D-lactic Production Research Articles

Paradigm of engineering recalcitrant non-model microorganism with dominant metabolic pathway as a biorefinery chassis

The development and implementation of microbial chassis cells have profound impacts on circular economy. Non-model bacterium Zymomonas mobilis is an excellent chassis owing to its extraordinary industrial characteristics. Here, the genome-scale metabolic model iZM516 is improved and updated by integrating enzyme constraints to simulate the dynamics of flux distribution and guide pathway design. We show that the innate dominant ethanol pathway of Z. mobilis restricts the titer and rate of these biochemicals. A dominant-metabolism compromised intermediate-chassis (DMCI) strategy is then developed through introducing low toxicity but cofactor imbalanced 2,3-butanediol pathway, and a recombinant D-lactate producer is constructed to produce more than 140.92 g/L and 104.6 g/L D-lactate (yield > 0.97 g/g) from glucose and corncob residue hydrolysate, respectively. Additionally, techno-economic analysis (TEA) and life cycle assessment (LCA) demonstrate the commercialization feasibility and greenhouse gas reduction capability of lignocellulosic D-lactate. This work thus establishes a paradigm for engineering recalcitrant microorganisms as biorefinery chassis.

Assessment of the safety and hepatic lipid-lowering effects of Lactobacillus delbrueckii subsp. lactis CKDB001

Probiotics have been shown to provide health benefits for several metabolic diseases, including obesity, type 2 diabetes, and metabolic dysfunction-associated steatotic liver disease (MASLD), by modulating the gut microbiota. In this study, we evaluated the safety and efficacy of Lactobacillus delbrueckii subsp. lactis CKDB001 as a potential therapeutic candidate for the treatment of MASLD. We evaluated antibiotic resistance, hemolytic, gelatinase, and bile salt hydrolase activities, and the production of biogenic amines and D-lactate using in vitro analyses. We found that L. lactis CKDB001 treatment resulted in significant anti-adipogenic properties in the HepG2 cell line, reducing lipid accumulation and improving lipid profiles through mechanisms involving the upregulation of SIRT1 and PPARα, and downregulation of CD36 and ELOVL6. These results suggest that L. lactis CKDB001 is a safe and effective probiotic for managing MASLD. Further in vivo studies and clinical trials are required to validate these effects and fully elucidate their therapeutic potential and safety profiles.

Probiotic and Postbiotic Potentials of Enterococcus faecalis EF-2001: A Safety Assessment.

Probiotics, which are live microorganisms that, when given in sufficient quantities, promote the host's health, have drawn a lot of interest for their ability to enhance gut health. Enterococcus faecalis, a member of the human gut microbiota, has shown promise as a probiotic candidate due to its functional attributes. However, safety concerns associated with certain strains warrant comprehensive evaluation before therapeutic application. In this study, E. faecalis EF-2001, originally isolated from fecal samples of a healthy human infant, was subjected to a multi-faceted assessment for its safety and probiotic potential. In silico analysis, CAZyme, biosynthetic, and stress-responsive proteins were identified. The genome lacked biogenic amine genes but contained some essential amino acid and vitamin synthetic genes, and carbohydrate-related enzymes essential for probiotic properties. The negligible difference of 0.03% between the 1st and 25th generations indicates that the genetic information of the E. faecalis EF-2001 genome remained stable. The live E. faecalis EF-2001 (E. faecalis EF-2001L) demonstrated low or no virulence potential, minimal D-Lactate production, and susceptibility to most antibiotics except some aminoglycosides. No bile salt deconjugation or biogenic amine production was observed in an in vitro assay. Hemolytic activity assessment showed a β-hemolytic pattern, indicating no red blood cell lysis. Furthermore, the EF-2001L did not produce gelatinase and tolerated simulated gastric and intestinal fluids in an in vitro study. Similarly, heat-killed E. faecalis EF-2001 (E. faecalis EF-2001HK) exhibits tolerance in both acid and base conditions in vitro. Further, no cytotoxicity of postbiotic EF-2001HK was observed in human colorectal adenocarcinoma HT-29 cells. These potential properties suggest that probiotic and postbiotic E. faecalis EF-2001 could be considered safe and retain metabolic activity suitable for human consumption.

