Recent developments in antimicrobial growth promoters in chicken health: Opportunities and challenges.

With a continuously increasing human population is an increasing global demand for food. People in countries with a higher socioeconomic status tend to switch their preferences from grains to meat and high-value foods. Their preference for chicken as a source of protein has grown by 70% over the last three decades. Many studies have shown the role of feed in regulating the animal gut microbiome and its impact on host health. The microbiome absorbs nutrients, digests foods, induces a mucosal immune response, maintains homeostasis, and regulates bioactive metabolites. These metabolic activities are influenced by the microbiota and diet. An imbalance in microbiota affects host physiology and progressively causes disorders and diseases. With the use of antibiotics, a shift from dysbiosis with a higher density of pathogens to homeostasis can occur. However, the progressive use of higher doses of antibiotics proved harmful and resulted in the emergence of multidrug-resistant microbes. As a result, the use of antibiotics as feed additives has been banned. Researchers, regulatory authorities, and managers in the poultry industry have assessed the challenges associated with these restrictions. Research has sought to identify alternatives to antibiotic growth promoters for poultry that do not have any adverse effects. Modulating the host intestinal microbiome by regulating dietary factors is much easier than manipulating host genetics. Research efforts have led to the identification of feed additives, including bacteriocins, immunostimulants, organic acids, phytogenics, prebiotics, probiotics, phytoncides, and bacteriophages. In contrast to focusing on one or more of these alternative bioadditives, an improved feed conversion ratio with enhanced poultry products is possible by employing a combination of feed additives. This article may be helpful in future research towards developing a sustainable poultry industry through the use of the proposed alternatives.

A safe and sustainable bacterial cellulose nanofiber separator for lithium rechargeable batteries.

Bacterial cellulose nanofiber (BCNF) with high thermal stability produced by an ecofriendly process has emerged as a promising solution to realize safe and sustainable materials in the large-scale battery. However, an understanding of the actual thermal behavior of the BCNF in the full-cell battery has been lacking, and the yield is still limited for commercialization. Here, we report the entire process of BCNF production and battery manufacture. We systematically constructed a strain with the highest yield (31.5%) by increasing metabolic flux and improved safety by introducing a Lewis base to overcome thermochemical degradation in the battery. This report will open ways of exploiting the BCNF as a "single-layer" separator, a good alternative to the existing chemical-derived one, and thus can greatly contribute to solving the environmental and safety issues.

Genomic and metabolic analysis of Komagataeibacter xylinus DSM 2325 producing bacterial cellulose nanofiber.

Bacterial cellulose nanofiber (CNF) is a polymer with a wide range of potential industrial applications. Several Komagataeibacter species, including Komagataeibacter xylinus as a model organism, produce CNF. However, the industrial application of CNF has been hampered by inefficient CNF production, necessitating metabolic engineering for the enhanced CNF production. Here, we present complete genome sequence and a genome-scale metabolic model KxyMBEL1810 of K. xylinus DSM 2325 for metabolic engineering applications. Genome analysis of this bacterium revealed that a set of genes associated with CNF biosynthesis and regulation were present in this bacterium, which were also conserved in another six representative Komagataeibacter species having complete genome information. To better understand the metabolic characteristics of K. xylinus DSM 2325, KxyMBEL1810 was reconstructed using genome annotation data, relevant computational resources and experimental growth data generated in this study. Random sampling and correlation analysis of the KxyMBEL1810 predicted pgi and gnd genes as novel overexpression targets for the enhanced CNF production. Among engineered K. xylinus strains individually overexpressing heterologous pgi and gnd genes, either from Escherichia coli or Corynebacterium glutamicum, batch fermentation of a strain overexpressing the E. coli pgi gene produced 3.15 g/L of CNF in a complex medium containing glucose, which was the best CNF concentration achieved in this study, and 115.8% higher than that (1.46 g/L) obtained from the control strain. Genome sequence data and KxyMBEL1810 generated in this study should be useful resources for metabolic engineering of K. xylinus for the enhanced CNF production.

Construction of genetically engineered Candida tropicalis for conversion of l-arabinose to l-ribulose.

