Lactose Utilization Research Articles

Non-canonical start codons confer context-dependent advantages in carbohydrate utilization for commensal E. coli in the murine gut.

Resource competition is a driver of gut microbiota composition. Bacteria can outcompete metabolically similar rivals through the limitation of shared growth-fuelling nutrients. The mechanisms underlying this remain unclear for bacteria with identical sets of metabolic genes. Here we analysed the lactose utilization operon in the murine commensal Escherichia coli 8178. Using in vitro and in vivo approaches, we showed that translation of the lactose utilization repressor gene lacI from its native non-canonical GTG start codon increases the basal expression of the lactose utilization cluster, enhancing adaptation to lactose consumption. Consequently, a strain carrying the wild type lacI GTG start codon outperformed the lacI ATG start codon mutant in the mouse intestine. This advantage was attenuated upon limiting host lactose intake through diet shift or altering the mutant frequency, emphasizing the context-dependent effect of a single nucleotide change on the bacterial fitness of a common member of the gut microbiota. Coupled with a genomic analysis highlighting the selection of non-ATG start codons in sugar utilization regulator genes across the Enterobacteriaceae family, our data exposed an unsuspected function of non-canonical start codons in metabolic competition.

Galacto-oligosaccharides as functional foods and their properties

Galacto-oligosaccharides (GOS) are classified as prebiotics due to their indigestible nature. GOS are regarded as resistant to salivary digestion and are not utilized by the mouth. Thus, they can be utilized as substitutes for low-cariogenic sugar. The structure of GOS mixtures is determined by the differentiation between the transferase reaction and hydrolysis. Transgalactosylation can be achieved intramolecularly when the glycosidic bond in lactose cleaves and reassembles itself on another glucose molecule. Microbes can be used as a source for the β-galactosidase enzyme or as agents to produce GOS units. Commercial β-galactosidase enzymes likewise do have a great potential for their use in GOS synthesis. These transgalactosyl reactions could find useful applications in dairy. Since 70% of the world's population cannot tolerate lactose, lactose utilization could only be enhanced through hydrolysis into its components, the monomer sugars of D-glucose and D-galactose. GOS can be produced industrially using whey or lactose as the substrate. Trisaccharide (β1-4 or β1-6 galactosyllactose) are generally the major products of this process. In comparison with lactose and other saccharide molecules, GOS has been shown to have low carcinogenicity, low calorific value, and low sweetness. This review focuses on GOS production, and the physicochemical characteristics, physiological effects, and applications of these prebiotics are summarized.

The SP1/SNHG16/GLUT1 axis promotes prostate cancer proliferation and invasion by regulating glucose metabolism

IntroductionThe SP1/SNHG16/GLUT1 is involved in diverse cancer-related process. This study was designed to study the role of SP1/SNHG16/GLUT1 axis in prostate cancer (PCa) via modulation of glucose metabolism.Material and methodsThe expression profile of SNHG16 in PCa tissues was obtained from the online public database GEPIA (http://gepia.cancer-pku.cn/). Real-time qPCR was used to ascertain the mRNA expression. To find cellular proliferation and invasion, respectively, CCK-8, transwell, and glycolysis were used to monitor proliferation, invasion, and glycolysis.ResultsAnalysis of GEPIA2 data revealed upregulation of SNHG16 in PCa tissues and a positive association between GLUT1 (SLC2A1) and SP1/SNHG16 expression in the correlation study. Consistently, SPI and SNHG16 were either overexpressed or knocked down in PCa cells to unveil the role of the SP1/SNHG16/GLUT1 axis. Results demonstrated that PC-3 and DU145 cell proliferation were promoted by the overexpression of either SPI or SNHG16. On the other hand, PC-3 and DU145 cell proliferation were reduced upon knockdown of SP1 or SNHG16. A real-time qPCR study revealed that GLUT1 mRNA was upregulated by SP1/SNHG16 overexpression and downregulated by SP1/SNHG16 knockdown. In PCa cells, overexpression of SNHG16/SP1 resulted in enhanced utilization of glucose, lactose, and ATP production, whereas SNHG16/SP1 knockdown had the reverse effect. Lastly, transwell assay results showed that overexpression of SNHG16/SP1 promoted, while knockdown of SNHG16/SP1 inhibited the invasiveness of PC-3 and DU145 PCa cells.ConclusionsCollectively, the SP1/SNHG16/GLUT1 axis regulates the proliferation of PCa cells via the glycolytic route and thus may act as a therapeutic target for PCa treatment.