Characterization of Human Breast Milk-Derived Limosilactobacillus reuteri MBHC 10138 with Respect to Purine Degradation, Anti-Biofilm, and Anti-Lipid Accumulation Activities.

Background: Human breast milk is a valuable source of potential probiotic candidates. The bacteria isolated from human breast milk play an important role in the development of the infant gut microbiota, exhibiting diverse biological functions. Methods: In this study, Limosilactobacillus reuteri MBHC 10138 isolated from breast milk was characterized in terms of its probiotic safety characteristics and potential efficacy in hyperuricemia, obesity, lipid liver, and dental caries, conditions which Korean consumers seek to manage using probiotics. Results: Strain MBHC 10138 demonstrated a lack of D-lactate and biogenic amine production as well as a lack of bile salt deconjugation and hemolytic activity. It also exhibited susceptibility to common antibiotics, tolerance to simulated oral-gastric-intestinal conditions, and superior biological activity compared to three L. reuteri reference strains, including KACC 11452 and MJ-1, isolated from feces, and a commercial strain isolated from human breast milk. Notably, L. reuteri MBHC 10138 showed high capabilities in assimilating guanosine (69.48%), inosine (81.92%), and adenosine (95.8%), strongly inhibited 92.74% of biofilm formation by Streptococcus mutans, and reduced lipid accumulation by 32% in HepG2 cells. Conclusions: These findings suggest that strain MBHC 10138, isolated from human breast milk, has potential to be developed as a probiotic for managing hyperuricemia, obesity, and dental caries after appropriate in vivo studies.

A new Zymomonas mobilis platform strain for the efficient production of chemicals

BackgroundZymomonas mobilis is well known for its outstanding ability to produce ethanol with both high specific productivity and with high yield close to the theoretical maximum. The key enzyme in the ethanol production pathway is the pyruvate decarboxylase (PDC) which is converting pyruvate to acetaldehyde. Since it is widely considered that its gene pdc is essential, metabolic engineering strategies aiming to produce other compounds derived from pyruvate need to find ways to reduce PDC activity.ResultsHere, we present a new platform strain (sGB027) of Z. mobilis in which the native promoter of pdc was replaced with the IPTG-inducible PT7A1, allowing for a controllable expression of pdc. Expression of lactate dehydrogenase from E. coli in sGB027 allowed the production of D-lactate with, to the best of our knowledge, the highest reported specific productivity of any microbial lactate producer as well as with the highest reported lactate yield for Z. mobilis so far. Additionally, by expressing the L-alanine dehydrogenase of Geobacillus stearothermophilus in sGB027 we produced L-alanine, further demonstrating the potential of sGB027 as a base for the production of compounds other than ethanol.ConclusionWe demonstrated that our new platform strain can be an excellent starting point for the efficient production of various compounds derived from pyruvate with Z. mobilis and can thus enhance the establishment of this organism as a workhorse for biotechnological production processes.

Biosensor-assisted CRISPRi high-throughput screening to identify genetic targets in Zymomonas mobilis for high d-lactate production

Lactate is an important monomer for the synthesis of poly-lactate (PLA), which is a substitute for the petrochemical plastics. To achieve the goal of high lactate titer, rate, and yield for commercial production, efficient lactate production pathway is needed as well as genetic targets that affect high lactate production and tolerance. In this study, an LldR-based d-lactate biosensor with a broad dynamic range was first applied into Zymomonas mobilis to select mutant strains with strong GFP fluorescence, which could be the mutant strains with increased d-lactate production. Then, LldR-based d-lactate biosensor was combined with a genome-wide CRISPR interference (CRISPRi) library targeting the entire genome to generate thousands of mutants with gRNA targeting different genetic targets across the whole genome. Specifically, two mutant libraries were selected containing 105 and 104 mutants with different interference sites from two rounds of fluorescence-activated cell sorting (FACS), respectively. Two genetic targets of ZMO1323 and ZMO1530 were characterized and confirmed to be associated with the increased d-lactate production, further knockout of ZMO1323 and ZMO1530 resulted in a 15% and 21% increase of d-lactate production, respectively. This work thus not only established a high-throughput approach that combines genome-scale CRISPRi and biosensor-assisted screening to identify genetic targets associated with d-lactate production in Z. mobilis, but also provided a feasible high-throughput screening approach for rapid identification of genetic targets associated with strain performance for other industrial microorganisms.

