Biomanufacture of L-homoserine lactone building block: A strategy for preparing γ-substituted L-amino acids by modular reaction.

A strain high-performance of esterase producing bacteria was screened from soil, which could selectively hydrolyze D-homoserine lactone from its racemate to achieve the resolution of L- homoserine lactone with more than 99% e.e. in 48% yield. L-homoserine lactone building block was then converted to L-α-amino-γ-bromobutyronic acid chiral blocks, which reacted with various nucleophilic reagent modules could to be applied to prepare L-γ- substituted α-amino acids such as L-selenomethionine, L-methionine, L-glufosinate and L-selenocystine. Its advantages included high selectivity of biocatalytic resolution reactions, high optical purity of products, racemic recycle of D-substrates and modular reaction, which simplified the production process of these products and highlighted the power of biological manufacturing.

Enantioselective resolution of (R,S)-DMPM to prepare (R)-DMPM by an adsorbed-covalent crosslinked esterase PAE07 from Pseudochrobactrum asaccharolyticum WZZ003.

(R)-N-(2,6-dimethylphenyl) aminopropionic acid methyl ester ((R)-DMPM) is an important chiral intermediate of the fungicide N-(2,6-Dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester ((R)-Metalaxyl). In this study, (1) D3520 (macroporous acrylic anion resin), selected from the ten resins, was used to immobilize the esterase from Pseudochrobactrum asaccharolyticum WZZ003 (PAE07) for resoluting the (R,S)-DMPM to obtain (R)-DMPM. (2) Up to 20 g/L PAE07 could be immobilized onto D3520 with a high enzymatic activity of 32.4 U/g. Moreover, the Km and Vmax values of 19.1 mM and 2.8 mM/min for D3520-immobilized PAE07 indicated its high activity and stereoselectivity. (3) The optimal temperature and pH for the immobilized PAE07 were 40 ℃ and 8.0, and substrate concentration was up to 0.35 M. After 15 h reaction, the conversion rate from (R,S)-DMPM to (R)-DMPM was 48.0% and the e.e.p and E values were 99.5% and 1393.0, respectively. In scale-up resolution, 200 g/L substrate and 12.5 g immobilized esterase PAE07 condition, a conversion rate from substrate to product of 48.1% and a product e.e.p of 98% were obtained within 12 h, with the activity of immobilized PAE07 retained 80.2% after 5 cycles of reactions. These results indicated that the D3520-immobilized esterase PAE07 had great potential for enzymatic resolution of (R,S)-DMPM to prepare (R)-Metalaxyl.

Mechano-Enzymatic Degradation of the Chitin from Crustacea Shells for Efficient Production of N-acetylglucosamine (GlcNAc).

Chitin, the second richest polymer in nature, is composed of the monomer N-acetylglucosamine (GlcNAc), which has numerous functions and is widely applied in the medical, food, and chemical industries. However, due to the highly crystalline configuration and low accessibility in water of the chitin resources, such as shrimp and crab shells, the chitin is difficult utilize, and the traditional chemical method causes serious environment pollution and a waste of resources. In the present study, three genes encoding chitinolytic enzymes, including the N-acetylglucosaminidase from Ostrinia furnacalis (OfHex1), endo-chitinase from Trichoderma viride (TvChi1), and multifunctional chitinase from Chitinolyticbacter meiyuanensis (CmChi1), were expressed in the Pichia pastoris system, and the positive transformants with multiple copies were isolated by the PTVA (post-transformational vector amplification) method, respectively. The three recombinants OfHex1, TvChi1, and CmChi1 were induced by methanol and purified by the chitin affinity adsorption method. The purified recombinants OfHex1 and TvChi1 were characterized, and they were further used together for degrading chitin from shrimp and crab shells to produce GlcNAc through liquid-assisted grinding (LAG) under a water-less condition. The substrate chitin concentration reached up to 300 g/L, and the highest yield of the product GlcNAc reached up to 61.3 g/L using the mechano-enzymatic method. A yield rate of up to 102.2 g GlcNAc per 1 g enzyme was obtained.

Production, Biosynthesis, and Commercial Applications of Fatty Acids From Oleaginous Fungi.

