Microbial-based Products Research Articles

Shikimate Metabolic Pathway Engineering in Corynebacterium glutamicum.

Shikimate is a key high-demand metabolite for synthesizing valuable antiviral drugs, such as the anti-influenza drug, oseltamivir (Tamiflu). Microbial-based strategies for shikimate production have been developed to overcome the unstable and expensive supply of shikimate derived from traditional plant extraction processes. In this study, a microbial cell factory using Corynebacterium glutamicum was designed to overproduce shikimate in a fed-batch culture system. First, the shikimate kinase gene (aroK) responsible for converting shikimate to the next step was disrupted to facilitate the accumulation of shikimate. Several genes encoding the shikimate bypass route, such as dehydroshikimate dehydratase (QsuB), pyruvate kinase (Pyk1), and quinate/shikimate dehydrogenase (QsuD), were disrupted sequentially. An artificial operon containing several shikimate pathway genes, including aroE, aroB, aroF, and aroG were overexpressed to maximize the glucose uptake and intermediate flux. The rationally designed shikimate-overproducing C. glutamicum strain grown in an optimized medium produced approximately 37.3 g/l of shikimate in 7-L fed-batch fermentation. Overall, rational cell factory design and culture process optimization for the microbial-based production of shikimate will play a key role in complementing traditional plant-derived shikimate production processes.

Recent advancements for microorganisms and their natural compounds useful in agriculture.

During the past years, microorganisms have been the cause of many problems for human's health. However, today with the development of many techniques of microbiology, the researchers have studied several roles of microorganisms which may help the society. Microbial-based products are expected to play important role in agriculture-enhancing plant production and therefore increasing crop's yieldeswani et al. . Microorganisms can act by several action mechanisms including antibiosis or mechanisms in plant-microbe interactions underlining the dual function of microbial strains toward plant nutrition and protection. The market has increased with the development of microbial-based products. Currently, it is normal to think that microorganisms help us in agriculture by applying them as biological control. In this mini review, we collect the last findings about this topic including very recent literature. KEY POINTS: • Microorganisms play a beneficial role in agriculture by different mechanisms. • One of these mechanisms is the secretion of chemical compounds with different activities.

Invited Review: Strategic use of microbial-based probiotics and prebiotics in dairy calf rearing

The objective of this narrative review was to describe the effect of microbial-based products, specifically pro- and prebiotics, on gut health, function, and disease prevention during early life and at weaning in dairy calves. The main source of data and information compiled for this review was peer-reviewed literature. Diarrhea is responsible for the majority of mortality and morbidity early in life. Pro- and prebiotics have recently been explored as mechanisms to promote gut health and decrease diarrhea in young calves. In addition, the change in calf diet, where there is a transition from a predominantly milk diet to a solid diet in a relatively short period of time, may also provide an opportunity to use microbial-based products. Based on the current studies that have supplemented calves with pro- and prebiotics, the majority of responses in growth, feed efficiency, and health have either been nonsignificant (39/68, 32/70, and 15/68, respectively) or positive (22/68, 9/70, and 31/68, respectively). The results presented in this review highlight that health and growth were the most positively affected responses to supplementation and that most of the beneficial effects were observed when these products were supplemented during a bout of illness. It appears that pro- and prebiotic supplementation to calves is low risk with potentially positive benefits that are worthy of further investigation. Supplementation of pro- and prebiotics to young ruminants requires further investigation to better understand the underlying mechanisms responsible for the phenotypical responses observed to implement better supplementation strategies.

Novel outlook in engineering synthetic methylotrophs and formatotrophs: a course for advancing C1-based chemicals production.

The world is at a critical stage to switch from fossil and agriculture feedstocks with sustainable alternatives for the production of biobased chemicals of everyday use. This has spurred interest in using carbon one compounds; methanol and formate as a substrate or cosubstrate for microbial-based production. However, considering that native methylotrophs and formatotrophs utilize methanol and formate respectively, their capabilities to efficiently produce high value-added chemicals are limited. Therefore, shifting from these native C1 microbes to metabolically engineered non-native C1 model strains has attracted increasing attention thanks to many advantages such as the availability of well-established tools and strategies for metabolic engineering, and in addition to its high cell growth rate. Herein, we discussed recent trends in developing synthetic methylotrophs and formatotrophs for methanol and formate-based biomanufacturing. Finally, we highlighted barriers and provided broad prospects on possible avenues for optimizing synthetic methylotrophic and formatotrophic strains with respect to the recent advances in biology.