Multilevel regulation of lactose operon in Bacillus licheniformis: Coordination of LacR, CcpA and TnrA regulators

Previous research has confirmed the involvement of the lac1 gene cluster in lactose metabolism in Bacillus licheniformis, which is able to co-utilize lactose and low concentrations of glucose or certain carbon sources. However, the internal regulatory mechanisms underlying specific lactose utilization remain elusive. In this study, we conducted a comprehensive investigation to elucidate the modulation mechanism of the lac1 gene cluster. We discovered a 162 bp bidirectional promoter located between the lacDCAB and lacR genes, which encodes a GntR family transcriptional regulatory factor. A 38 bp perfectly palindromic sequence (BOX1) within this promoter is critical for inducing lac1 expression. LacR and CcpA were shown to bind specifically to BOX1, and their novel binding motifs were identified. In BL2ΔlacR, BL2ΔccpA, and BL2ΔlacRΔccpA mutant strains, we confirmed the differential inhibitory levels of LacR and CcpA on bidirectional promoters. Furthermore, it was demonstrated that TnrA also binds BOX1 and exerts a positive regulatory effect on the lac1 cluster. Finally, we proposed a novel model to unravel the complex molecular interactions controlling the expression of the lac1 gene. These insights provide a foundation for exploring rational design strategies to optimize lactose fermentation processes and improve the efficiency of industrial lactose utilization.

Transcriptomic and metabolic changes in Trichoderma reesei caused by mutation in xylanase regulator 1 (xyr1)

BackgroundTrichoderma reesei is known for its ability to produce large amounts of extracellular proteins and is one of the most important industrially used filamentous fungus. Xylanase regulator 1 (XYR1) is the master regulator responsible for the activation of cellulase and hemicellulase gene expression under inducing conditions. It has been reported that strains with point mutations in certain areas of xyr1 bypass the need for inducing carbon source, allowing high (hemi)cellulase production even in the presence of glucose. These mutations also change the profile of produced proteins, shifting it more towards xylanase production, and increase the overall protein production in inducing conditions. However, how these mutations alter the metabolism and other cellular processes to cause these changes remains unclear.ResultsIn this study, we aimed to explore changes caused by a point mutation in xyr1 on transcriptomic and metabolic level to better understand the reasons behind the increased protein production in both repressing glucose and inducing lactose conditions. As expected, the expression of many carbohydrate-active enzyme (CAZy) genes was increased in the xyr1 mutant in both conditions. However, their induction was higher under inducing conditions. The xyr1 mutant strain built more biomass and produced more extracellular proteins during growth on lactose compared to the wild type xyr1 strain. Genes involved in oxidoreductive D-galactose catabolism pathway were upregulated in the xyr1 mutant strain, potentially contributing to the more efficient utilization of lactose. In addition to CAZy genes, clustering and enrichment analysis showed over-representation of mitochondria-related Gene Ontology terms in clusters where gene expression was higher in the xyr1 mutant, indicating that mitochondria play a role in the altered metabolic state associated with the xyr1 mutation. Metabolomics revealed that free tyrosine was more abundant in the xyr1 mutant strain in all measured timepoints, whereas multiple fatty acids were less abundant in the mutant strain on glucose.ConclusionsThe results contribute to more in-depth knowledge on T. reesei physiology growing under inducing and repressing carbon sources and gives new insights on the function of the master regulator XYR1. The vast data generated serve as a source for new targets for improved protein production.