In vitro evaluation of microbial D- and L-lactate production as biomarkers of infection.

Mammalian cells produce and metabolize almost exclusively L-lactate, bacterial species have the capacity to produce both D-lactate and L-lactate. The aim of this study was to evaluate the intrinsic production of D- and L-lactate in the most common pathogenic microorganisms causing septic arthritis (SA) and periprosthetic joint infection (PJI) as a potential biomarker for the diagnosis of infection. Following microorganisms were grown according to ATCC culture guides and tested for production of D- and L-lactate: Staphylococcus aureus (ATCC 43300), Staphylococcus epidermidis (ATCC 35984), Enterococcus faecalis (ATCC 19433), Streptococcus pyogenes (ATCC 19615), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Cutibacterium acnes (ATCC 11827), and Candida albicans (ATCC 90028). Pathogens were inoculated in 8 ml of appropriate liquid media and incubated as planktonic or biofilm form in either aerobic, anaerobic or CO2 atmosphere up to 312 h. D- and L-lactate measurements were performed at different time points: 0, 6, 9, 12 and 24 h, then once per day for slow-growing pathogens. Samples were serially diluted and plated for colony counting. Liquid culture media without microorganisms served as a negative control. Production of D-lactate was observed in all tested microorganisms, whereas no L-lactate was detected in E. coli, P. aeruginosa, and C. albicans. Maximal concentration of D-lactate was produced by S. aureus (10.99 mmol/L), followed by E. coli (1.22 mmol/L), and S. epidermidis (0.48 mmol/L). Maximal L-lactate concentration was observed in S. pyogenes (10.12 mmol/L), followed by S. aureus (9.71 mmol/L), E. faecalis (2.64 mmol/L), and S. epidermidis (2.50 mmol/L). S. epidermidis bacterial biofilm produced significantly higher amount of D- and L-lactate compared to planktonic form (p = 0.015 and p = 0.002, respectively). Our study has demonstrated that the most common pathogenic microorganisms causing SA and PJI have the capability to generate measurable amounts of D-lactate in both planktonic and biofilm form, highlighting the practical value of this biomarker as an indicator for bacterial and fungal infections. In contrast to D-lactate, the absence of L-lactate production in certain tested bacteria, as well as in fungi, suggests that L-lactate is not eligible as a biomarker for diagnosing microbial infections.

Exclusive D-lactate-isomer production during a reactor-microbiome conversion of lactose-rich waste by controlling pH and temperature

Lactate is among the top-ten-biobased products. It occurs naturally as D- or L-isomer and as a racemic mixture (DL-lactate). Generally, lactate with a high optical purity is more valuable. In searching for suitable renewable feedstocks for lactate production, unutilized organic waste streams are increasingly coming into focus. Here, we investigated acid whey, which is a lactose-rich byproduct of yogurt production, that represents a considerable environmental footprint for the dairy industry. We investigated the steering of the lactate-isomer composition in a continuous and open culture system (HRT = 0.6 d) at different pH values (pH 5.0 vs. pH 6.5) and process temperatures (38°C to 50°C). The process startup was achieved by autoinoculation. At a pH of 5.0 and a temperature of 47°C-50°C, exclusive D-lactate production occurred because of the dominance of Lactobacillus spp. (> 95% of relative abundance). The highest volumetric D-lactate production rate of 722 ± 94.6 mmol C L−1 d−1 (0.90 ± 0.12 g L−1 h−1), yielding 0.93 ± 0.15 mmol C mmol C−1, was achieved at a pH of 5.0 and a temperature of 44°C (n = 18). At a pH of 6.5 and a temperature of 44°C, we found a mixture of DL-lactate (average D-to-L-lactate production rate ratio of 1.69 ± 0.90), which correlated with a high abundance of Streptococcus spp. and Enterococcus spp. However, exclusive L-lactate production could not be achieved. Our results show that for the continuous conversion of lactose-rich dairy waste streams, the pH was a critical process parameter to control the yield of lactate isomers by influencing the composition of the microbiota. In contrast, temperature adjustments allowed the improvement of bioprocess kinetics.

