Pioneering Astaxanthin-Tumor Cell Membrane Nanoparticles for Innovative Targeted Drug Delivery on Melanoma.

Background: Recently, the use of the tumor or its secretions as drug carriers has gradually become popular, with the advantages of high biocompatibility and enhanced drug delivery to specific cells. Melanoma is the most malignant tumor of all skin cancers; it is the most metastatic and, therefore, the most difficult to treat. The main purpose of this study is to develop nanovesicles with tumor cell membrane secretion properties to encapsulate target substances to enhance the therapeutic effect of cancer. Methods: Astaxanthin was selected as an anticancer drug due to our previous research finding that astaxanthin has extremely high antioxidant, anti-ultraviolet damage, and anti-tumor properties. The manufacturing method of the astaxanthin nanovesicle carrier is to mix melanoma cells and astaxanthin in an appropriate ratio and then remove the genetic material and inflammatory factors of cancer cells by extrusion. Results: In terms of results, after the co-culture of astaxanthin nanovesicles and melanoma cancer cells, it was confirmed that the ability of astaxanthin nanovesicles to inhibit the growth and metastasis of melanoma cancer cells was significantly better than the same amount of astaxanthin alone, and it had no effect on normal Human cells are also effective. There was no apparent harm on normal cells, indicating the ability of the vesicles to be selectively transported. Conclusion: Our findings illustrated the potential of astaxanthin nanovesicles as an anticancer drug.

Structure-based development of a canine TNF-α-specific antibody using adalimumab as a template.

The canine anti-tumor necrosis factor-alpha (TNF-α) monoclonal antibody is a potential therapeutic option for treating canine arthritis. The current treatments for arthritis in dogs have limitations due to side effects, emphasizing the need for safer and more effective therapies. The crystal structure of canine TNF-α (cTNF-α) was successfully determined at a resolution of 1.85 Å, and the protein was shown to assemble as a trimer, with high similarity to the functional quaternary structure of human TNF-α (hTNF-α). Adalimumab (Humira), a known TNF-α inhibitor, effectively targets and neutralizes TNF-α to reduce inflammation and has been used to manage autoimmune conditions such as rheumatoid arthritis. By comparing the structure of cTNF-α with the complex structure of hTNF-α and adalimumab-Fab, the epitope of adalimumab on cTNF-α was identified. The significant structural similarities of epitopes in cTNF-α and hTNF-α indicate the potential of using adalimumab to target cTNF-α. Therefore, a canine/human chimeric antibody, Humivet-R1, was created by grafting the variable domain of adalimumab onto a canine antibody framework derived from ranevetmab. Humivet-R1 exhibits potent neutralizing ability (IC50 = 0.05 nM) and high binding affinity (EC50 = 0.416 nM) to cTNF-α, comparable to that of adalimumab for both hTNF-α and cTNF-α. These results strongly suggest that Humivet-R1 has the potential to provide effective treatment for canine arthritis with reduced side effects. Here, we propose a structure-guided antibody design for the use of a chimeric antibody to treat canine inflammatory disease. Our successful development strategy can speed up therapeutic antibody discovery for animals and has the potential to revolutionize veterinary medicine.

Specific recognition of cyclic oligonucleotides by Cap4 for phage infection.