Combinatorial Engineering of Escherichia coli for Enhancing 3-Fucosyllactose Production.

3-Fucosyllactose (3-FL) is an important fucosylated human milk oligosaccharide (HMO) with biological functions such as promoting immunity and brain development. Therefore, the construction of microbial cell factories is a promising approach to synthesizing 3-FL from renewable feedstocks. In this study, a combinatorial engineering strategy was used to achieve efficient de novo 3-FL production in Escherichia coli. α-1,3-Fucosyltransferase (futM2) from Bacteroides gallinaceum was introduced into E. coli and optimized to create a 3-FL-producing chassis strain. Subsequently, the 3-FL titer increased to 5.2 g/L by improving the utilization of the precursor lactose and down-regulating the endogenous competitive pathways. Furthermore, a synthetic membraneless organelle system based on intrinsically disordered proteins was designed to spatially regulate the pathway enzymes, producing 7.3 g/L 3-FL. The supply of the cofactors NADPH and GTP was also enhanced, after which the 3-FL titer of engineered strain E26 was improved to 8.2 g/L in a shake flask and 10.8 g/L in a 3 L fermenter. In this study, we developed a valuable approach for constructing an efficient 3-FL-producing cell factory and provided a versatile workflow for other chassis cells and HMOs.

Activation of a silent lactose utilization pathway in an evolved Listeria monocytogenes F2365 outbreak isolate

Listeria monocytogenes, a widespread food-borne pathogen, utilizes diverse growth substrates including mono- and di-saccharides via PEP-phosphotransferase (PTS) systems. We evaluated a collection of L. monocytogenes isolates of different origins for their ability to utilize lactose, a disaccharide composed of galactose and glucose and the main carbon source in milk and dairy products. Notably, the dairy-associated outbreak strain F2365 could not utilize lactose efficiently, conceivably due to a frameshift mutation (lacR887del) resulting in a truncated LacR. Transcriptional activator LacR is involved in the expression of two PTS systems, encoded by the lpo operon lmo1718-1720 in combination with lmo2708 and the lmo2683-2685 operon, and linked to lactose and/or cellobiose metabolism in L. monocytogenes. Via experimental evolution of the ancestral strain F2365, an evolved isolate F2365 EV was obtained which showed enhanced growth and metabolism of lactose. Using the lactose-positive model strain L. monocytogenes EGDe as a control, HPLC experiments showed that EGDe and F2365 EV could consume lactose and utilize the glucose moiety, while the galactose moiety was exported from the cells. Genome sequencing of F2365 EV found the original lacR887del mutation was still present but an additional point mutation lmo2766C415T had occurred, resulting in an amino acid substitution in the putative regulator Lmo2766. The lmo2766 gene is located next to operon lmo2761-2765 with putative PTS genes in the genome. Notably, comparative RNAseq analysis confirmed that the lmo2761-2765 operon was strongly upregulated in F2365 EV in the presence of lactose but not in EGDe and F2365. Conversely, the LacR-regulated lpo operon, lmo2708, and lmo2683-2685 operon were only upregulated in EGDe. Additional growth and HPLC experiments, using mutants constructed in lactose-positive L. monocytogenes EGDe, showed reduced growth of the EGDe lacR887del mutant with no utilization of lactose, while the double mutant EGDe lacR887dellmo2766C415T showed enhanced growth and lactose utilization. Hence, these results demonstrate that an amino acid substitution in the Lmo2766 regulator activates a previously silent lactose utilization pathway encoded by PTS operon lmo2761-2765, facilitating the growth and metabolism of L. monocytogenes with lactose as a substrate. This finding enhances our understanding of the metabolic capabilities and adaptability of L. monocytogenes, offering a broader view of the lactose utilization capacity of this pathogen.

