Astaxanthin (AX) is a carotenoid pigment with antioxidant properties widely used as a feed supplement. Wild‐type strains of Phaffia rhodozyma naturally produce low AX yields, but we increased AX ...yields 50‐fold in previous research using random mutagenesis of P. rhodozyma CBS6938 and fermentation optimization. On that study, genome changes were linked with phenotype, but relevant metabolic changes were not resolved. In this study, the wild‐type and the superior P. rhodozyma mutant strains were grown in chemically defined media and instrumented fermenters. Differential kinetic, metabolomics, and transcriptomics data were collected. Our results suggest that carotenoid production was mainly associated with cell growth and had a positive regulation of central carbon metabolism metabolites, amino acids, and fatty acids. In the stationary phase, amino acids associated with the TCA cycle increased, but most of the fatty acids and central carbon metabolism metabolites decreased. TCA cycle metabolites were in abundance and media supplementation of citrate, malate, α‐ketoglutarate, succinate, or fumarate increased AX production in the mutant strain. Transcriptomic data correlated with the metabolic and genomic data and found a positive regulation of genes associated with the electron transport chain suggesting this to be the main driver for improved AX production in the mutant strain.
Integrative metabolic analysis of improved astaxanthin production in Phaffia rhodozyma. Luna‐Flores and co‐workers grew their mutant and wild‐type P. rhodozyma strains in instrumented fermenters and obtained kinetics, metabolomics, and transcriptomics data for comparative analysis. By integration with genomic data, the authors found correlation between genome changes and metabolic data suggesting an improved electron transport chain as the main driver for improved astaxanthin production in the mutant strain.
Pichia pastoris (Komagataella phaffii) is a fast-growing methylotrophic yeast with the ability to assimilate several carbon sources such as methanol, glucose, or glycerol. It has been shown to have ...outstanding secretion capability with a variety of heterologous proteins. In previous studies, we engineered P. pastoris to co-express Escherichia coli AppA phytase and the HAC1 transcriptional activator using a bidirectional promoter. Phytase production was characterized in shake flasks and did not reflect industrial conditions. In the present study, phytase expression was explored and optimized using instrumented fermenters in continuous and fed-batch modes. First, the production of phytase was investigated under glucose de-repression in continuous culture at three dilution factors, 0.5 d
, 1 d
, and 1.5 d
. The fermenter parameters of these cultures were used to inform a kinetic model in batch and fed-batch modes for growth and phytase production. The kinetic model developed aided to design the glucose-feeding profile of a fed-batch culture. Kinetic model simulations under glucose de-repression and fed-batch conditions identified optimal phytase productivity at the specific growth rate of 0.041 h
. Validation of the model simulation with experimental data confirmed the feasibility of the model to predict phytase production in our newly engineered strain. Methanol was used only to induce the expression of phytase at high cell densities. Our results showed that high phytase production required two stages, the first stage used glucose under de-repression conditions to generate biomass while expressing phytase, and stage two used methanol to induce phytase expression. The production of phytase was improved 3.5-fold by methanol induction compared to the expression with glucose alone under de-repression conditions to a final phytase activity of 12.65 MU/L. This final volumetric phytase production represented an approximate 36-fold change compared to the flask fermentations. Finally, the phytase protein produced was assayed to confirm its molecular weight, and pH and temperature profiles. This study highlights the importance of optimizing protein production in P. pastoris when using novel promoters and presents a general approach to performing bioprocess optimization in this important production host.
Pichia pastoris (Komagataella phaffii) is an important platform for heterologous protein production due to its growth to high cell density and outstanding secretory capabilities. Recent developments ...in synthetic biology have extended the toolbox for genetic engineering of P. pastoris to improve production strains. Yet, overloading the folding and secretion capacity of the cell by over-expression of recombinant proteins is still an issue and rational design of strains is critical to achieve cost-effective industrial manufacture. Several enzymes are commercially produced in P. pastoris, with phytases being one of the biggest on the global market. Phytases are ubiquitously used as a dietary supplement for swine and poultry to increase digestibility of phytic acid, the main form of phosphorous storage in grains.
Potential bottlenecks for expression of E. coli AppA phytase in P. pastoris were explored by applying bidirectional promoters (BDPs) to express AppA together with folding chaperones, disulfide bond isomerases, trafficking proteins and a cytosolic redox metabolism protein. Additionally, transcriptional studies were used to provide insights into the expression profile of BDPs. A flavoprotein encoded by ERV2 that has not been characterised in P. pastoris was used to improve the expression of the phytase, indicating its role as an alternative pathway to ERO1. Subsequent AppA production increased by 2.90-fold compared to the expression from the state of the AOX1 promoter.