Oleaginous fungi (including fungus-like protists) are attractive in lipid production due to their short growth cycle, large biomass and high yield of lipids. Some typical oleaginous fungi including Galactomyces geotrichum, Thraustochytrids, Mortierella isabellina, and Mucor circinelloides, have been well studied for the ability to accumulate fatty acids with commercial application. Here, we review recent progress toward fermentation, extraction, of fungal fatty acids. To reduce cost of the fatty acids, fatty acid productions from raw materials were also summarized. Then, the synthesis mechanism of fatty acids was introduced. We also review recent studies of the metabolic engineering strategies have been developed as efficient tools in oleaginous fungi to overcome the biochemical limit and to improve production efficiency of the special fatty acids. It also can be predictable that metabolic engineering can further enhance biosynthesis of fatty acids and change the storage mode of fatty acids.

The Transcriptomic Mechanism of a Novel Autolysis Induced by a Recombinant Antibacterial Peptide from Chicken Expressed in Pichia pastoris.

Autolysis is a common physiological process in eukaryotic cells that is often prevented or applied, especially in yeast expression systems. In this study, an antimicrobial peptide from chicken (AMP) was recombinantly expressed in the Pichia pastoris expression system, which induced a series of cellular autolysis phenotypes after methanol treatment, such as the aggregated, lysed, irregular, and enlarged cell morphology, while the cells expressing a recombinant aflatoxin-detoxifizyme (ADTZ) were not autolyzed. A comparative transcriptomic analysis showed that the transcriptomic profiles of cells derived from the autolysis and non-autolysis groups were well discriminated, suggesting that the mechanisms of autolysis were at the transcriptional level. A further differential expression gene (DEG) analysis showed that the DEGs from the two groups were involved mainly in autophagy, the MAPK signaling pathway, transcriptional factors, the central carbon metabolism, anti-stress functions, and so on. In the autolysis group, the cell activity was significantly reduced with the MAPK signaling pathway, the central carbon metabolism was down-regulated, and components of the cytoplasm-to-vacuole targeting (CVT) and mitophagy pathways were up-regulated, suggesting that the autophagy involved in the trafficking of intracellular molecules in the vacuole and mitochondrion contributed to autolysis, which was regulated by transcriptional factors and signal pathways at the transcriptional level. This study provides a theoretical basis for genetic modifications to prevent or utilize cell autolysis in the recombinant protein expression system.

One-pot fermentation for erythritol production from distillers grains by the co-cultivation of Yarrowia lipolytica and Trichoderma reesei.

A co-fermentation process involving Yarrowia lipolytica and Trichoderma reesei was studied, using distillers grains (DGS) as feedstocks for erythritol production. DGS can be effectively hydrolyzed by cellulase in the single-strain culture of T. reesei. One-pot solid state fermentation for erythritol production was then established by co-cultivating Y. lipolytica M53-S with the 12 h delay inoculated T. reesei Rut C-30, in which efficient saccharification of DGS and improved production of erythritol were simultaneously achieved. The 10:1 inoculation proportion of Y. lipolytica and T. reesei contributed to the maximum erythritol production of 267.1 mg/gds under the optimal conditions including initial moisture of 55%, pH of 5.0, NaCl addition of 0.02 g/gds and DGS mass of 200 g in 144 h co-cultivation. Being compared with the attempts to produce erythritol from other raw materials, the one-pot SSF with DGS is proposed to be a potential strategy for efficient and economical erythritol production.

Semi-rational protein engineering of a novel esterase from Bacillus aryabhattai (BaCE) for resolution of (R,S)-ethyl indoline-2-carboxylate to prepare (S)-indoline-2-carboxylic acid.

A gene encoding an esterase from Bacillus aryabhattai (BaCE) was identified, synthesized and efficiently expressed in the Escherichia coli system. A semi-rational protein engineering was applied to further improve the enzyme's enantioselectivity. Under the guidance of the molecular docking result, a single mutant BaCE-L86Q and a double mutant BaCE-L86Q/G284E were obtained, with its Emax value 6.4 times and 13.9 times of the wild-type BaCE, respectively. The recombinant BaCEs were purified and characterized. The overwhelming E value demonstrated that BaCE-L86Q/G284E was a promising biocatalyst for the biological resolution to prepare (S)-indoline-2-carboxylic acid.