Under selective pressure, bacteria have evolved diverse defense systems against phage infections. The SMODS-associated and fused to various effector domains (SAVED)-domain containing proteins were identified as major downstream effectors in cyclic oligonucleotide-based antiphage signaling system (CBASS) for bacterial defense. Recent study structurally characterizes a cGAS/DncV-like nucleotidyltransferase (CD-NTase)-associated protein 4 from Acinetobacter baumannii (AbCap4) in complex with 2'3'3'-cyclic AMP-AMP-AMP (cAAA). However, the homologue Cap4 from Enterobacter cloacae (EcCap4) is activated by 3'3'3'-cyclic AMP-AMP-GMP (cAAG). To elucidate the ligand specificity of Cap4 proteins, we determined the crystal structures of full-length wild-type and K74A mutant of EcCap4 to 2.18 and 2.42 Å resolution, respectively. The DNA endonuclease domain of EcCap4 shares similar catalytic mechanism with type II restriction endonuclease. Mutating the key residue K74 in the conserved DXn(D/E)XK motif completely abolishes its DNA degradation activity. The potential ligand-binding cavity of EcCap4 SAVED domain is located adjacent to its N-terminal domain, significantly differing from the centrally located cavity of AbCap4 SAVED domain which recognizes cAAA. Based on structural and bioinformatic analysis, we found that Cap4 proteins can be classified into two types: the type I Cap4, like AbCap4, recognize cAAA and the type II Cap4, like EcCap4, bind cAAG. Several conserved residues identified at the surface of potential ligand-binding pocket of EcCap4 SAVED domain are confirmed by ITC experiment for their direct binding roles for cAAG. Changing Q351, T391 and R392 to alanine abolished the binding of cAAG by EcCap4 and significantly reduced the anti-phage ability of the E. cloacae CBASS system constituting EcCdnD (CD-NTase in clade D) and EcCap4. In summary, we revealed the molecular basis for specific cAAG recognition by the C-terminal SAVED domain of EcCap4 and demonstrates the structural differences for ligand discrimination among different SAVED-domain containing proteins.

Short-term effects of Chlorhexidine mouthwash and Listerine on oral microbiome in hospitalized patients.

Introduction: Chlorhexidine (CHX) and essential oil containing mouthwashes like Listerine® can improve oral hygiene via suppressing oral microbes. In hospitalized patients, CHX mouthwash reduces the incidence of ventilator-associated pneumonia. However, CHX use was also associated with increased mortality, which might be related to nitrate-reducing bacteria. Currently, no study determines oral bacteria targeted by essential oils mouthwash in hospitalized patients using a metagenomic approach. Methods: We recruited 87 hospitalized patients from a previous randomized control study, and assigned them to three mouthwash groups: CHX, Listerine, and normal saline (control). Before and after gargling the mouthwash twice a day for 5-7 days, oral bacteria were examined using a 16S rDNA approach. Results: Alpha diversities at the genus level decreased significantly only for the CHX and Listerine groups. Only for the two groups, oral microbiota before and after gargling were significantly different, but not clearly distinct. Paired analysis eliminated the substantial individual differences and revealed eight bacterial genera (including Prevotella, Fusobacterium, and Selenomonas) with a decreased relative abundance, while Rothia increased after gargling the CHX mouthwash. After gargling Listerine, seven genera (including Parvimonas, Eubacterium, and Selenomonas) showed a decreased relative abundance, and the magnitudes were smaller compared to the CHX group. Fewer bacteria targeted by Listerine were reported to be nitrate-reducing compared to the CHX mouthwash. Discussion: In conclusion, short-term gargling of the CHX mouthwash and Listerine altered oral microbiota in our hospitalized patients. The bacterial genera targeted by the CHX mouthwash and Listerine were largely different and the magnitudes of changes were smaller using Listerine. Functional alterations of gargling CHX and Listerine were also different. These findings can be considered for managing oral hygiene of hospitalized patients.

Improving red-color performance, immune response and resistance to Vibrio parahaemolyticus on white shrimp Penaeus vannamei by an engineered astaxanthin yeast.