The microbial production of important industrial enzymes in recombinant systems can be improved by applying newly available molecular tools. Overall, the work presented here on the optimisation of phytase production in P. pastoris contributes to the improved understanding of recombinant protein folding and secretion in this important yeast microbial production host.
Efficacy of orally used of red yeast (Phaffia rhodozyma) (1.6 mg astaxanthin/g product) at 15.5 g (P1), 37.3 g (P2) and 47 (P3) g per kg diet were studied on performance, carcass composition, ...antioxidant activity and fillet pigmentation in rainbow trout (Oncorhynchus mykiss) weighing 208–212 g for eight weeks at 11 °C. Synthetic astaxanthin (AX) (0.5 g/kg diet) and basal diet were used as controls. With an increase in P. rhodozyma concentration in diet, weight gain and specific growth rate exhibited an increase compared to basal diet (P < 0.05%). A significant difference was seen among the treatments but only P3 diet significantly demonstrated a better growth than AX diet (P < 0.05). Activity of antioxidant enzymes; superoxidase dismutase, glutathione peroxidase, total antioxidant activity, glutathione reductase, and catalase exhibited an enhancement in serum or liver samples of fish fed P. rhodozyma compared to basal diet (P < 0.05). Protein content in fillet of fish fed P3 diet was higher than both basal and AX diets (P < 0.05), but no significant difference was seen in contents of lipid, moisture, ash and pH values either among the treatments or between treatments and basal diet (P > 0.05). Water holding capacity and lipid loss in fillets of treatments were lower than fish fed basal diet (P < 0.05). By increasing P. rhodozyma concentration in fish diets, redness value exhibited a progressive increase, and the highest value was seen in P3 diet compared to other treatments (P < 0.05). These data show application of P. rhodozyma at 47 g/kg diet in trout could provide a better performance, antioxidant activities, and fillet pigmentation.
•Growth and antioxidant capacity were notably improved in trout fed P. rhodozyma.•Dietary P. rhodozyma improved the fillet pigmentation and quality effectively.•Inclusion of 47 g P. rhodozyma /kg diet exhibited a better effect than lower concentrations.•Inclusion of 47 g P. rhodozyma /kg diet was comparable with synthetic astaxanthin in trout diet.
Probiotics provided from hatch have a major influence on microbiota development, and together with environmental and bedding microbiota, shape the microbial community of the litter. We investigated ...the influence of probiotic supplementation and a leaky gut challenge induced using dexamethasone (DEX) on the litter microbial community and litter parameters. The probiotic product was a mix of three Bacillus amyloliquefaciens strains. The litter microbiota were compared to the microbial communities from other gut sections. The litter samples had higher microbial diversity compared to the caecum, gizzard, jejunum, and jejunal mucosa. The high similarity between the litter phylum-level microbiota and gizzard microbiota detected in our study could be a consequence of ingested feed and litter passing through the gizzard. Moreover, the litter microbial community is fundamentally distinct from the intestinal microbiota, as evidenced by the number of genera present in the litter but absent from all the intestinal sections and vice versa. Furthermore, LEfSe analysis identified distinct microbial taxa across different groups, with specific genera associated with different treatments. In terms of litter quality, the birds in the DEX groups had a significantly higher moisture content, indicating successful leaky gut challenge, while probiotic supplementation did not significantly affect the moisture levels. These findings provide comprehensive insights into the distinct microbiota characteristics of litter.
The supplementation of antimicrobial growth promoters (AGPs) has been banned in many countries because of the emergence of antimicrobial-resistant pathogens in poultry products and the environment. ...Probiotics have been broadly studied and demonstrated as a promising AGP substitute. Our study is centred on the effects of a multi-strain
-based probiotic product on broiler production performance and gut microbial profile in a dexamethasone-induced leaky gut challenge. Two hundred and fifty-six broiler chicks were hatched and randomly assigned into four groups (wheat-soybean meal basal diet (BD) = non-supplemented control (C), BD supplemented with dexamethasone in week 4 (CD), BD containing a probiotic from day one (P), and BD containing a probiotic from day one and supplemented with dexamethasone during challenge week 4 (PD)). The production performance and caecal, gizzard, jejunal lumen and jejunal mucosa swab microbiota were studied by 16S rRNA gene sequencing. The
probiotic product significantly improved production performance and altered caecal gut microbiota (
≤ 0.05), but no significant impact on microbiota was observed in other gut sections.