Combining thermosonication microstress and pineapple peel extract addition to achieve quality and post-acidification control in yogurt fermentation

This work investigated the effects of the combined use of thermosonication-preconditioned lactic acid bacteria (LAB) with the addition of ultrasound-assisted pineapple peel extracts (UU group) on the post-acidification potential, physicochemical and functional qualities of yogurt products, aimed at achieving prolonged preservation and enhancing functional attributes. Accordingly, the physical–chemical features, adhesion properties, and sensory profiles, acidification kinetics, the contents of major organic acids, and antioxidant activities of the differentially processed yogurts during refrigeration were characterized. Following a 14-day chilled storage process, UU group exhibited acidity levels of 0.5–2 oT lower than the control group and a higher lactose content of 0.07 mg/ml as well as unmodified adhesion potential, indicating that the proposed combination method efficiently inhibited post-acidification and delayed lactose metabolism without leading to significant impairment of the probiotic properties. The results of physicochemical analysis showed no significant changes in viscosity, hardness, and color of yogurt. Furthermore, the total phenolic content of UU-treated samples was 98 μg/mL, 1.78 times higher than that of the control, corresponding with the significantly lower IC50 values of DPPH and ABTS radical scavenging activities of the UU group than those of the control group. Observations by fluorescence inverted microscopy demonstrated the obvious adhesion phenomenon with no significant difference found among differentially prepared yogurts. The results of targeted metabolomics indicated the proposed combination strategy significantly modified the microbial metabolism, leading to the delayed utilization of lactose and the inhibited conversion into glucose during post-fermentation, as well as the decreased lactic acid production and a notable shift towards the formation of relatively weak acids such as succinic acid and citric acid. This study confirmed the feasibility of thermosonication-preconditioned LAB inocula, in combination with the use of natural active components from fruit processing byproducts, to alleviate post-acidification in yogurt and to enhance its antioxidant activities as well as simultaneously maintaining sensory features.

Genomic and phenotypic characterisation of Enterococcus mundtii AM_AQ_BC8 for its anti-biofilm, antimicrobial and probiotic potential.

Enterococcus mundtii AM_AQ_BC8 isolated from biofouled filtration membrane was characterised as a potential probiotic bacterium showing strong L-lactic acid-producing capability. Experimental studies revealed that E. mundtii AM_AQ_BC8 possess antibiofilm and antimicrobial ability too, as tested against strong biofilm-forming bacteria like Pseudomonas spp. The present study has evaluated the genetic potential of E. mundtii AM_AQ_BC8 through genome sequencing. Whole genome analysis revealed the presence of key genes like ldh_1 and ldh_2 responsible for lactic acid production along with genes encoding probiotic features such as acid and bile salt resistance (dnaK, dnaJ, argS), fatty acid synthesis (fabD, fabE) and lactose utilisation (lacG, lacD). The phylogenomic analysis based on OrthoANI (99.85%) and dDDH (96.8%) values revealed that the strain AM_AQ_BC8 shared the highest homology with E. mundtii. The genome sequence of strain AM_AQ_BC8 has been deposited to NCBI and released with GenBank accession no. SAMN32531201. The study primarily demonstrated the probiotic potential of E. mundtii AM_AQ_BC8 isolate, for L-lactate synthesis in high concentration (8.98g/L/day), which also showed anti-biofilm and antimicrobial activities.

Lactobacillus amylovorus Promotes Lactose Utilization in Small Intestine and Enhances Intestinal Barrier Function in Intrauterine Growth Restricted Piglets