Enzymatic extraction of pectic oligosaccharides from finger citron (Citrus medica L. var. sarcodactylis Swingle) pomace with antioxidant potential.

Finger citron pomace is a cheap and renewable by-product of the citrus processing industry, representing up to 60% of the fruit biomass. In this study, a pectinase-based and ultrasonic-assisted method was firstly used to extract pectic oligosaccharides (POS) from finger citron pomace. Using the orthogonal experiment design (OED), the maximum conversion rate of up to 64.5% from pomace to POS was obtained under the extraction conditions of 0.25 mg mL-1 pectinase and 50 mg mL-1 pectin at 45 °C and pH 4.5 for 2 h. The extracted POS was then fractionated and purified to homogeneous oligosaccharides (FCPOS-1) with a molecular weight of 2.15 kDa, and the analyses of monosaccharide composition, FTIR, NMR and ESI-MS indicated that FCPOS-1 consisted of GalA and a small amount of mannose, galactose and arabinose. Multiple antioxidant activity assays in vitro revealed that FCPOS-1 possessed remarkable antioxidant properties, especially scavenging activity against DPPH radicals up to 94.07%. FCPOS-1 has the potential to be an effective natural antioxidant for applications in the food and pharmaceutical industries.

The strategies to reduce cost and improve productivity in DHA production by Aurantiochytrium sp.: from biochemical to genetic respects.

The marine oleaginous protist Aurantiochytrium sp. (Schizochytrium sp.) is a well-known docosahexaenoic acid (DHA) producer and its different DHA products are the ideal substitute for the traditional fish oil resource. However, the cost of the DHA products derived from Aurantiochytrium sp. (Schizochytrium sp.) is still high, limiting their wide applications. In order to reduce the cost or improve the productivity of DHA from the microbial resource, many researches are focusing on exploring the renewable and low-cost materials as feedbacks, and/or the stimulators for biomass and DHA production. In addition, the genetic engineering is also being used in the Aurantiochytrium sp. (Schizochytrium sp.) system for further improvement. These break the bottleneck of the DHA production by Aurantiochytrium sp. (Schizochytrium sp.) in some degree. In this review, the strategies used currently to reduce cost and improve DHA productivity, mainly from the utilizations of low-cost materials and effective stimulators to the genetic engineering perspectives, are summarized, and the availabilities from the cost perspective are also evaluated. This review provides an overview about the strategies to revolve the production cost and yield of the DHA by Aurantiochytrium sp. (Schizochytrium sp.), a theoretical basis for genetic modification of Aurantiochytrium sp. (Schizochytrium sp.), and a practical basis for the development of DHA industry. KEY POINTS : • Utilizations of various low-cost materials for DHA production • Inducing the growth and DHA biosynthesis by the effective stimulators • Reducing cost and improving DHA productivity by genetic modification • The availability from cost perspective is evaluated.

One-step utilization of inulin for docosahexaenoic acid (DHA) production by recombinant Aurantiochytrium sp. carrying Kluyveromyces marxianus inulinase.

This study aimed to express an inulinase gene from the yeast Kluyveromyces marxianus (KmINU) in Aurantiochytrium sp. and realized one-step utilization of inulin resource for DHA production without any chemical pretreatment. An expression cassette with a length of 6052 bp for expressing the inulinase gene was constructed by a fast two-step PCR method and then was transferred into the Aurantiochytrium sp. cells. The Aurantiochytrium sp. recombinant T39 was selected with an inulinase activity up to 50.1 U/mL in 72 h. In a 5-l fed-batch fermentation, as high as 148.9 g/L of inulin was directly used within 120 h, and only 1.2 g/L of total sugar was left in the medium at the end of fermentation. The biomass of 51.4 g/L with a lipid content of 69.2% DCW and a DHA yield of 14.9 g/L was obtained.

Effect of Quercetin on PC12 Alzheimer's Disease Cell Model Induced by Aß 25-35 and Its Mechanism Based on Sirtuin1/Nrf2/HO-1 Pathway.

