12-HEPE, EPA, arachidonic acid (ARA), and docosahexaenoic acid (DHA) in vas deferens than wild-caught shrimp. To improve total sperm counts in domesticated shrimp, these broodstocks were fed with polychaetes, which contained higher levels of PUFAs than commercial feed pellets. Polychaete-fed shrimp produced higher total sperm counts and higher levels of PGE(2) in vas deferens than pellet-fed shrimp. In contrast, pellet-fed shrimp contained higher levels of (±)12-HEPE, (±)18-HEPE, and EPA in testes and higher levels of (±)12-HEPE in vas deferens than polychaete-fed shrimp. These data suggest a positive correlation between high levels of PGE(2) in vas deferens and high total sperm counts as well as a negative correlation between (±)12-HEPE in both shrimp testis and vas deferens and total sperm counts. Our analysis not only confirms the presence of PUFAs and eicosanoids in crustacean male reproductive organs, but also suggests that the eicosanoid biosynthesis pathway may serve as a potential target to improve sperm production in shrimp

<i>In vitro</i> prostaglandin synthase activity assay using shrimp ovary homogenates.

<p>Shrimp ovary homogenates were incubated with 25 µM AA at 28 °C and collected at different time points (0, 30, 60, 120, 240 and 360 min). The homogenates were spun down and concentrations of PGE₂ (A) and PGF_2α (B) in the homogenate supernatant were estimated using EIA. The experiment was performed in triplicate and error bars indicate the standard deviation from the means. Asterisk indicates significant difference between the prostaglandin concentration at 0 h and the marked time point (<i>P</i><0.05).</p

Mapping of essential residues in the predicted <i>P. monodon</i> PGFS enzyme.

<p>Multiple sequence alignments of <i>P. monodon</i> PGFS protein and their homologs were performed, revealing residues that are important for substrate (black arrows) and the NADP⁺ cofactor (white arrow head) binding. Genus and species used in this alignment are abbreviated as followed: <i>Penaeus</i> − <i>P. monodon</i>, Litopenaeus − <i>L. vannamei</i>, Canis − <i>Canis lupus familiaris</i>, Ovis − <i>Ovis aries</i>, Bos − <i>B. taurus</i>, Equus − <i>Equus caballus</i> and Homo − <i>H. sapiens</i>.</p