BackgroundIntrauterine growth restriction (IUGR) resulted in high mortality and many physiological defects of piglets, causing huge economic loss in the swine industry. Lactobacillus amylovorus (L. amylovorus) was identified as one of the main differential bacteria between IUGR and normal piglets. However, the effects of L. amylovorus on the growth performance and intestinal health in IUGR piglets remained unclear. ObjectivesThis study aimed to investigate the promoting effects of L. amylovorus Mafic1501, a new strain isolated from normal piglets, on the growth performance and intestinal barrier functions in IUGR piglets. MethodsNewborn mice or piglets were assigned into 3 groups: CON (normal birth weight, control), IUGR (low birth weight), and IUGR+L. amy (low birth weight), administered with sterile saline or L. amylovorus Mafic1501, respectively. Growth performance, lactose content in the digesta, intestinal lactose transporter, and barrier function parameters were profiled. IPEC-J2 cells were cultured to verify the effects of L. amylovorus Mafic1501 on lactose utilization and intestinal barrier functions. ResultsL. amylovorus Mafic1501 elevated body weight and average daily gain of IUGR mice and piglets (P < 0.05). The lactose content in the ileum was decreased, whereas gene expression of glucose transporter 2 (GLUT2) was increased by L. amylovorus Mafic1501 in IUGR piglets during suckling period (P < 0.05). Besides, L. amylovorus Mafic1501 promoted intestinal barrier functions by increasing the villus height and relative gene expressions of tight junctions (P < 0.05). L. amylovorus Mafic1501 and its culture supernatant decreased the lactose level in the medium and upregulated gene expressions of transporter GLUT2 and tight junction protein Claudin-1 of IPEC-J2 cells (P < 0.05). ConclusionL. amylovorus Mafic1501 improved the growth performance of IUGR piglets by promoting the lactose utilization in small intestine and enhancing intestinal barrier functions. Our results provided the new evidence of L. amylovorus Mafic1501 for its application in the swine industry.

The potential of lactose to inhibit cereulide biosynthesis of emetic Bacillus cereus in milk

This study aims to investigate the potential role of lactose on cereulide biosynthesis by emetic Bacillus cereus in dairy matrices. The cereulide yields in whole milk and lactose-free milk were investigated using the emetic reference strain F4810/72. To eliminate the influence of complex food substrates, the LB medium model was further used to characterize the effect of lactose on cereulide produced by F4810/72 and five other emetic B. cereus strains. Results showed that the lactose-free milk displayed a 13-fold higher amount of cereulide than whole milk, but the cereulide level could be reduced by 91 % when the lactose content was restored. The significant inhibition of lactose on cereulide yields of all tested B. cereus strains was observed in LB medium, showing a dose-dependent manner with inhibition rates ranging of 89–98 %. The growth curves and lactose utilization patterns of all strains demonstrated that B. cereus cannot utilize lactose as a carbon source and lactose might act as a signal molecule to regulate cereulide production. Moreover, lactose strongly repressed the expression of cereulide synthetase genes (ces), possibly by inhibiting the key regulator Spo0A at the transcriptional level. Our findings highlight the potential of lactose as an effective strategy to control cereulide production in food.

A major facilitator superfamily transporter PlMFS1 contributes to growth, oosporogenesis, and pathogenesis of Peronophythora litchii

Major facilitator superfamily (MFS) transporters are secondary active membrane transporters that play an important role in solute interchange and energy metabolism. Peronophythora litchii causes the most destructive disease on lichi, litchi downy blight. PlM90 was reported as a key oosporogenesis regulator. Here, we identified an MFS transporter gene PlMFS1, which is up-regulated during oospore formation at the late infection stage, while down-regulated in the PlM90 mutant. To investigate PlMFS1 function, we generated PlMFS1 knockout mutants using CRISPR/Cas9-mediated genome editing technology. Compared with the wild-type strain SHS3, PlMFS1 deletion impaired mycelium growth, zoospore release, oospore production and pathogenicity. Furthermore, PlMFS1 deletion significantly affected P. litchii utilization of fructose, lactose and maltose, and may be the PlMFS1 mechanism involved in mycelial growth. PlMFS1 gene deletion also led to deceased laccase activity, laccase-encoding gene downregulation and impaired P. litchii pathogenicity. To our knowledge, this is the first report of an MFS transporter involved in sugar utilization, sexual reproduction, asexual reproduction and pathogenesis in oomycetes.

Increase in Lactulose Content in a Hot-Alkaline-Based System through Fermentation with a Selected Lactic Acid Bacteria Strain Followed by the β-Galactosidase Catalysis Process.