Towards Nutrition-Sensitive Agriculture: An evaluation of biocontrol effects, nutritional value, and ecological impact of bacterial inoculants

Nutrition-Sensitive Agriculture (NSA) is a novel concept in agriculture that considers not only yield, but also nutritional value of produce, sustainability of production, and ecological impact of agriculture. In accordance with its goals, NSA would benefit from applying microbial-based products as they are deemed more sustainable than their synthetic counterparts.This study characterized 3 plant-beneficial bacterial strains (Paenibacillus pasadenensis strain R16, Pseudomonas syringae strain 260-02, Bacillus amyloliquefaciens strain CC2) on their biocontrol activity and effect on nutritional and texture quality of romaine lettuce plants (Lactuca sativa) in greenhouse. The pathogens used in the trials are Rhizoctonia solani and Pythium ultimum.The obtained results indicate that strain R16 had a significant ability to cause a statistically significant reduction in the symptoms caused by both P. ultimum (reduction of 32%) and R. solani (reduction of 42%), while the other two strains showed a less efficient biocontrol ability.Indices of the nutritional quality (content in phenols, carotenoids and chlorophyll) were unaffected by the treatments, indicating that the product was equivalent to that obtained without using the bacteria, while the texture of the leaves benefits from the biocontrol treatments. In particular, the mechanical resistance of the leaves was significantly higher in non-treated plants affected by R. solani but was restored to the values of healthy plants when the bacterial inoculants were present as well.The ecological impact was evaluated by characterizing the bacterial microbiota in bulk soil, rhizosphere, and root in the presence or absence of the inoculants.The composition of the microbiota, analyzed with a Unifrac model to describe beta-diversity, was radically different in the rhizosphere and the root endosphere among treatments, while the bulk soil formed a single cluster regardless of treatment, indicating that the use of these treatments did not have an ecological impact outside of the plant.

Invited Review: Nutritional regulation of gut function in dairy calves: From colostrum to weaning

PurposePublished literature regarding calf nutrition was reviewed to create an information base for the implementation of proper nutritional management to maximize health and productivity.SourcesThe main source of data and information for this review was peer-reviewed literature.SynthesisFeeding a sufficient volume of colostrum during the first hours of life is crucial to calf health and survival; however, less is known about transition milk feeding and the potential benefits of the myriad of bioactive compounds it contains. After feeding colostrum and transition milk, calves are susceptible to diarrhea when moved onto high amounts of milk, and antibiotic use is often necessary to decrease disease. Feeding an elevated plane of milk nutrition results in increased ADG and, in some studies, increased future milk production. Thus, this nutritional strategy is recommended; however, weaning calves from high volumes of milk represents massive changes in the structure and microbiology of the gastrointestinal tract.Conclusions and ApplicationsColostrum and transition milk contain an abundance of bioactive molecules that can positively affect gut development and microbiota. There is significant potential for the use of novel feeding strategies and microbial-based products as alternatives to antibiotics. Calves fed an elevated plane of nutrition in the first month of life have greater productivity and growth. However, weaning should take place later in life. Moreover, applying a proper step-down feeding protocol is recommended, as it allows calves to intake and digest sufficient solid feed for growth and minimize distress at weaning.