OBJECTIVE: This study is aimed at studying the effect of quercetin on the Alzheimer disease cell model induced by Aß 25-35 in PC12 cells and its mechanism of action. METHODS: The AD cell model was established by Aß 25-35. Quercetin was used at different concentrations (0, 10, 20, 40, and 80 µmol/L). The morphology of cells was observed, and the effect on cell survival rate was detected by the MTT method. Cell proliferation was detected by the SRB method. The contents of LDH, SOD, MDA, GSH-Px, AChE, CAT, and T-AOC were detected by kits. The expression of sirtuin1/Nrf2/HO-1 was detected by RT-qPCR and Western blot. RESULTS: PC12 cells in the control group grew quickly and adhered well to the wall, most of which had extended long axons and easily grew into clusters. In the model group, cells were significantly damaged and the number of cells was significantly reduced. It was found that PC12 cells were swollen, rounded, protruding, and retracting, with reduced adherent function and floating phenomenon. Quercetin could increase the survival rate and proliferation rate of PC12 cells; reduce the levels of LDH, AChE, MDA, and HO-1 protein; and increase the levels of SOD, GSH-Px, CAT, T-AOC, sirtuin1, and Nrf2 protein. CONCLUSION: Quercetin can increase the survival rate of PC12 injured by Aß 25-35, promote cell proliferation, and antagonize the toxicity of Aß; it also has certain neuroprotective effects. Therefore, quercetin is expected to become a drug for the treatment of AD.

Protein Engineering of a Pyridoxal-5'-Phosphate-Dependent l-Aspartate-α-Decarboxylase from Tribolium castaneum for ß-Alanine Production.

In the present study, a pyridoxal-5'-phosphate (PLP)-dependent L-aspartate-α-decarboxylase from Tribolium castaneum (TcPanD) was selected for protein engineering to efficiently produce ß-alanine. A mutant TcPanD-R98H/K305S with a 2.45-fold higher activity than the wide type was selected through error-prone PCR, site-saturation mutagenesis, and 96-well plate screening technologies. The characterization of purified enzyme TcPanD-R98H/K305S showed that the optimal cofactor PLP concentration, temperature, and pH were 0.04% (m/v), 50 °C, and 7.0, respectively. The 1mM of Na+, Ni2+, Co2+, K+, and Ca2+ stimulated the activity of TcPanD-R98H/K305S, while only 5 mM of Ni2+ and Na+ could increase its activity. The kinetic analysis indicated that TcPanD-R98H/K305S had a higher substrate affinity and enzymatic reaction rate than the wild enzyme. A total of 267 g/L substrate l-aspartic acid was consumed and 170.5 g/L of ß-alanine with a molar conversion of 95.5% was obtained under the optimal condition and 5-L reactor fermentation.

Novel strategy of incorporating biochar in solid-state fermentation for enhancing erythritol production by forming "microzones".

Biochar is increasingly considered in addressing bioprocess issues due to its strong adsorbability and excellent compatibility to microbes. Here, biochar was first applied in aerobic solid-state fermentation (SSF) for erythritol production. Biochars derived from different agricultural wastes under various pyrolysis temperatures were evaluated, and wheat straw pyrolyzed at 300 °C (WSc) performed the best in enhancing fermentative erythritol production, with a dosage of 4% (w/w). In this procedure, cell-biochar-substrate "microzones" were formed, which was conductive to cell growth and attachment, and hence contributed enhanced enzyme activities, oil consumption, and erythritol production. The resultant erythritol productions of batch and fed-batch fermentations were 207.3 and 222.5 mg/gds, respectively. In repeated-batch fermentation, high cell viability and robust erythritol synthesis were maintained throughout seven cycles. This study demonstrates that SSF can be remarkably facilitated by biochar addition, suggesting a new perspective of biochar application in microbiological processes.

Synergism of cellulase, pectinase and xylanase on hydrolyzing differently pretreated sweet potato residues.

The synergism of cellulase (C), pectinase (P), and xylanase (X) for the saccharification of sweet potato residues (SPR) was investigated. The removal of starch from SPR was easily achieved by using amylase, but the cellulose conversion of de-starched SPR was relatively low, thus dilute H2SO4, NaOH, and H2O2 pretreatment was conducted to improve the enzymatic digestibility. The lignin content of NaOH pretreated SPR was the lowest, whereas H2SO4 pretreatment resulted in the lowest contents of hemicellulose and pectin. The combination of C, P, and X exhibited different sugar production patterns, C-P displayed synergistic action on glucose and galactose production from each type of SPR, C-X also exhibited synergistic effect on glucose production except when H2SO4 pretreated SPR was used, whereas no synergism between P-X on monosaccharide production was observed. The presence of synergism between cellulase and mixed accessory enzymes [C-(PX)] on glucose formation was determined by C-X, and the degree of synergism between C-P and C-(PX) on glucose production had a positive relationship with pectin content. The highest cellulose conversion of 96.2% was obtained from NaOH pretreated SPR using mixed enzymes comprising C, P, and X with the ratio of 8:1:1.