Genome engineering of Kluyveromyces marxianus for high D-( -)-lactic acid production under low pH conditions.

Saccharomyces cerevisiae is the workhorse of fermentation industry. Upon engineering for D-lactate production by a series of gene deletions, this yeast had deficiencies in cell growth and D-lactate production at high substrate concentrations. Complex nutrients or high cell density were thus required to support growth and D-lactate production with a potential to increase medium and process cost of industrial-scale D-lactate production. As an alternative microbial biocatalyst, a Crabtree-negative and thermotolerant yeast Kluyveromyces marxianus was engineered in this study to produce high titer and yield of D-lactate at a lower pH without growth defects. Only pyruvate decarboxylase 1 (PDC1) gene was replaced by a codon-optimized bacterial D-lactate dehydrogenase (ldhA). Ethanol, glycerol, or acetic acid was not produced by the resulting strain, KMΔpdc1::ldhA. Aeration rate at 1.5 vvm and culture pH 5.0 at 30°C provided the highest D-lactate titer of 42.97 ± 0.48g/L from glucose. Yield and productivity of D-lactate, and glucose-consumption rate were 0.85 ± 0.01g/g, 0.90 ± 0.01g/(L·h), and 1.06 ± 0.00g/(L·h), respectively. Surprisingly, D-lactate titer, productivity, and glucose-consumption rate of 52.29 ± 0.68g/L, 1.38 ± 0.05g/(L·h), and 1.22 ± 0.00g/(L·h), respectively, were higher at 42°C compared to 30°C. Sugarcane molasses, a low-value carbon, led to the highest D-lactate titer and yield of 66.26 ± 0.81g/L and 0.91 ± 0.01g/g, respectively, in a medium without additional nutrients. This study is a pioneer work of engineering K. marxianus to produce D-lactate at the yield approaching theoretical maximum using simple batch process. Our results support the potential of an engineered K. marxianus for D-lactate production on an industrial scale. KEY POINTS: • K. marxianus was engineered by deleting PDC1 and expressing codon-optimized D-ldhA. • The strain allowed high D-lactate titer and yield under pH ranging from 3.5 to 5.0. • The strain produced 66g/L D-lactate at 30°C from molasses without any additional nutrients.

D-Lactate: Implications for Gastrointestinal Diseases.

D-lactate is produced in very low amounts in human tissues. However, certain bacteria in the human intestine produce D-lactate. In some gastrointestinal diseases, increased bacterial D-lactate production and uptake from the gut into the bloodstream take place. In its extreme, excessive accumulation of D-lactate in humans can lead to potentially life-threatening D-lactic acidosis. This metabolic phenomenon is well described in pediatric patients with short bowel syndrome. Less is known about a subclinical rise in D-lactate. We discuss in this review the pathophysiology of D-lactate in the human body. We cover D-lactic acidosis in patients with short bowel syndrome as well as subclinical elevations of D-lactate in other diseases affecting the gastrointestinal tract. Furthermore, we argue for the potential of D-lactate as a marker of intestinal barrier integrity in the context of dysbiosis. Subsequently, we conclude that there is a research need to establish D-lactate as a minimally invasive biomarker in gastrointestinal diseases.