Astaxanthin (AST), a super antioxidant with coloring and medical properties, renders it a beneficial feed additive for shrimp. This study conducted a white shrimp feeding trial of 3S, 3'S isoform AST, which was derived from metabolic-engineered Kluyveromyces marxianus fermented broth (TB) and its extract (TE) compared to sources from two chemically synthetic ASTs (Carophyll Pink [CP] and Lucantin Pink [LP]), which contain 3S, 3'S, 3R, 3'S (3S, 3'R) and 3R, 3'R isoforms ratio of 1:2:1. The effects on red coloration, immune parameters and resistance to Vibrio infection were evaluated. Four AST sources were incorporated into the diets at concentrations of 0 (control), 100 mg kg-1 (TB100, TE100, CP100, and LP100), and 200 mg kg-1 (TB200, TE200, CP200, and LP200). Results revealed that in week 4, shrimps that received AST-supplemented feeds, especially TB100, TB200, and TE200, significantly increased redness (a*) values. Immune responses including phagocytosis activity, superoxide-anion production, phenoloxidase activity, and immune-related genes were examined on days 0, 1, 2, 4, 7, 14, 21, and 28. Generally, shrimps that received AST-supplemented feeds exhibited higher immune responses on days 7 and 14 than the control feed. Gene expression levels of superoxide dismutase and glutathione peroxidase were significantly upregulated on days 7 and 14 in shrimps that received AST-supplemented feeds, while genes of penaeidins, antilipopolysaccharide factor, and lysozyme were upregulated on days 4, 7, and 14, especially received TB200 and TE200. Furthermore, shrimps that received TB100, TE100, CP100, and LP100 7 days were then challenged with Vibrio parahaemolyticus and the result demonstrated higher survival rates especially TB100 at 168 h than the control feed. In conclusion, incorporating AST into the diets enhanced shrimp red coloration, immune parameters, and resistance against V. parahaemolyticus infection. The K. marxianus-derived AST exhibited higher performance than did chemical AST to be a potential feed additive in shrimp aquaculture.

Crystal structure of the capsular polysaccharide-synthesis enzyme CapG from Staphylococcus aureus.

Bacterial capsular polysaccharides provide protection against environmental stress and immune evasion from the host immune system, and are therefore considered to be attractive therapeutic targets for the development of anti-infectious reagents. Here, we focused on CapG, one of the key enzymes in the synthesis pathway of capsular polysaccharides type 5 (CP5) from the opportunistic pathogen Staphylococcus aureus. SaCapG catalyses the 2-epimerization of UDP-N-acetyl-D-talosamine (UDP-TalNAc) to UDP-N-acetyl-D-fucosamine (UDP-FucNAc), which is one of the nucleotide-activated precursors for the synthesis of the trisaccharide repeating units of CP5. Here, the cloning, expression and purification of recombinant SaCapG are reported. After extensive efforts, single crystals of SaCapG were successfully obtained which belonged to space group C2 and exhibited unit-cell parameters a = 302.91, b = 84.34, c = 145.09 Å, ß = 110.65°. The structure was solved by molecular replacement and was refined to 3.2 Šresolution. The asymmetric unit revealed a homohexameric assembly of SaCapG, which was consistent with gel-filtration analysis. Structural comparison with UDP-N-acetyl-D-glucosamine 2-epimerase from Methanocaldococcus jannaschii identified α2, the α2-α3 loop and α10 as a gate-regulated switch controlling substrate entry and/or product release.

Safety Assessment of 3S, 3'S Astaxanthin Derived from Metabolically Engineered K. marxianus.

Previous reviews have already explored the safety and bioavailability of astaxanthin, as well as its beneficial effects on human body. The great commercial potential in a variety of industries, such as the pharmaceutical and health supplement industries, has led to a skyrocketing demand for natural astaxanthin. In this study, we have successfully optimized the astaxanthin yield up to 12.8 mg/g DCW in a probiotic yeast and purity to 97%. We also verified that it is the desired free-form 3S, 3'S configurational stereoisomer by NMR and FITR that can significantly increase the bioavailability of astaxanthin. In addition, we have proven that our extracted astaxanthin crystals have higher antioxidant capabilities compared with natural esterified astaxanthin from H. pluvialis. We also screened for potential adverse effects of the pure astaxanthin crystals extracted from the engineered probiotic yeast by dosing SD rats with 6, 12, and 24 mg/kg/day of astaxanthin crystals via oral gavages for a 13-week period and have found no significant biological differences between the control and treatment groups in rats of both genders, further confirming the safety of astaxanthin crystals. This study demonstrates that developing metabolically engineered microorganisms provides a safe and feasible approach for the bio-based production of many beneficial compounds, including astaxanthin.

Dietary of Lactobacillus paracasei and Bifidobacterium longum improve nonspecific immune responses, growth performance, and resistance against Vibrio parahaemolyticus in Penaeus vannamei.

