Photosynthesis-fermentation hybrid system to produce lipid feedstock for algal biofuel

<div><p>To avoid bacterial contamination due to medium replacement in the expanded application of a photosynthesis–fermentation model, an integrated photosynthesis-fermentation hybrid system was set up and evaluated for algal lipid production using <i>Chlorella protothecoides</i>. In this system, the CO₂-rich off-gas from the fermentation process was recycled to agitate medium in the photobioreactor, which could provide initial cells for the heterotrophic fermentation. The cell concentration reached 1.03±0.07 g/L during photoautotrophic growth and then the concentrated green cells were switched to heterotrophic fermentation after removing over 99.5% of nitrogen in the medium by a nitrogen removal device. At the end of fermentation in the system, the cell concentration could reach as high as 100.51±2.03 g/L, and 60.05±1.38% lipid content was achieved simultaneously. The lipid yield (60.36±2.63 g/L) in the hybrid system was over 700 times higher than that in a photobioreactor and exceeded that by fermentation alone (47.56±7.31 g/L). The developed photosynthesis-fermentation hybrid system in this study was not only a feasible option to enhance microalgal lipid production, but also an environment-friendly approach to produce biofuel feedstock through concurrent utilization of ammonia nitrogen, CO₂, and organic carbons.</p> </div

Wicket: A Versatile Tool for the Integration and Optimization of Exogenous Pathways in <i>Saccharomyces cerevisiae</i>

Yeast can be used as a microbial cell factory to produce valuable chemicals. However, introducing an exogenous pathway into particular or different chromosomal locations for stable expression is still a daunting task. To address this issue, we designed a DNA cassette called a “wicket”, which can be integrated into the yeast genome at designated loci to accept exogenous DNA upon excision by a nuclease. Using this system, we demonstrated that, in strains with “wickets”, we could achieve near 100% efficiency for integration of the β-carotene pathway with no need for selective markers. Furthermore, it allowed independent and simultaneous integration of different genes in a pathway, resulting in a large variety of strains with variable copy numbers of each gene. This system could be a useful tool to modulate the integration of multiple copies of genes within a metabolic pathway and to optimize the yield of the target products

Partial sequence alignment of ATG proteins maintaining conserved catalytic and binding sites.

<p>The abbreviations of species are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041826#pone-0041826-t001" target="_blank">Table 1</a> and the sequence accession numbers are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041826#pone-0041826-t004" target="_blank">Table 4</a>. The asterisks indicate similar residues. Residues in the catalytic and binding sites are in red boxes and the numbers indicate the positions in <i>S.cerevisiae</i> (Sc) ATG proteins.</p

Distribution of ATG proteins and domains in microalgae and <i>M. brevicollis</i>: Other ATG proteins pexophagy.

<p>Na, not present or not identifiable; numbers indicate GenBank DNA accession numbers.</p

Distribution of ATG proteins and domains in microalgae and <i>M. brevicollis</i>: ATG proteins exclusively involved in CVT.

<p>Na, not present or not identifiable; numbers indicate GenBank DNA accession numbers.</p

Domain organization of putative ATG17 proteins.

<p>Species and sequences are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041826#pone-0041826-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041826#pone-0041826-t007" target="_blank">Table 7</a>. Fused domains that form a single polypeptide chain are connected by a horizontal line. Figures are not drawn to scale.</p

Distribution of autophagy components in microalgae and choanoflagellate.

<p>Blank box: no homologues or putative orthologs could be found in any of the seven microalgae genomes; green box: predicted orthologs were detected in each of the genomes studied; blue box: putative orthologs were detected in some algal genomes; yellow box: homologues could only be found in <i>Monosiga brevicollis</i>. A: Distribution of putative ATG proteins in “core autophagic machinery”. B: The existence of homologues of non-yeast ATG proteins. C: The existence of the orthologs of additional pathway-specific requirement.</p

Distribution of ATG proteins and domains in microalgae and <i>M. brevicollis</i>: ATG proteins involved in PI3K complex.

<p>Na, not present or not identifiable; numbers indicate GenBank DNA accession numbers.</p

Species analyzed in this study.

<p>Na, not present.</p

Phylogenetic tree of ATG8 proteins.

<p>ATG8 from <i>M. brevicollis</i> is used as outgroups. The phylogenetic tree was constructed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041826#s4" target="_blank">Methods</a> section (1000 bootstrap replicates). Protein accession numbers and the strain names are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041826#pone-0041826-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041826#pone-0041826-t004" target="_blank">Table 4</a>, respectively.</p