Transcriptome Mechanism of Utilizing Corn Steep Liquor as the Sole Nitrogen Resource for Lipid and DHA Biosynthesis in Marine Oleaginous Protist Aurantiochytrium sp.

In the current study, corn steep liquor (CSL) is evaluated as an ideal raw agro-material for efficient lipid and docosahexaenoic acid DHA production by Aurantiochytrium sp. Low CSL level in medium (nitrogen deficiency) stimulated the biosynthesis of lipids and DHA while inhibiting cellular growth. The transcriptomic profiles of the Aurantiochytrium sp. cells are analyzed and compared when cultured under high (H group), normal (N group), and low (L group) levels of CSL in the medium. The discriminated transcriptomic profiles from the three groups indicates that changes in CSL level in medium result in a global change in transcriptome of Aurantiochytrium sp. The overall de novo assembly of cDNA sequence data generated 61,163 unigenes, and 18,129 of them were annotated in at least one database. A total of 5105 differently expressed (DE) genes were found in the N group versus the H group, with 2218 downregulated and 2887 upregulated. A total of 3625 DE genes were found in the N group versus the L group, with 1904 downregulated and 1721 upregulated. The analysis and categorization of the DE genes indicates that the regulation mechanism of CSL involved in the perception and transduction of the limited nitrogen signal, the interactions between the transcription factors (TFs) and multiple downstream genes, and the variations in downstream genes and metabolites, in sequence, are illuminated for the first time in the current study.

Oil crop wastes as substrate candidates for enhancing erythritol production by modified Yarrowia lipolytica via one-step solid state fermentation.

Oil crop wastes are attractive feedstocks in microbial processes due to their low cost. However, the product yields can be limited by their undesirable nitrogen surplus. Present study proposed a one-step solid state fermentation (SSF) method for producing erythritol from unrefined oil crop wastes using a modified strain Y. lipolytica M53-S. Enhanced erythritol production (185.4 mg/gds) was obtained from peanut press cake mixed with 40% sesame meal and 10% waste cooking oil. The process was performed at pH 4.0 in 5 L flasks, with initial moisture content, NaCl addition, and inoculum size of 70%, 0.02 g/gds, and 7.5 × 104 cells/gds, respectively. This procedure showed advantages in terms of lower material cost than that of submerged fermentation and shorter culture cycle (96 h) than other SSF processes. In repeated-batch fermentation, erythritol was continuously produced for seven cycles. This study presents a feasible approach in developing an efficient erythritol cultivation from nitrogen-rich wastes.

Integrated Approaches to Reveal Genes Crucial for Tannin Degradation in Aureobasidium melanogenum T9.

Tannins biodegradation by a microorganism is one of the most efficient ways to produce bioproducts of high value. However, the mechanism of tannins biodegradation by yeast has been little explored. In this study, Aureobasidium melanogenum T9 isolated from red wine starter showed the ability for tannins degradation and had its highest biomass when the initial tannic acid concentration was 20 g/L. Furthermore, the genes involved in the tannin degradation process were analyzed. Genes tan A, tan B and tan C encoding three different tannases respectively were identified in the A. melanogenum T9. Among these genes, tan A and tan B can be induced by tannin acid simultaneously at both gene transcription and protein expression levels. Our assay result showed that the deletion of tanA and tanB resulted in tannase activity decline with 51.3 ± 4.1 and 64.1 ± 1.9 U/mL, respectively, which is much lower than that of A. melanogenum T9 with 91.3 ± 5.8 U/mL. In addition, another gene coding gallic acid decarboxylase (gad) was knocked out to better clarify its function. Mutant Δgad completely lost gallic acid decarboxylase activity and no pyrogallic acid was seen during the entire cultivation process, confirming that there was a sole gene encoding decarboxylase in the A. melanogenum T9. These results demonstrated that tanA, tanB and gad were crucial for tannin degradation and provided new insights for the mechanism of tannins biodegradation by yeast. This finding showed that A. melanogenum has potential in the production of tannase and metabolites, such as gall acid and pyrogallol.