This study investigated the effects of two probiotics, namely Lactobacillus paracasei and Bifidobacterium longum, as feed additives on growth performance, nonspecific immunity, immune-related gene expression, and disease resistance against Vibrio parahaemolyticus in Penaeus vannamei. The experimental diets were prepared using L. paracasei and B. longum at concentrations of 105 and 107 CFU/g; these diets were referred to as P5, P7, B5, and B7. After 8 weeks of the diets, regarding growth performance, the B7 group showed the highest weight gain rate (890.34 ± 103.65%), special growth rate (4.08 ± 0.19%), and feed conversion rate (1.52 ± 0.19%) compared with the other groups. Moreover, the total hemocyte counts were significantly increased (p < 0.05) in the P7 groups on day 14 during the 28-day feeding trial. The phagocytosis rate in all experimental groups was increased on day 14 and was persistently significantly activated to day 21, especially in the P7 and B5 group. The phagocytic index of the P7 group showed a significant increase on day 14 and persistent activation to day 21. In the analysis of respiratory burst activity and phenoloxidase activity, the P7 and B5 groups showed a significant increase on day 7 and persistent activation to day 21. The expression level of the immune-related genes of superoxide dismutase, clotting protein, Penaeidin2, Penaeidin3, Penaeidin4, anti-LPS factor, crustin, and lysozyme was significantly increased in the experimental groups, especially in the P7 group. Furthermore, the optimum conditions of feed additives were determined in challenge trials conducted using P7 and B5. Shrimps fed P7 and B5 showed an increased survival rate (72.73% and 66.67%) after the V. parahaemolyticus challenge. In sum, the results revealed that B. longum, as a feed additive at 107 CFU/g, enhanced growth performance. L. paracasei at 107 CFU/g and B. longum at 105 CFU/g can enhance nonspecific immune responses and immune-related gene expression, and 107 CFU/g L. paracasei has the highest resistance ability for V. parahaemolyticus. Thus, dietary supplementation with L. paracasei and B. longum may be a valuable approach in white shrimp aquaculture.

Synergistic effects of dietary oxolinic acid combined with oxytetracycline on nonspecific immune responses and resistance against Vibrio parahaemolyticus infection of white shrimp (Penaeus vannamei).

This study investigated the synergistic effects of oxolinic acid (OA) combined with oxytetracycline (OTC) on white shrimp (Penaeus vannamei). Disk diffusion susceptibility testing was performed to analyze the sensitivity of Vibrio alginolyticus and Vibrio parahaemolyticus to different concentrations of OA and OTC. The results revealed that 50 mg OA/L combined with 50 mg OTC/L exhibited stronger antibacterial effects on V. alginolyticus and V. parahaemolyticus. The results of in vitro tests indicated that cotreatment with OA and OTC significantly reduced superoxide anion production and phenoloxidase activity, but not phagocytic activity. Subsequently, feeding trials were performed to investigate the immunomodulatory effects and bioaccumulation of dietary OA combined with OTC on shrimp. The healthy shrimp (15.13 ± 1.02 g) were divided into four groups: control, 100 mg OA/kg combined with 50 mg OTC/kg, 50 mg OA/kg combined with 100 mg OTC/kg, and 50 mg OA/kg combined with 50 mg OTC/kg. The shrimp were sampled to determine innate immunity parameters and residual OA and OTC levels in the muscle during a 28-day feeding regimen; the shrimp were fed the experimental diet from day 1 to day 5 and a commercial diet from day 6 to day 28. Residual OA levels were considerably higher in the group fed 100 mg OA/kg combined with 50 mg OTC/kg compared with the other groups and peaked on day 4. The residual OA levels of all the groups were below the detection limit after without providing OA. The residual OTC levels of the group fed 50 mg OA/kg combined with 100 mg OTC/kg were considerably higher from day 1 to day 4. The residual OTC levels in all the groups decreased rapidly and could not be detected on day 28. The administration of 50 mg OA/kg combined with 100 mg OTC/kg exerted the least effect on the white shrimp. Moreover, the survival rates of the treatment groups after the V. parahaemolyticus challenge were higher than those of the control group, especially the group fed 50 mg OA/kg combined with 100 mg OTC/kg. This result indicated that the synergistic effects of dietary OA and OTC are safe and effective. Combination therapy is a new method of antibiotic use in aquaculture.