Astaxanthin (AX) is a potent antioxidant with increasing biotechnological and commercial potential as a feed supplement, and gives salmonids and crustaceans their attractive characteristic pink ...color. The red yeast Phaffia rhodozyma naturally produces AX as its main fermentation product but wild-type strains and those previously generated through classical random mutagenesis produce low yields of AX. Existing strains do not meet commercial economic requirements, fundamentally due to a lack of understanding of the underlying mechanisms and genotype-phenotype associations regarding AX production in P. rhodozyma. In the present study, screening of P. rhodozyma CBS 6938 mutant strains generated through chemical and ultra violet radiation mutagenesis delivered increased AX production yields that were then maximized using culture media optimization and fed-batch culture kinetic modeling. The whole genomes of the wild-type and eight increased production strains were sequenced to identify genomic changes. The selected strains produced 50-fold more AX than the wild-type strain with a total biomass of around 100 gDCW/L and a carotenoid production of 1 g/L. Genomic variant analyses found 368 conserved mutations across the selected strains with important mutations found in protein coding regions associated with regulators and catalysts of AX precursors in the mevalonate pathway, the electron transport chain, oxidative stress mechanisms, and carotenogenesis.
•Random mutagenesis to improve astaxanthin production in Phaffia rhodozyma (also known as Xanthophyllomyces dendrorhous).•Kinetic fed-batch modeling for high cell densities fermentations to produce astaxanthin in Phaffia rhodozyma.•Next generation sequencing for whole genome sequencing and variant analyses of superior strains of Phaffia rhodozyma.•Culture media optimization and surface response to maximize astaxanthin production in Phaffia rhodozyma.
Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to ...withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market.
In this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in k
and K
for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme.
Overall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes.
The supplementation of antimicrobial growth promoters (AGPs) has been banned in many countries because of the emergence of antimicrobial-resistant pathogens in poultry products and the environment. ...Probiotics have been broadly studied and demonstrated as a promising AGP substitute. Our study is centred on the effects of a multi-strain Bacillus-based probiotic product on broiler production performance and gut microbial profile in a dexamethasone-induced leaky gut challenge. Two hundred and fifty-six broiler chicks were hatched and randomly assigned into four groups (wheat-soybean meal basal diet (BD) = non-supplemented control (C), BD supplemented with dexamethasone in week 4 (CD), BD containing a probiotic from day one (P), and BD containing a probiotic from day one and supplemented with dexamethasone during challenge week 4 (PD)). The production performance and caecal, gizzard, jejunal lumen and jejunal mucosa swab microbiota were studied by 16S rRNA gene sequencing. The Bacillus probiotic product significantly improved production performance and altered caecal gut microbiota (p ≤ 0.05), but no significant impact on microbiota was observed in other gut sections.
Considerable research has been conducted into the efficacy of individual probiotics in broiler production, however information on the most effective combinations of synergistic Bacillus probiotic is ...lacking. This study investigated the impact of different Bacillus strain combinations in broiler chickens, as well as in vitro enzyme production. In Exp. 1, a total of 576 one-day-old ROSS308 broilers were grown for 21 d across 6 treatments of maize-soybean diets (n = 12 pens per treatment) to compare three different strain combinations (formulation 1 F1: 3 strains Bacillus amyloliquefaciens, F2: Bacillus coagulans and 2 strains B. amyloliquefaciens, F3: B. coagulans, Bacillus licheniformis and 2 strains B. amyloliquefaciens, F5: Bacillus subtilis, B. licheniformis and 2 strains B. amyloliquefaciens), positive control (PC), and a negative control antibiotic treatment group (NC). In Exp. 2, a total of 360 one-day-old ROSS308 broilers were used to test five treatments (n = 9) including PC, NC, F1 and F5 (selected from Exp. 1), and F4 (Bacillus pumilis and 2 strains B. amyloliquefaciens) in a maize-soybean diet. B. amyloliquefaciens F1 demonstrated a significant improvement in feed conversion ratio (FCR) compared to F2 at d 14 (1.49 vs 2.10; P = 0.038) and the body weight (BW) at d 21 (847.0 g vs 787.4g) compared to other combinations (P = 0.027). The FCR at d 21 tended to be lower in birds fed F1 (1.46 vs 1.66) compared to the control (P = 0.068). Probiotic treatments had significantly improved nutrient digestibility compared to the PC and NC. Also, probiotic treatments supported the growth of Streptococcus, a common commensal genus and reduced the abundance of genera that correlated with low weight gain such as Akkermansia. Experiment two revealed that F4 improved FCR (P < 0.001) and BW at 28 d (P = 0.014). In vitro testing showed a high production of protease and amylase by Bacillus. Thus, the addition of Bacillus probiotics, particularly containing B. amyloliquefaciens strains and Bacillus pumilus, into the diet of broiler chickens improves production performance, nutrient digestibility, and allows the proliferation of beneficial gut microbiota.
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