Vascular traffic control of neutrophil recruitment to the liver by microbiota-endothelium crosstalk

During bloodstream infections, neutrophils home to the liver as part of an intravascular immune response to eradicate blood-borne pathogens, but the mechanisms regulating this crucial response are unknown. Using invivo imaging of neutrophil trafficking in germ-free and gnotobiotic mice, we demonstrate that the intestinal microbiota guides neutrophil homing to the liver in response to infection mediated by the microbial metabolite D-lactate. Commensal-derived D-lactate augments neutrophil adhesion in the liver independent of granulopoiesis in bone marrow or neutrophil maturation and activation in blood. Instead, gut-to-liver D-lactate signaling primes liver endothelial cells to upregulate adhesion molecule expression in response to infection and promote neutrophil adherence. Targeted correction of microbiota D-lactate production in a model of antibiotic-induced dysbiosis restores neutrophil homing to the liver and reduces bacteremia in a model of Staphylococcus aureus infection. These findings reveal long-distance traffic control of neutrophil recruitment to the liver by microbiota-endothelium crosstalk.

Immobilization of Lactobacillus bulgaricus with gellan gum and its application in continuous fermentation of D-lactic acid from corn straw

Biorefinery of chemicals from straw is an effective approach to alleviate the environmental pollution caused by straw burning. In this paper, we prepared gellan gum immobilized Lactobacillus bulgaricus T15 gel beads (LA-GAGR-T15 gel beads), characterized their properties, and established a continuous cell recycle fermentation process for D-lactate (D-LA) production using the LA-GAGR-T15 gel beads. The fracture stress of LA-GAGR-T15 gel beads was (91.68±0.11) kPa, which was 125.12% higher than that of the calcium alginate immobilized T15 gel beads (calcium alginate-T15 gel beads). This indicated that the strength of LA-GAGR-T15 gel beads was stronger, and the strain was less likely to leak out. The average D-LA production was (72.90±2.79) g/L after fermentation for ten recycles (720 h) using LA-GAGR-T15 gel beads as the starting strain and glucose as the substrate, which was 33.85% higher than that of calcium alginate-T15 gel beads and 37.70% higher than that of free T15. Subsequently, glucose was replaced by enzymatically hydrolyzed corn straw and fermented for ten recycles (240 h) using LA-GAGR-T15 gel beads. The yield of D-LA reached (1.74±0.79) g/(L·h), which was much higher than that of using free bacteria. The wear rate of gel beads was less than 5% after ten recycles, which indicated that LA-GAGR is a good carrier for cell immobilization and can be widely used in industrial fermentation. This study provides basic data for the industrial production of D-LA using cell-recycled fermentation, and provides a new way for the biorefinery of D-LA from corn straw.

Potential oral probiotic Lactobacillus pentosus MJM60383 inhibits Streptococcus mutans biofilm formation by inhibiting sucrose decomposition

ABSTRACT Streptococcus mutans is known as a contributor to dental caries. In this work, Lactobacillus pentosus MJM60383 was selected for its strong antagonistic activity against S. mutans and was characterized by good oral probiotic properties including lysozyme tolerance, adhesive ability to oral cells, good aggregation (auto-aggregation, co-aggregation) ability, hydrogen peroxide production and inhibition of biofilm formation of S. mutans. L. pentosus MJM60383 also exhibited safety as a probiotic characterized by no hemolytic activity, no D-lactate production, no biogenic amine production, and susceptibility to antibiotics. Furthermore, the biofilm formation of S. mutans was also significantly inhibited by the supernatant of L. pentosus MJM60383. An anti-biofilm mechanism study revealed that sucrose decomposition and the production of water-insoluble exopolysaccharides by S. mutans were inhibited by the treatment with L. pentosus MJM60383 supernatant. Real-time PCR analysis indicated that the supernatant of L. pentosus MJM60383 significantly inhibited the mRNA expression of S. mutans glycosyltransferases, which synthesize glucan to construct biofilm architecture and mediate bacterial adherence. Our study demonstrated L. pentosus MJM60383 as a potential oral probiotic and revealed its anti-biofilm mechanism.