Effect of dietary supplementation with Moringa oleifera leaf extract and Lactobacillus acidophilus on growth performance, intestinal microbiota, immune response, and disease resistance in whiteleg shrimp (Penaeus vannamei).

This study investigated the effect of the moringa (Moringa oleifera) leaf extract and Lactobacillus acidophilus individually or combined on growth performance, enzyme activity, intestinal and hepatopancreatic histology, intestinal microbiota, immune response, and resistance against Vibrio alginolyticus and Vibrio parahaemolyticus in whiteleg shrimp (Penaeus vannamei). Six diets were formulated: three diets without L. acidophilus containining 0 (control, ME0), 2.5 (ME2.5), and 5.0 g/kg of moringa (ME5.0) and the same three diets containing L. acidophilus at 1 × 107 CFU/g of diet (ME0+P, ME2.5 + P, and ME5.0 + P, respectively). Growth performance was measured after 60 days of the rearing period. On the final day, the shrimp were sampled to assess enzyme activity, intestinal and hepatopancreatic histology, and gut microbiota. Shrimp hemocytes were examined on Days 0, 1, 2, 4, 7, 14, 21, and 28 to measure the immune response in terms of the total hemocyte count, phenoloxidase activity, phagocytosis, and superoxide anion production. Furthermore, the shrimp were challenged with V. alginolyticus and V. parahaemolyticus. The results revealed that ME2.5 + P significantly increased (P < 0.05) final weight, weight gain, specific growth rate, enzyme activities, and villi height compared with ME2.5 and control. Wall thickness was increased in the shrimp fed diet supplemented with moringa and L. acidophilus compared with the control shrimp. Hepatopancreatic histology revealed that R cells were more abundant in the shrimp fed diet containing moringa and L. acidophilus compared with those fed diet containing moringa alone (P < 0.05) at the same concentration. High-throughput sequencing analysis indicated that the dietary supplementation with moringa and L. acidophilus affected the gut microbiota composition. All gene functions, members of KEGG level 2, related to metabolism were increased in diet supplemented with moringa with or without L. acidophilus compared with the control group. The immune assay revealed that the total hemocyte count, phenoloxidase activity, phagocytic rate, superoxide anion production, and immune-related gene expression (including those of prophenoloxidase II, alpha-2-macroglobulin, penaeidin2, antilipopolysaccharide factor, crustin, lysozyme, glutathione peroxidase, and superoxide dismutase) were higher in the experimental groups than in the control group on several observed days; however, the increases were observed more often in the ME2.5 + P group than in the other treatment groups. Furthermore, the ME2.5 + P group exhibited a significantly higher survival rate (P < 0.05) in the challenge test against V. alginolyticus and V. parahaemolyticus. In conclusion, supplementation with dietary moringa and L. acidophilus at ME2.5 + P improved growth performance, immune system, and resistance against Vibrio in the shrimp.

Microbial cell factories for the production of polyhydroxyalkanoates.

Pollution caused by persistent petro-plastics is the most pressing problem currently, with 8 million tons of plastic waste dumped annually in the oceans. Plastic waste management is not systematized in many countries, because it is laborious and expensive with secondary pollution hazards. Bioplastics, synthesized by microorganisms, are viable alternatives to petrochemical-based thermoplastics due to their biodegradable nature. Polyhydroxyalkanoates (PHAs) are a structurally and functionally diverse group of storage polymers synthesized by many microorganisms, including bacteria and Archaea. Some of the most important PHA accumulating bacteria include Cupriavidus necator, Burkholderia sacchari, Pseudomonas sp., Bacillus sp., recombinant Escherichia coli, and certain halophilic extremophiles. PHAs are synthesized by specialized PHA polymerases with assorted monomers derived from the cellular metabolite pool. In the natural cycle of cellular growth, PHAs are depolymerized by the native host for carbon and energy. The presence of these microbial PHA depolymerases in natural niches is responsible for the degradation of bioplastics. Polyhydroxybutyrate (PHB) is the most common PHA with desirable thermoplastic-like properties. PHAs have widespread applications in various industries including biomedicine, fine chemicals production, drug delivery, packaging, and agriculture. This review provides the updated knowledge on the metabolic pathways for PHAs synthesis in bacteria, and the major microbial hosts for PHAs production. Yeasts are presented as a potential candidate for industrial PHAs production, with their high amenability to genetic engineering and the availability of industrial-scale technology. The major bottlenecks in the commercialization of PHAs as an alternative for plastics and future perspectives are also critically discussed.