For the biological production of l-ribulose, conversion by enzymes or resting cells has been investigated. However, expensive or concentrated substrates, an additional purification step to remove borate and the requirement for cell cultivation and harvest steps before utilization of resting cells make the production process complex and unfavorable. Microbial fermentation may help overcome these limitations. In this study, we constructed a genetically engineered Candida tropicalis strain to produce l-ribulose by fermentation with a glucose/l-arabinose mixture. For the uptake of l-arabinose as a substrate and conversion of l-arabinose to l-ribulose, two heterologous genes coding for l-arabinose transporter and l-arabinose isomerase, were constitutively expressed in C. tropicalis under the GAPDH promoter. The Arabidopsis thaliana-originated l-arabinose transporter gene (STP2)-expressing strain exhibited a high l-arabinose uptake rate of 0.103 g/g cell/h and the expression of l-arabinose isomerase from Lactobacillus sakei 23 K showed 30% of conversion (9 g/L) from 30 g/L of l-arabinose. This genetically engineered strain can be used for l-ribulose production by fermentation using mixed sugars of glucose and l-arabinose.

Metabolic engineering of Pichia pastoris for production of hyaluronic acid with high molecular weight.

The high molecular weight (>1 MDa) of hyaluronic acid (HA) is important for its biological functions. The reported limiting factors for the production of HA with high molecular weight (MW) by microbial fermentation are the insufficient HA precursor pool and cell growth inhibition. To overcome these issues, the Xenopus laevis xhasA2 and xhasB genes encoding hyaluronan synthase 2 (xhasA2) and UDP-glucose dehydrogenase (xhasB), were expressed in Pichia pastoris widely used for production of heterologous proteins. In this study, expression vectors containing various combination cassettes of HA pathway genes including xhasA2 and xhasB from X. laevis as well as UDP-glucose pyrophosphorylase (hasC), UDP-N-acetylglucosamine pyrophosphorylase (hasD) and phosphoglucose isomerase (hasE) from P. pastoris were constructed and tested. First, HA pathway genes were overexpressed using pAO815 and pGAPZB vectors, resulting in the production of 1.2 MDa HA polymers. Second, in order to decrease hyaluronan synthase expression a strong AOX1 promoter in the xhasA2 gene was replaced by a weak AOX2 promoter which increased the mean MW of HA to 2.1 MDa. Finally, the MW of HA polymer was further increased to 2.5 MDa by low-temperature cultivation (26 °C) which reduced cell growth inhibition. The yield of HA production by the P. pastoris recombinant strains in 1L of fermentation culture was 0.8-1.7 g/L.

Effect of heterologous xylose transporter expression in Candida tropicalis on xylitol production rate.

Xylose utilization is inhibited by glucose uptake in xylose-assimilating yeasts, including Candida tropicalis, resulting in limitation of xylose uptake during the fermentation of glucose/xylose mixtures. In this study, a heterologous xylose transporter gene (At5g17010) from Arabidopsis thaliana was selected because of its high affinity for xylose and was codon-optimized for functional expression in C. tropicalis. The codon-optimized gene was placed under the control of the GAPDH promoter and was integrated into the genome of C. tropicalis strain LXU1 which is xyl2-disrupted and NXRG (codon-optimized Neurospora crassa xylose reductase) introduced. The xylose uptake rate was increased by 37-73 % in the transporter expression-enhanced strains depending on the glucose/xylose mixture ratio. The recombinant strain LXT2 in 500-mL flask culture using glucose/xylose mixtures showed a xylose uptake rate that was 29 % higher and a xylitol volumetric productivity (1.14 g/L/h) that was 25 % higher than the corresponding rates for control strain LXU1. Membrane protein extraction and Western blot analysis confirmed the successful heterologous expression and membrane localization of the xylose transporter in C. tropicalis.

Enhancement of xylitol production in glycerol kinase disrupted Candida tropicalis by co-expression of three genes involved in glycerol metabolic pathway.

Glycerol can be used as a primary carbon source by yeasts, little is known regarding glycerol metabolism in Candida tropicalis. In this study, glycerol kinase gene (gk) was disrupted from xylitol dehydrogenase gene (XYL2) knockout C. tropicalis strain BSXDH-3. The resultant gk knockout C. tropicalis strain was incapable to grow on glycerol. The cells growth on glycerol was resumed by co-expressing Scheffersomyces stipitis gcy1, 2 and 3 genes, which respectively encode NADP(+)-dependent glycerol dehydrogenase 1, 2 and 3, under the control of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. NADPH-dependent xylitol production was higher in the engineered strain, termed "GK", than in BSXDH-3. In fermentation experiments using glycerol as co-substrate with xylose, strain GK produced xylitol 0.85 and 1.28 g l(-1) h(-1) at the time periods of 16 and 24 h, respectively, which is 30 and 18 % higher at same time intervals in BSXDH-3. This is the first report of gk gene disruption and co-expression of gcy1, 2 and 3 genes for NADPH regeneration and enhanced xylitol production in C. tropicalis.