In vitro assessment of probiotic attributes for strains contained in commercial formulations

Although probiotics are often indiscriminately prescribed, they are not equal and their effects on the host may profoundly differ. In vitro determination of the attributes of probiotics should be a primary concern and be performed even before clinical studies are designed. In fact, knowledge on the biological properties a microbe possesses is crucial for selecting the most suitable bacteriotherapy for each individual. Herein, nine strains (Bacillus clausii NR, OC, SIN, T, Bacillus coagulans ATCC 7050, Bifidobacterium breve DSM 16604, Limosilactobacillus reuteri DSM 17938, Lacticaseibacillus rhamnosus ATCC 53103, and Saccharomyces boulardii CNCM I-745) declared to be contained in six commercial formulations were tested for their ability to tolerate simulated intestinal conditions, adhere to mucins, and produce β-galactosidase, antioxidant enzymes, riboflavin, and d-lactate. With the exception of B. breve, all microbes survived in simulated intestinal fluid. L. rhamnosus was unable to adhere to mucins and differences in mucin adhesion were evidenced for L. reuteri and S. boulardii depending on oxygen levels. All microorganisms produced antioxidant enzymes, but only B. clausii, B. coagulans, B. breve, and L. reuteri synthesize β-galactosidase. Riboflavin secretion was observed for Bacillus species and L. rhamnosus, while d-lactate production was restricted to L. reuteri and L. rhamnosus. Our findings indicate that the analyzed strains possess different in vitro biological properties, thus highlighting the usefulness of in vitro tests as prelude for clinical research.

The effect of a high-protein and high-carbohydrate diet on the content of D-lactate in the blood plasma and intestines of a model organism – rainbow trout

D-lactic acid stereoisomer (D-lactate) is produced by the intestinal microflora and can enter the bloodstream and cause in some cases a condition of acute D-lactic acidemia known as short gut syndrome. The level of D-lactate in blood and in the contents of the intestine is considered as a promising marker of the development of inflammation associated with microflora disorders, as well as with the development of a bacterial infection, while the mechanism of its entry into the blood of vertebrates from the intestine has not been studied in detail.The aim of the study. To investigate the relationship between the level of D-lactate in blood and in the intestine, taking into account the permeability of the intestinal epithelium.Materials and methods. As a model object of the study, we used juvenile rainbow trout O. mykiss. For 54 days, they were high-carbohydrate or high-protein fed. Since different types of bacteria prefer different substrates, it was expected that at the end of the experiment, the composition of the intestinal microflora would be significantly different in fish fed with different diets. The content of D-lactate in blood plasma in vitro was assessed by the Larsen method with modifications; intestinal permeability was assessed by the intensity of fluorescence of the FITС-Dextran stain in the blood of fish. The analysis of the metagenome of samples of the contents and epithelium of the fore and hind intestine was carried out. The hematological profile was partially characterized using blood smears taken immediately after fish blood sampling. By the means of a different diet, it was possible to obtain two groups of fish that differ significantly in the permeability of the intestinal epithelium and in the content of D-lactate in the intestine. At the same time, despite the differences between the experimental groups in the content of D-lactate in the intestine and in intestinal permeability, no significant differences in D-lactate level in blood were found between them. Analysis of the composition of the intestinal microbiome by metabarcoding for the 16S rRNA gene revealed the absence of lactobacilli in the production of D-lactate in fish.Results. It was shown that the mechanism of accumulation of D-lactate in the blood plasma in fish is less associated with increased intestinal permeability or hyperproduction of this metabolite by the intestinal microflora and is more associated with the utilization of D-lactate in the body. In the experiment, it was not possible to achieve a significant change in the species composition of the intestinal microflora of trout under the influence of a highcarbohydrate diet for 54 days compared to fish that received high-protein diet. Some tendencies towards changes in the composition of the microflora were found in the contents of the hindgut, and perhaps with a longer exposure, these changes could reach a statistically significant level.

Identification of HAGHL as a novel metabolic oncogene regulating human colorectal cancer progression.