Constructing a human complex type N-linked glycosylation pathway in Kluyveromyces marxianus.

Glycosylation can affect various protein properties such as stability, biological activity, and immunogenicity. To produce human therapeutic proteins, a host that can produce glycoproteins with correct glycan structures is required. Microbial expression systems offer economical, rapid and serum-free production and are more amenable to genetic manipulation. In this study, we developed a protocol for CRISPR/Cas9 multiple gene knockouts and knockins in Kluyveromyces marxianus, a probiotic yeast with a rapid growth rate. As hyper-mannosylation is a common problem in yeast, we first knocked out the α-1,3-mannosyltransferase (ALG3) and α-1,6-mannosyltransferase (OCH1) genes to reduce mannosylation. We also knocked out the subunit of the telomeric Ku domain (KU70) to increase the homologous recombination efficiency of K. marxianus. In addition, we knocked in the MdsI (α-1,2-mannosidase) gene to reduce mannosylation and the GnTI (ß-1,2-N-acetylglucosaminyltransferase I) and GnTII genes to produce human N-glycan structures. We finally obtained two strains that can produce low amounts of the core N-glycan Man3GlcNAc2 and the human complex N-glycan Man3GlcNAc4, where Man is mannose and GlcNAc is N-acetylglucosamine. This study lays a cornerstone of glycosylation engineering in K. marxianus toward producing human glycoproteins.

Constructing a yeast to express the largest cellulosome complex on the cell surface.

Cellulosomes, which are multienzyme complexes from anaerobic bacteria, are considered nature's finest cellulolytic machinery. Thus, constructing a cellulosome in an industrial yeast has long been a goal pursued by scientists. However, it remains highly challenging due to the size and complexity of cellulosomal genes. Here, we overcame the difficulties by synthesizing the Clostridium thermocellum scaffoldin gene (CipA) and the anchoring protein gene (OlpB) using advanced synthetic biology techniques. The engineered Kluyveromyces marxianus, a probiotic yeast, secreted a mixture of dockerin-fused fungal cellulases, including an endoglucanase (TrEgIII), exoglucanase (CBHII), ß-glucosidase (NpaBGS), and cellulase boosters (TaLPMO and MtCDH). The confocal microscopy results confirmed the cell-surface display of OlpB-ScGPI and fluorescence-activated cell sorting analysis results revealed that almost 81% of yeast cells displayed OlpB-ScGPI. We have also demonstrated the cellulosome complex formation using purified and crude cellulosomal proteins. Native polyacrylamide gel electrophoresis and mass spectrometric analysis further confirmed the cellulosome complex formation. Our engineered cellulosome can accommodate up to 63 enzymes, whereas the largest engineered cellulosome reported thus far could accommodate only 12 enzymes and was expressed by a plasmid instead of chromosomal integration. Interestingly, CipA 2B9C (with two cellulose binding modules, CBM) released significantly higher quantities of reducing sugars compared with other CipA variants, thus confirming the importance of cohesin numbers and CBM domain on cellulosome complex. The engineered yeast host efficiently degraded cellulosic substrates and released 3.09 g/L and 8.61 g/L of ethanol from avicel and phosphoric acid-swollen cellulose, respectively, which is higher than any previously constructed yeast cellulosome.

Metabolic engineering probiotic yeast produces 3S, 3'S-astaxanthin to inhibit B16F10 metastasis.

