Modular Metabolic Engineering of Mortierella alpina by the 2A Peptide Platform to Improve Arachidonic Acid Production.

Arachidonic acid (ARA) is an essential fatty acid in human nutrition. Mortierella alpina, a filamentous fungus, has been widely used for the production of ARA. Here, we report a modular engineering approach that systematically eliminates metabolic bottlenecks in the multigene elongase/desaturase pathway and has led to significant improvements of the ARA titer. The elongase/desaturase pathway in Mortierella alpina was recast into two modules, namely, push and pull modules, based on its function in the ARA synthesis. Combinatorial optimization of these two modules has balanced the production and consumption of intermediate metabolites. A 2A peptide-based facile assembly platform that can achieve multigene expression as a polycistron was first established. The platform was then applied to express the push and pull modules in Mortierella alpina. In the shake-flask fermentation, the lipid and ARA contents of the engineered strain MA5 were increased by 1.2-fold and 77.6%, respectively, resulting in about fivefold increase of the ARA yield. The final ARA titer reached 4.4 g L-1 in shake-flask fermentation. The modular engineering strategies presented in this study demonstrate a generalized approach for the engineering of cell factories in the production of valuable metabolites.

Integrated lactic acid production from lignocellulosic agricultural wastes under thermal conditions.

The production of lactic acid (LA) from agricultural wastes attracts great attention because of the sustainability and abundance of lignocellulosic feedstocks, as well as the increasing demand for biodegradable polylactic acid. In this study, we isolated a thermophilic strain Geobacillus stearothermophilus 2H-3 for use in robust production of L-(+)LA under the optimal conditions of 60 °C, pH 6.5, which were consistent with the whole-cell-based consolidated bio-saccharification (CBS) process. Sugar-rich CBS hydrolysates derived from various agricultural wastes, including corn stover, corncob residue, and wheat straw, were used as the carbon sources for 2H-3 fermentation by directly inoculating 2H-3 cells into the CBS system, without intermediate sterilization, nutrient supplementation, or adjustment of fermentation conditions. Thus, we successfully combined two whole-cell-based steps into a one-pot successive fermentation process to efficiently produce LA with high optical purity (99.5%), titer (51.36 g/L), and yield (0.74 g/gbiomass). This study provides a promising strategy for LA production from lignocellulose through CBS and 2H-3 fermentation integration.

Cocktail biosynthesis of triacylglycerol by rational modulation of diacylglycerol acyltransferases in industrial oleaginous Aurantiochytrium.

BACKGROUND: Triacylglycerol (TAG) is an important storage lipid in organisms, depending on the degree of unsaturation of fatty acid molecules attached to glycerol; it is usually used as the feedstock for nutrition or biodiesel. However, the mechanism of assembly of saturated fatty acids (SFAs) or polyunsaturated fatty acids (PUFAs) into TAGs remains unclear for industrial oleaginous microorganism. RESULTS: Diacylglycerol acyltransferase (DGAT) is a key enzyme for TAG synthesis. Hence, ex vivo (in yeast), and in vivo functions of four DGAT2s (DGAT2A, DGAT2B, DGAT2C, and DGAT2D) in industrial oleaginous thraustochytrid Aurantiochytrium sp. SD116 were analyzed. Results revealed that DGAT2C was mainly responsible for connecting PUFA to the sn-3 position of TAG molecules. However, DGAT2A and DGAT2D target SFA and/or MUFA. CONCLUSIONS: There are two specific TAG assembly routes in Aurantiochytrium. The "saturated fatty acid (SFA) TAG lane" primarily produces SFA-TAGs mainly mediated by DGAT2D whose function is complemented by DGAT2A. And, the "polyunsaturated fatty acid (PUFA) TAG lane" primarily produces PUFA-TAGs via DGAT2C. In this study, we demonstrated the functional distribution pattern of four DGAT2s in oleaginous thraustochytrid Aurantiochytrium, and provided a promising target to rationally design TAG molecular with the desired characteristics.

Obtaining High-Purity Docosahexaenoic Acid Oil in Thraustochytrid Aurantiochytrium through a Combined Metabolic Engineering Strategy.

High-purity docosahexaenoic acid (DHA) resources are insufficient in the pharmaceutical and food industries. Although many efforts have attempted to obtain the high-purity DHA production, few reports have been successful. Here, a combined metabolic engineering strategy was employed to increase the DHA purity in the oleaginous thraustochytrid Aurantiochytrium. The strategy includes both partial deactivation of the competing pathway of DHA biosynthesis, by disrupting one copy of the fatty acid synthase gene, and strengthening of substrate supply and triacylglycerol synthesis, by the overexpression of acetyl-CoA carboxylase and diacylglycerol acyltransferase. With this strategy, a final mutant was obtained with a DHA purity of 61% in total fatty acids and a content of 331 mg/g dry cell weight. This study provides an advanced strategy for sustainable high-purity DHA production and highlights the strategy for producing designer oils in industrial oleaginous microorganisms.