Whole Conversion of Soybean Molasses into Isomaltulose and Ethanol by Combining Enzymatic Hydrolysis and Successive Selective Fermentations.

Isomaltulose is mainly produced from sucrose by microbial fermentation, when the utilization of sucrose contributes a high production cost. To achieve a low-cost isomaltulose production, soy molasses was introduced as an alternative substrate. Firstly, α-galactosidase gene from Rhizomucor miehei was expressed in Yarrowia lipolytica, which then showed a galactosidase activity of 121.6 U/mL. Under the effects of the recombinant α-galactosidase, most of the raffinose-family oligosaccharides in soy molasses were hydrolyzed into sucrose. Then the soy molasses hydrolysate with high sucrose content (22.04%, w/w) was supplemented into the medium, with an isomaltulose production of 209.4 g/L, and the yield of 0.95 g/g. Finally, by virtue of the bioremoval process using Pichia stipitis, sugar byproducts in broth were transformed into ethanol at the end of fermentation, thus resulting in high isomaltulose purity (97.8%). The bioprocess employed in this study provides a novel strategy for low-cost and efficient isomaltulose production from soybean molasses.

High-Throughput Biochemical Fingerprinting of Oleaginous Aurantiochytrium sp. Strains by Fourier Transform Infrared Spectroscopy (FT-IR) for Lipid and Carbohydrate Productions.

The traditional biochemical methods for analyzing cellular composition of oleaginous microorganisms are time-consuming, polluting, and expensive. In the present study, an FT-IR method was used to analyze the cellular composition of the marine oleaginous protist Aurantiochytrium sp. during various research processes, such as strains screening, medium optimization, and fermentation, and was evaluated as a green, low-cost, high throughput, and accurate method compared with the traditional methods. A total of 109 Aurantiochytrium sp. strains were screened for lipid and carbohydrate production and the best results were found for the strains No. 6 and No. 32. The yields and productivities could reach up to 47.2 g/L and 0.72 g/L/h for lipid, 21.6 g/L and 0.33 g/L/h for docosahexaenoic acid (DHA) in the strain No. 6, and 15.4 g/L and 0.18 g/L/h for carbohydrate in the strain No. 32, under the optimal conditions, respectively. These results confirmed potentials of the two Aurantiochytrium sp. strains for lipid, DHA, and carbohydrate productions at industrial scales. The FT-IR method in this study will facilitate research on the oleaginous Aurantiochytrium sp., and the obtained two strains for lipid and carbohydrate productions will provide the foundations for their applications in medical, food, and feed industries.

Transcriptomic Mechanism of the Phytohormone 6-Benzylaminopurine (6-BAP) Stimulating Lipid and DHA Synthesis in Aurantiochytrium sp.

The phytohormone 6-benzylaminopurine (6-BAP) significantly improves lipid synthesis of oleaginous microorganisms with the great potential applied in lipid production. In the current study, the lipid and DHA productions in oleaginous Aurantiochytrium sp. were found to be improved by 48.7% and 55.3%, respectively, induced by 6-BAP treatments. Then, using high-throughput RNA-seq technology, the overall de novo assembly of the cDNA sequence data generated 53871 unigenes, and 15902 of these were annotated in at least one database. The comparative transcriptomic profiles of cells with and without 6-BAP treatments revealed that a total of 717 were differently expressed genes (DE), with 472 upregulated and 245 downregulated. Further annotation and categorization indicated that some DE genes were involved in pathways crucial to lipid and DHA productions, such as fatty acid synthesis, central carbon metabolism, transcriptional factor, signal transduction, and mevalonate pathway. A regulation mode of 6-BAP, in turn, perception and transduction of 6-BAP signal, transcription factor, expression regulations of the downstream genes, and metabolic changes, respectively, was put forward for the first time in the present study. This research illuminates the transcriptomic mechanism of phytohormone stimulation of lipid and DHA production in an oleaginous microorganism and provides the potential targets modified using genetic engineering for improving lipid and DHA productivity.