3S, 3'S-Astaxanthin is the most powerful antioxidant to scavenge free radicals in the world. In this study, a 3S, 3'S-astaxanthin biosynthesis pathway was constructed in a probiotic yeast, Kluveromyces marxianus, denoted YEAST, and its bioactive metabolites were extracted for biofunctional assessments. The bio-safety examination was achieved by two animal models as following: First, no significant toxic effects on YEAST groups were found in zebrafish; Second, after feeding YEAST for 4 weeks, the rat-groups showed no visible abnormality, and no significant change of the body weight and blood biochemistry tests. The inhibition of lung metastasis of melanoma cells and the increment of the survival rate were demonstrated by feeding YEAST and injecting the intravenous commercial astaxanthin in vivo rodent model. Based on in vitro assays of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) scavenging analysis, ferrous ion chelating ability, reducing power assessment, and mushroom tyrosinase inhibition evaluation, YEAST-astaxanthin showed anti-oxidative and tyrosinase suppressive properties. Taken together, the 3S, 3'S-astaxanthin producing probiotic yeast is safe to be used in the bio-synthesis of functional and pharmaceutical compounds, which have broad industrial applications on cosmetic, food and feed additive and healthcare.

Characterizing an engineered carotenoid-producing yeast as an anti-stress chassis for building cell factories.

BACKGROUND: A microorganism engineered for non-native tasks may suffer stresses it never met before. Therefore, we examined whether a Kluyveromyces marxianus strain engineered with a carotenoid biosynthesis pathway can serve as an anti-stress chassis for building cell factories. RESULTS: Carotenoids, a family of antioxidants, are valuable natural products with high commercial potential. We showed that the free radical removal ability of carotenoids can confer the engineered host with a higher tolerance to ethanol, so that it can produce more bio-ethanol than the wild type. Moreover, we found that this engineered strain has improved tolerance to other toxic effects including furfurals, heavy metals such as arsenate (biomass contaminant) and isobutanol (end product). Furthermore, the enhanced ethanol tolerance of the host can be applied to bioconversion of a natural medicine that needs to use ethanol as the delivery solvent of hydrophobic precursors. The result suggested that the engineered yeast showed enhanced tolerance to ethanol-dissolved hydrophobic 10-deacetylbaccatin III, which is considered a sustainable precursor for paclitaxel (taxol) bioconversion. CONCLUSIONS: The stress tolerances of the engineered yeast strain showed tolerance to several toxins, so it may serve as a chassis for cell factories to produce target products, and the co-production of carotenoids may make the biorefinary more cost-effective.

Engineering the oleaginous red yeast Rhodotorula glutinis for simultaneous ß-carotene and cellulase production.

Rhodotorula glutinis, an oleaginous red yeast, intrinsically produces several bio-products (i.e., lipids, carotenoids and enzymes) and is regarded as a potential host for biorefinery. In view of the limited available genetic engineering tools for this yeast, we have developed a useful genetic transformation method and transformed the ß-carotene biosynthesis genes (crtI, crtE, crtYB and tHMG1) and cellulase genes (CBHI, CBHII, EgI, EgIII, EglA and BGS) into R. glutinis genome. The transformant P4-10-9-63Y-14B produced significantly higher ß-carotene (27.13 ± 0.66 mg/g) than the wild type and also exhibited cellulase activity. Furthermore, the lipid production and salt tolerance ability of the transformants were unaffected. This is the first study to engineer the R. glutinis for simultaneous ß-carotene and cellulase production. As R. glutinis can grow in sea water and can be engineered to utilize the cheaper substrates (i.e. biomass) for the production of biofuels or valuable compounds, it is a promising host for biorefinery.

Biomimetic strategy for constructing Clostridium thermocellum cellulosomal operons in Bacillus subtilis.