A Two-Stage Adaptive Laboratory Evolution Strategy to Enhance Docosahexaenoic Acid Synthesis in Oleaginous Thraustochytrid.

Thraustochytrid is a promising algal oil resource with the potential to meet the demand for docosahexaenoic acid (DHA). However, oils with high DHA content produced by genetic modified thraustochytrids are not accepted by the food and pharmaceutical industries in many countries. Therefore, in order to obtain non-transgenic strains with high DHA content, a two-stage adaptive laboratory evolution (ALE) strategy was applied to the thraustochytrid Aurantiochytrium sp. Heavy-ion irradiation technique was first used before the ALE to increase the genetic diversity of strains, and then two-step ALE: low temperature based ALE and ACCase inhibitor quizalofop-p-ethyl based ALE were employed in enhancing the DHA production. Using this strategy, the end-point strain E-81 with a DHA content 51% higher than that of the parental strain was obtained. The performance of E-81 strain was further analyzed by component analysis and quantitative real-time PCR. The results showed that the enhanced in lipid content was due to the up-regulated expression of key enzymes in lipid accumulation, while the increase in DHA content was due to the increased transcriptional levels of polyunsaturated fatty acid synthase. This study demonstrated a non-genetic approach to enhance lipid and DHA content in non-model industrial oleaginous strains.

PUFA-synthase-specific PPTase enhanced the polyunsaturated fatty acid biosynthesis via the polyketide synthase pathway in Aurantiochytrium.

BACKGROUND: Phosphopantetheinyl transferase (PPTase) can change the acyl-carrier protein (ACP) from an inactive apo-ACP to an active holo-ACP that plays a key role in fatty acids biosynthesis. Currently, the PPTase has been proved to be involved in the biosynthesis of polyunsaturated fatty acids (PUFAs) via a polyketide synthase (PKS) pathway in Thraustochytrids, while its characteristics are not clarified. RESULTS: Here, the heterologous PPTase gene (pfaE) from bacteria was first co-expressed with the PKS system (orfA-orfC) from Thraustochytrid Aurantiochytrium. Then, a new endogenous PPTase (ppt_a) in Aurantiochytrium was identified by homologous alignment and its function was verified in E. coli. Moreover, the endogenous ppt_a was then overexpressed in Aurantiochytrium, and results showed that the production and proportion of PUFAs, especially docosahexaenoic acid (DHA), in the transformant SD116::PPT_A were increased by 35.5% and 17.6%, respectively. Finally, higher DHA and PUFA proportion (53.9% and 64.5% of TFA, respectively) were obtained in SD116::PPT_A using a cerulenin feeding strategy. CONCLUSIONS: This study has illustrated a PUFAs-synthase-specific PPTase in PKS system and provided a new strategy to improve the PUFA production in Thraustochytrids.

Optimizing Eicosapentaenoic Acid Production by Grafting a Heterologous Polyketide Synthase Pathway in the Thraustochytrid Aurantiochytrium.

Eicosapentaenoic acid (EPA) is an essential nutritional supplement for human health. The most prominent dietary source of EPA is fish oil, which is unsustainable because of the decline in fishery resources and serious environmental pollution. Alternatively, a heterologous polyketide synthase pathway for EPA biosynthesis was assembled in Thraustochytrid Aurantiochytrium. A 2A peptide-based facile assembly platform that can achieve multigene expression as a polycistron was first established. The platform was then applied to express the EPA biosynthetic gene cluster from Shewanella japonica in Aurantiochytrium. In the shake flask fermentation, the lipid and PUFA yields of the mutant were increased by 26.9 and 36.0%, respectively, and led to about 5-fold increase of the EPA yield. The final EPA titer reached 2.7 g/L in fed-batch fermentation. This study provides a novel metabolic engineering strategy to regulate the EPA ratio in microalgal oil for human nutritional supplementation.

Effect of Urchin-Like Mullite Whiskers on the High-Temperature Performance of Porous SiO2-Based Ceramic Molds.

Urchin-like mullite whiskers synthesized by the vapor-liquid-solid growth method were used to improve the high-temperature performance of porous gelcast SiO2-based ceramic molds. Aluminum was used to facilitate the synthesis of polycrystal urchin-like mullite whiskers which acted as bridges between particles. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) were used to investigate the microstructures and phase compositions of the sintered ceramic samples, respectively. Urchin-like mullite whiskers with diameters of 0.2~1.0 µm and lengths of 1.0~8.0 µm were successfully synthesized in SiO2-based ceramic. When 15 vol% Al was added, the high-temperature strength at 1200 °C was improved from 8.5 to 27.5 MPa, and the creep deformation was decreased to 0.56 mm. Meanwhile, a sintering shrinkage below 0.3% was obtained, and the de-coring rate was accelerated by 67% compared to that of the pure SiO2-based ceramic. This method showed excellent high-temperature strength and high precision, having remarkable potential in the fabrication of hollow turbine blades.