In this study, lactic acid bacteria (LAB) fermentation and β-galactosidase catalysis methods were combined to increase the lactulose concentration and reduce the galactose and lactose content in a hot-alkaline-based system. The optimal conditions for chemical isomerization were 70 °C for 50 min for lactulose production, in which the concentration of lactulose was 31.3 ± 1.2%. Then, the selection and identification of LAB, which can utilize lactose and cannot affect lactulose content, were determined from 451 strains in the laboratory. It was found that Lactobacillus salivarius TM-2-8 had weak lactulose utilization and more robust lactose utilization. Lactobacillus rhamnosus grx.21 was weak in terms of lactulose utilization and strong in terms of galactose utilization. These two strains fermented the chemical isomerization system of lactulose to reduce the content of lactose and galactose. The results showed that the lactose concentration was 48.96 ± 2.92 g/L and the lactulose concentration was 59.73 ± 1. 8 g/L for fermentation lasting 18 h. The β-galactosidase was used to increase the content of lactulose in the fermented system at this time. The highest concentration of 74.89 ± 1.68 g/L lactulose was obtained at an enzymatic concentration of 3 U/mL and catalyzed at 50 °C for 3 h by β-galactosidase.

Set1/COMPASS regulates growth, pathogenicity, and patulin biosynthesis of Penicillium expansum via H3K4 methylation and the interaction with PeVelB

IntroductionPenicillium expansum is a harmful plant fungal pathogen that causes blue mold disease and produces mycotoxin patulin, leading to huge economic losses and food safety hazard. Set1 associated complex Set1/COMPASS deposits the methylation at lysine 4 of histone H3, which is associated with gene expression in diverse biological processes of fungi. However, the function and underlying mechanisms of Set1/COMPASS are poorly defined in P. expansum. ObjectivesThe study aimed to identify Set1/COMPASS and investigate its regulation mechanisms on growth, pathogenicity, and patulin biosynthesis of P. expansum. MethodsAnalyses of phylogenetic relationship, conserved structural domain, and gene deletion were used to identify components of Set1/COMPASS. Phenotype analysis and stress tolerance test of gene deletion mutants were conducted to analyze the function of these components. Yeast two-hybrid, Co-Immunoprecipitation (Co-IP), and point mutation were performed to verify the protein interaction. Western blot was conducted for detection of H3K4 methylation levels. ResultsP. expansum owns six components of Set1/COMPASS besides PeSet1. Absence of each component resulted in reduction of H3K4 methylation levels and impaired growth, pathogenicity, and patulin biosynthesis, as well as altered stress responses of P. expansum. One component PeBre2p was found to interact with the conserved global regulator PeVelB (VelvetLike protein B) at Asp294 of PeBre2p. This interaction affected fungal growth and utilization of fructose, lactose, glycine, and proline in P. expansum. ConclusionThis study revealed the important roles of Set1/COMPASS in P. expansum and clarified for the first time the combined regulation of PeBre2p and PeVelB in fungal growth and nutrition utilization. These results will provide potential targets for the control of blue mold disease.

Propionibacteria as promising tools for the production of pro-bioactive scotta: a proof-of-concept study.