BACKGROUND: Enzymatic conversion of lignocellulosic biomass into soluble sugars is a major bottleneck in the plant biomass utilization. Several anaerobic organisms cope these issues via multiple-enzyme complex system so called 'cellulosome'. Hence, we proposed a "biomimic operon" concept for making an artificial cellulosome which can be used as a promising tool for the expression of cellulosomal enzymes in Bacillus subtilis. RESULTS: According to the proteomic analysis of Clostridium thermocellum ATCC27405 induced by Avicel or cellobiose, we selected eight highly expressed cellulosomal genes including a scaffoldin protein gene (cipA), a cell-surface anchor gene (sdbA), two exoglucanase genes (celK and celS), two endoglucanase genes (celA and celR), and two xylanase genes (xynC and xynZ). Arranging these eight genes in two different orders, we constructed two different polycistronic operons using the ordered gene assembly in Bacillus method. This is the first study to express the whole CipA along with cellulolytic enzymes in B. subtilis. Each operon was successfully expressed in B. subtilis RM125, and the protein complex assembly, cellulose-binding ability, thermostability, and cellulolytic activity were demonstrated. The operon with a higher xylanase activity showed greater saccharification on complex cellulosic substrates such as Napier grass than the other operon. CONCLUSIONS: In this study, a strategy for constructing an efficient cellulosome system was developed and two different artificial cellulosomal operons were constructed. Both operons could efficiently express the cellulosomal enzymes and exhibited cellulose saccharification. This strategy can be applied to different industries with cellulose-containing materials, such as papermaking, biofuel, agricultural compost, mushroom cultivation, and waste processing industries.

Genome-wide prediction of CRISPR/Cas9 targets in Kluyveromyces marxianus and its application to obtain a stable haploid strain.

Kluyveromyces marxianus, a probiotic yeast, is important in industrial applications because it has a broad substrate spectrum, a rapid growth rate and high thermotolerance. To date, however, there has been little effort in its genetic engineering by the CRISPR/Cas9 system. Therefore, we aimed at establishing the CRISPR/Cas9 system in K. marxianus and creating stable haploid strains, which will make genome engineering simpler. First, we predicted the genome-wide target sites of CRISPR/Cas9 that have been conserved among the eight sequenced genomes of K. marxianus strains. Second, we established the CRISPR/Cas9 system in the K. marxianus 4G5 strain, which was selected for its high thermotolerance, rapid growth, a pH range of pH3-9, utilization of xylose, cellobiose and glycerol, and toxin tolerance, and we knocked out its MATα3 to prevent mating-type switching. Finally, we used K. marxianus MATα3 knockout diploid strains to obtain stable haploid strains with a growth rate comparable to that of the diploid 4G5 strain. In summary, we present the workflow from identifying conserved CRISPR/Cas9 targets in the genome to knock out the MATα3 genes in K. marxianus to obtain a stable haploid strain, which can facilitate genome engineering applications.

Constructing a cellulosic yeast host with an efficient cellulase cocktail.

Cellulose is a renewable feedstock for green industry. It is therefore important to develop a technique to construct a host with a high cellulolytic efficiency to digest cellulose. In this study, we developed a convenient host-engineering technique to adjust the expression levels of heterologous genes in the host by promoter rearrangement and gene copy number adjustment. Using genes from different glycoside hydrolase (GH) families including GH2, GH3, GH5, GH6, GH7, and GH12 from Aspergillus niger, Trichoderma reesei, and Neocallimastix patriciarum, we constructed a cellulolytic Kluyveromyces marxianus with eight cellulase gene-cassettes that produced a cellulase cocktail with a high cellulolytic efficiency, leading to a significant reduction in enzyme cost in a rice straw saccharification process. Our technique can be used to design a host that can efficiently convert biomass feedstock to biofuel.

Metabolic engineering a yeast to produce astaxanthin.

In this study, an astaxanthin-biosynthesis Kluyveromyces marxianus strain Sm23 was first constructed, which could produce 31µg/g DCW astaxanthin. Then, repeated genome integration of the key astaxanthin biosynthesis genes Hpchyb and bkt was done to increase gene copy number and astaxanthin yield. Four improved strains were obtained and the yield of astaxanthin and the total yield of carotenoids in a strain increased with the copy numbers of Hpchyb and bkt. To improve the yield further, the gene Hpchyb from Haematococcus pluvialis was modified by site-directed mutagenesis to increase the enzyme efficiency or/and to prevent the heterologous protein degradation by ubiquitination. Using repeated-integration approach of bkt and the mutated Hpchyb into Sm23, the S3-2 strain was obtained and shown to produce the 3S, 3'S-astaxanthin at 9972µg/g DCW in a 5L fermentor.