Cancer development remains the most challenging obstacle in colorectal cancer (CRC) treatment. The current study aims to identify and demonstrate novel oncogenes for CRC. The CRC data of the Cancer Genome Atlas database and the Gene Expression Omnibus database were subjected to bioinformatics analysis to identify the novel potential diagnostic and prognostic biomarkers for CRC. Immunohistochemical assay, western blot, and quantitative PCR (qPCR) were used to analyze hydroxyacylglutathione hydrolase-like (HAGHL) gene expression in CRC tissues and cultured CRC cells. D-Lactate colorimetric assay was applied to determine concentration of D-lactate in supernatants from CRC tissues and cell culture medium. Cell counting kit-8 (CCK-8) assay, flow cytometry, tumor xenografts experiment, and TUNEL staining analysis were performed to evaluate the function of HAGHL in CRC. We comprehensively analyzed the CRC data of the Cancer Genome Atlas database and the Gene Expression Omnibus database, and identified several novel potential diagnostic and prognostic biomarkers for CRC, including HAGHL, DNTTIP1, DHX34, and AP1S3. The expression of HAGHL, the strongest oncogenic activity gene, is positively related to D-lactate levels in CRC tissues and negatively associated with patient prognosis. HAGHL downregulation suppressed the production of D-lactate and induced apoptosis, resulting in inhibition of cell proliferation in vitro. In vivo experiment showed that knockdown of HAGHL induced cell apoptosis and inhibited tumor growth. These findings suggest that HAGHL acts as a novel metabolic oncogene and demonstrate the underlying mechanism by which HAGHL regulates CRC progression, highlighting its utility as a diagnostic and prognostic factor and as a potential therapeutic target for the treatment of CRC.

Galactitol Transport Factor GatA Relieves ATP Supply Restriction to Enhance Acid Tolerance of Escherichia coli in the Two-Stage Fermentation Production of D-Lactate

Escherichia coli is a major contributor to the industrial production of organic acids, but its production capacity and cost are limited by its acid sensitivity. Enhancing acid resistance in E. coli is essential for improving cell performance and production value. Here, we propose a feasible strategy for improving cellular acid tolerance by reducing ATP supply restriction. Transcriptome assays of acid-tolerant evolved strains revealed that the galactitol phosphotransferase system transporter protein GatA is an acid-tolerance factor that assists E. coli in improving its resistance to a variety of organic acids. Enhanced GatA expression increased cell survival under conditions of lethal stress due to D-lactic acid, itaconic acid and succinic acid by 101.8-fold, 29.4-fold and 41.6-fold, respectively. In addition, fermentation patterns for aerobic growth and oxygen-limited production of D-lactic acid were identified, and suitable transition and induction stages were evaluated. GatA effectively compensated for the lack of cellular energy during oxygen limitation and enabled the D-lactic acid producing strain to exhibit more sustainable productivity in acidic fermentation environments with a 55.7% increase in D-lactic acid titer from 9.5 g·L−1 to 14.8 g·L−1 and reduced generation of by-product. Thus, this study developed a method to improve the acid resistance of E. coli cells by compensating for the energy gap without affecting normal cell metabolism while reducing the cost of organic acid production.

Safety Evaluation by Phenotypic and Genomic Characterization of Four Lactobacilli Strains with Probiotic Properties.

Probiotic Lactobacillus species are known to exert health benefits in hosts when administered in adequate quantities. A systematic safety assessment of the strains must be performed before the Lactobacillus strains can be designated as probiotics for human consumption. In this study, we selected Lactobacillus fermentum IDCC 3901, L. gasseri IDCC 3101, L. helveticus IDCC 3801, and L. salivarius IDCC 3551 as representative Lactobacilli probiotic strains and investigated their probiotic properties and potential risks through phenotypic and genomic characterization. Various assays including antimicrobial resistance, biogenic amine production, L-/D-lactate production, acute oral toxicity, and antipathogenic effect were performed to evaluate the safety of the four Lactobacillus strains. Genomic analysis using whole genome sequencing was performed to investigate virulence and antibiotic resistance genes in the genomes of the selected probiotic strains. The phenotypes of the strains such as enzymatic activity and carbohydrate utilization were also investigated. As a result, antibiotic resistances of the four Lactobacillus species were detected; however, neither antibiotic resistance-related genes nor virulence genes were found by genomic analysis. Moreover, the four Lactobacillus species did not exhibit hemolytic activity or β-glucuronidase activity. The biogenic amine production and oral acute toxicity were not shown in the four Lactobacillus species, whereas they produced D-lactate with minor ratio. The four Lactobacillus species exhibited antipathogenic effect to five pathogenic microorganisms. This study provides a way to assess the potential risks of four different Lactobacillus species and validates the safety of all four strains as probiotics for human consumption.