Dairy propionibacteria are Gram positive Actinomycetota, routinely utilized as starters in Swiss type cheese making and highly appreciated for their probiotic properties and health promoting effects. In this work, within the frame of a circular economy approach, 47 Propionibacterium and Acidipropionibacterium spp. were isolated from goat cheese and milk, and ewe rumen liquor, and characterized in view of their possible utilization for the production of novel pro-bioactive food and feed on scotta, a lactose rich substrate and one of the main by-products of the dairy industry. The evaluation of the Minimum Inhibitory Concentration (MIC) of 13 among the most common antibiotics in clinical practice revealed a general susceptibility to ampicillin, gentamycin, streptomycin, vancomycin, chloramphenicol, and clindamycin while confirming a lower susceptibility to aminoglycosides and ciprofloxacin. Twenty-five isolates, that proved capable of lactose utilization as the sole carbon source, were then characterized for functional and biotechnological properties. Four of them, ascribed to Propionibacterium freudenreichii species, and harboring resistance to bile salts (growth at 0.7-1.56 mM of unconjugated bile salts), acid stress (>80% survival after 1 h at pH 2), osmostress (growth at up to 6.5% NaCl) and lyophilization (survival rate > 80%), were selected and inoculated in scotta. On this substrate the four isolates reached cell densities ranging from 8.11 ± 0.14 to 9.45 ± 0.06 Log CFU mL-1 and proved capable of producing different vitamin B9 vitamers after 72 h incubation at 30°C. In addition, the semi-quantitative analysis following the metabolomics profiling revealed a total production of cobalamin derivatives (vitamin B12) in the range 0.49-1.31 mg L-1, thus suggesting a full activity of the corresponding biosynthetic pathways, likely involving a complex interplay between folate cycle and methylation cycle required in vitamin B12 biosynthesis. These isolates appear interesting candidates for further ad-hoc investigation regarding the production of pro-bioactive scotta.

Role of cellulose response transporter-like protein CRT2 in cellulase induction in Trichoderma reesei.

Induction of cellulase in cellulolytic fungi Trichoderma reesei is strongly activated by cellulosic carbon sources. The transport of cellulosic inducer and the perception of inducing signal is generally considered as the critical process for cellulase induction, that the inducing signal would be perceived by a sugar transporter/transceptor in T. reesei. Several sugar transporters are coexpressed during the induction stage, but which function they serve and how they work collaboratively are still difficult to elucidate. In this study, we found that the constitutive expression of the cellulose response transporter-like protein CRT2 (previously identified as putative lactose permease TRE77517) improves cellulase induction on a cellulose, cellobiose or lactose medium. Functional studies indicate that the membrane-bound CRT2 is not a transporter of cellobiose, lactose or glucose in a yeast system, and it also does not affect cellobiose and lactose utilization in T. reesei. Further study reveals that CRT2 has a slightly similar function to the cellobiose transporter CRT1 in cellulase induction. Overexpression of CRT2 led to upregulation of CRT1 and the key transcription factor XYR1. Moreover, overexpression of CRT2 could partially compensate for the function loss of CRT1 on cellulase induction. Our study uncovers the novel function of CRT2 in cellulase induction collaborated with CRT1 and XYR1, possibly as a signal transductor. These results deepen the understanding of the influence of sugar transporters in cellulase production.

Study on a galactose decomposition strain of Lactiplantibacillus plantarum MWLp-4 from human milk: Safety evaluating and key genes analysis of potential probiotic function

Galactose intolerance, associated with bloating and abdominal pain, is similar to lactose intolerance. Congenital galactose malabsorption in a child can lead to severe diarrhea. To avoid the consequences of galactose malabsorption, we obtained a galactose decomposition strain of Lactiplantibacillus plantarum MWLp-4 from human milk. Importantly, this strain had almost overlapping dynamics of galactose and lactose utilization, showing a great galactose catabolic ability. In this paper, we studied safety evaluation including hemolysis test, drug sensitivity test, and determination of amine in vitro of L. plantarum MWLp-4. In addition, we evaluated the probiotic function such as antimicrobial properties, adhesion, acid resistance, and bile salt resistance, etc. Meanwhile, we have analyzed the key genes on function and safety. As a conclusion, this strain possess great safety and probiotic function. The genes related to galactose metabolism, bacteriocin synthesis, surface protein synthesis, bile salt hydrolysis and stress response are found in L. plantarum MWLp-4. Analysis of the consistency of the phenotype with its genotype via whole genome sequencing showed that the phenotype of its physiological function is due to the large number of related genes existing in it. Therefore, this study would provide potential degrading galactose probiotics for producing special functional food.

Conversion of Deproteinized Cheese Whey to Lactobionate by an Engineered Neurospora crassa Strain F5.