Enhancement of xylitol production in Candida tropicalis by co-expression of two genes involved in pentose phosphate pathway.

The yeast Candida tropicalis produces xylitol, a natural, low-calorie sweetener whose metabolism does not require insulin, by catalytic activity of NADPH-dependent xylose reductase. The oxidative pentose phosphate pathway (PPP) is a major basis for NADPH biosynthesis in C. tropicalis. In order to increase xylitol production rate, xylitol dehydrogenase gene (XYL2)disrupted C. tropicalis strain BSXDH-3 was engineered to co-express zwf and gnd genes which, respectively encodes glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6-PGDH), under the control of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. NADPH-dependent xylitol production was higher in the engineered strain, termed "PP", than in BSXDH-3. In fermentation experiments using glycerol as a co-substrate with xylose, strain PP showed volumetric xylitol productivity of 1.25 g l(-1) h(-1), 21% higher than the rate (1.04 g l(-1) h(-1)) in BSXDH-3. This is the first report of increased metabolic flux toward PPP in C. tropicalis for NADPH regeneration and enhanced xylitol production.

Xylitol production is increased by expression of codon-optimized Neurospora crassa xylose reductase gene in Candida tropicalis.

Xylose reductase (XR) is the first enzyme in D: -xylose metabolism, catalyzing the reduction of D: -xylose to xylitol. Formation of XR in the yeast Candida tropicalis is significantly repressed in cells grown on medium that contains glucose as carbon and energy source, because of the repressive effect of glucose. This is one reason why glucose is not a suitable co-substrate for cell growth in industrial xylitol production. XR from the ascomycete Neurospora crassa (NcXR) has high catalytic efficiency; however, NcXR is not expressed in C. tropicalis because of difference in codon usage between the two species. In this study, NcXR codons were changed to those preferred in C. tropicalis. This codon-optimized NcXR gene (termed NXRG) was placed under control of a constitutive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter derived from C. tropicalis, and integrated into the genome of xylitol dehydrogenase gene (XYL2)-disrupted C. tropicalis. High expression level of NXRG was confirmed by determining XR activity in cells grown on glucose medium. The resulting recombinant strain, LNG2, showed high XR activity (2.86 U (mg of protein)(-1)), whereas parent strain BSXDH-3 showed no activity. In xylitol fermentation using glucose as a co-substrate with xylose, LNG2 showed xylitol production rate 1.44 g L(-1) h(-1) and xylitol yield of 96% at 44 h, which were 73 and 62%, respectively, higher than corresponding values for BSXDH-3 (rate 0.83 g L(-1) h(-1); yield 59%).

L-arabinose pathway engineering for arabitol-free xylitol production in Candida tropicalis.

Xylose reductase (XR) is a key enzyme in biological xylitol production, and most XRs have broad substrate specificities. During xylitol production from biomass hydrolysate, non-specific XRs can reduce L-arabinose, which is the second-most abundant hemicellulosic sugar, to the undesirable byproduct arabitol, which interferes with xylitol crystallization in downstream processing. To minimize the flux from L-arabinose to arabitol, the L-arabinose-preferring, endogenous XR was replaced by a D-xylose-preferring heterologous XR in Candida tropicalis. Then, Bacillus licheniformis araA and Escherichia coli araB and araD were codon-optimized and expressed functionally in C. tropicalis for the efficient assimilation of L-arabinose. During xylitol fermentation, the control strains BSXDH-3 and KNV converted 9.9 g L-arabinose l(-1) into 9.5 and 8.3 g arabitol l(-1), respectively, whereas the recombinant strain JY consumed 10.5 g L-arabinose l(-1) for cell growth without forming arabitol. Moreover, JY produced xylitol with 42 and 16% higher productivity than BSXDH-3 and KNV, respectively.