Quantification of cytosol and membrane proteins in rumen epithelium of sheep with low or high CH4 emission phenotype.

BackgroundRuminant livestock are a major contributor to Australian agricultural sector carbon emissions. Variation in methane (CH4) produced from enteric microbial fermentation of feed in the reticulo-rumen of sheep differs with different digestive functions.MethodWe isolated rumen epithelium enzymatically to extract membrane and cytosol proteins from sheep with high (H) and low (L) CH4 emission. Protein abundance was quantified using SWATH-mass spectrometry.ResultsThe research found differences related to the metabolism of glucose, lactate and processes of cell defence against microbes in sheep from each phenotype. Enzymes in the methylglyoxal pathway, a side path of glycolysis, resulting in D-lactate production, differed in abundance. In the H CH4 rumen epithelium the enzyme hydroxyacylglutathione hydrolase (HAGH) was 2.56 fold higher in abundance, whereas in the L CH4 epithelium lactate dehydrogenase D (LDHD) was 1.93 fold higher. Malic enzyme 1 which converts D-lactate to pyruvate via the tricarboxylic cycle was 1.57 fold higher in the L CH4 phenotype. Other proteins that are known to regulate cell defence against microbes had differential abundance in the epithelium of each phenotype.ConclusionDifferences in the abundance of enzymes involved in the metabolism of glucose were associated with H and L CH4 phenotype sheep. Potentially this represents an opportunity to use protein markers in the rumen epithelium to select low CH4 emitting sheep.

Fermentation Quality of Silages Produced from Wilted Sown Tropical Perennial Grass Pastures with or without a Bacterial Inoculant

High growth rates and rapid reproductive development and associated decline in feed quality of sown tropical perennial grass pastures present management challenges for livestock producers. Conservation of surplus forage as silage could be an effective management tool. Experiments were conducted to evaluate the fermentation quality of silages produced from tropical grasses. Five species (Chloris gayana, Megathyrsus maximus, Panicum coloratum, Digitaria eriantha and Cenchrus clandestinus) were ensiled without additives after a short, effective wilt at dry matter (DM) contents ranging from 302.4 to 650.1 g kg−1. The fermentation profile of all silages in 2019 was typical for high DM silages, but in 2020 ammonia (% of total nitrogen: NH3-N), acetic acid and pH levels were higher. In 2020 M. maximus (302.4 g kg−1 DM) was poorly preserved with 20.2% NH3-N. The DM content of all other silages exceeded 350 g kg−1 and fermentation quality was generally good. In a second experiment, M. maximus was ensiled at 365 g kg−1 chopped and 447 g kg−1 DM chopped and unchopped, either without or with Pioneer 1171® (Lactobacillus plantarum and Enterococcus faecium) or Lallemand Magniva Classic® (L. plantarum and Pediococcus pentasaceus) bacterial inoculant. Inoculants increased lactic acid production, reduced pH and improved fermentation compared to Control, but D-lactate, L-lactate and acetic acid production differed between inoculants. Unchopped silages had higher pH and NH3-N and better preserved protein fraction than chopped silages at the same DM content. In both experiments, wilting increased water soluble carbohydrates by 0.5–31.5 g kg−1 DM and ensiling increased degradation of the protein fraction. We concluded that a rapid and effective wilt combined with a bacterial additive resulted in well preserved tropical grass silages.