We report a novel production process for lactobionic acid (LBA) production using an engineered Neurospora crassa strain F5. The wild-type N. crassa strain produces cellobiose dehydrogenase (CDH) and uses lactose as a carbon source. N. crassa strain F5, which was constructed by deleting six out of the seven β-glucosidases in the wild type, showed a much slower lactose utilization rate and produced a much higher level of cellobiose dehydrogenase (CDH) than the wild type. Strain N. crassa F5 produced CDH and laccase simultaneously on the pretreated wheat straw with 3µM of cycloheximide added as the laccase inducer. The deproteinized cheese whey was added directly to the shake flasks with the fungus present to achieve LBA production. Strain F5 produced about 37g/L of LBA from 45g/L of lactose in 27h since deproteinized cheese whey addition. The yield of LBA from consumed lactose was about 85%, and the LBA productivity achieved was about 1.37g/L/h.

Food-grade expression system of Lactobacillus plantarum using β-galactosidase small subunit as selection marker and lactose as screening condition

Lactobacillus plantarum, one of the most important and widely studied lactic acid bacteria, has been used in many fields. In recent years, the construction and application of food-grade expression systems in L. plantarum has shown significant advantages in the fields of food and medicine. In this study, a food-grade expression system of L. plantarum with β-galactosidase as a screening marker was constructed by exploring the complementary properties of the gene lacLM of β-galactosidase in L. plantarum. Firstly, L. plantarum knockout strains ΔlacM and ΔlacAlacM were constructed, and it was determined that the knockout strain ΔlacAlacM was endowed with the phenotype of being unable to metabolize lactose. The knockout strain ΔlacAlacM was selected as the food-grade host for the food-grade expression system. The lacM gene was further expressed by plasmid pMG36e and transformed into L. plantarum ΔlacAlacM. The complementary strain restored lactose utilization ability, and its biomass reached 87.3% of that of L. plantarum LP01. Based on this phenomenon, a food-grade vector pMG36m with lacM gene as a selection marker was obtained by removing the antibiotic resistance gene. The genetic stability test showed that the plasmid existence rate was still close to 100% after 98 generations of L. plantarum ΔlacAlacM/pMG36m. Subsequently, an expression vector pMG36m-gfp was constructed with green fluorescent protein gene (gfp) as the reporter gene. These results indicated that a novel food-grade expression system of L. plantarum with lacM gene as a selection marker and lactose as a screening condition was successfully established, laying a foundation for the application of L. plantarum.

A metagenome-level analysis of a microbial community fermenting ultra-filtered milk permeate.

Fermentative microbial communities have the potential to serve as biocatalysts for the conversion of low-value dairy coproducts into renewable chemicals, contributing to a more sustainable global economy. To develop predictive tools for the design and operation of industrially relevant strategies that utilize fermentative microbial communities, there is a need to determine the genomic features of community members that are characteristic to the accumulation of different products. To address this knowledge gap, we performed a 282-day bioreactor experiment with a microbial community that was fed ultra-filtered milk permeate, a low-value coproduct from the dairy industry. The bioreactor was inoculated with a microbial community from an acid-phase digester. A metagenomic analysis was used to assess microbial community dynamics, construct metagenome-assembled genomes (MAGs), and evaluate the potential for lactose utilization and fermentation product synthesis of community members represented by the assembled MAGs. This analysis led us to propose that, in this reactor, members of the Actinobacteriota phylum are important in the degradation of lactose, via the Leloir pathway and the bifid shunt, and the production of acetic, lactic, and succinic acids. In addition, members of the Firmicutes phylum contribute to the chain-elongation-mediated production of butyric, hexanoic, and octanoic acids, with different microbes using either lactose, ethanol, or lactic acid as the growth substrate. We conclude that genes encoding carbohydrate utilization pathways, and genes encoding lactic acid transport into the cell, electron confurcating lactate dehydrogenase, and its associated electron transfer flavoproteins, are genomic features whose presence in Firmicutes needs to be established to infer the growth substrate used for chain elongation.