Development and application of a transcriptional sensor for detection of heterologous acrylic acid production in E. coli

BackgroundAcrylic acid (AA) is a widely used commodity chemical derived from non-renewable fossil fuel sources. Alternative microbial-based production methodologies are being developed with the aim of providing “green” acrylic acid. These initiatives will benefit from component sensing tools that facilitate rapid and easy detection of in vivo AA production.ResultsWe developed a novel transcriptional sensor facilitating in vivo detection of acrylic acid (AA). RNAseq analysis of Escherichia coli exposed to sub-lethal doses of acrylic acid identified a selectively responsive promoter (PyhcN) that was cloned upstream of the eGFP gene. In the presence of AA, eGFP expression in E. coli cells harbouring the sensing construct was readily observable by fluorescence read-out. Low concentrations of AA (500 μM) could be detected whilst the closely related lactic and 3-hydroxy propionic acids failed to activate the sensor. We further used the developed AA-biosensor for in vivo FACS-based screening and identification of amidase mutants with improved catalytic properties for deamination of acrylamide to acrylic acid.ConclusionsThe transcriptional AA sensor developed in this study will benefit strain, enzyme and pathway engineering initiatives targeting the efficient formation of bio-acrylic acid.

Evaluation of denaturing gradient gel electrophoresis (DGGE) and next generation sequencing (NGS) in combination with enrichment culture techniques to identify bacteria in commercial microbial-based products

Identification of bacteria in new or existing commercial microbial-based products (MBPs) is important for compliance with government regulations and for human and environmental risk assessment. Research was performed to develop effective methods to identify bacteria present in a MBP using a combined approach of conventional enrichment culture technique and denaturing gradient gel electrophoresis (DGGE) followed by clonal sequencing or next generation sequencing (NGS). Genomic DNA obtained from un-enriched or enriched MBP in MacConkey broth, Azide Dextrose broth, Peptone Water mixed with Polymixine B and Gram Negative (GN) media under three different temperatures (22 °C, 28 °C and 37 °C) were sequenced in two methods for the V3 and V6 hypersensitive regions of 16S ribosomal DNA (rDNA) and compared.Enrichment followed by DGGE and clonal sequence analysis identified 20 bacterial genera in all enriched and un-enriched media. In contrast, NGS was able to identify 114 bacterial families and 134 genera both in V3 and V6 regions. In clonal sequence analysis, in comparison to the un-enriched MBP, the MacConkey broth enriched for Escherichia or Shigella and Morganella species, GN medium enriched for Proteus and Morganella species and Azide Dextrose broth enriched for Vagococcus and Enterococcus species at both 28 °C and 37 °C. Moreover, the enrichment facilitated NGS to record higher numbers of families and genera in all enrichment cultures, comparatively higher variations in V3 region than in V6. More prominently, NGS identified 14 genera and 9 species in the family Enterobacteriaceae compared to only 5 genera identified in the un-enriched control using V6 region variance in MacConkey broth at 28 °C. Increasing the temperature without enrichment identified specific families by V3 and V6 regions.This study indicates that the polyphasic approach with appropriate enrichment and incubation at different temperatures followed by NGS analysis is a promising method for the identification of viable, non-pathogenic or potential pathogenic bacteria in complex MBPs.

Laboratory Assays Against Adult and Larval Sap Beetles (Coleoptera: Nitidulidae) Using Entomopathogenic Nematodes, Microbial-Based Insecticides, and Synthetic Insecticides

Sap beetles, Carpophilus spp. (Coleoptera: Nitidulidae), damage peach fruit leading to the need for efficacious control measures. We assayed different species and strains of entomopathogenic nematodes (Heterorhabditis bacteriophora Vs strain, H. indica HOM1 strain, H. megidis UK211 strain, Steinernema carpocapsae All strain, S. feltiae Sn strain, and S. riobrave 355 strain) against larval Carpophilus spp. and insecticides (microbialbased Grandevo® and Venerate™ bioinsecticides, along with the synthetic thiamethoxam, acetamiprid, indoxacarb, β-cyfluthrin, clothianidin, carbaryl, chlorantraniliprole, fenpropathrin, spinetoram, phosmet, malathion, and imidacloprid) against adult Carpophilus spp. in the laboratory. All entomopathogenic nematodes assayed caused significantly higher larval mortality than the control. How the insecticides were presented to the adult beetles affected whether beetles were rated as nonfeeding (dead+moribund). Fewer insecticides were active against the adults when applied to filter paper than when applied to a plug of pear that beetles fed upon. Overall, indoxacarb and phosmet provided consistently better control, regardless of the exposure method. These two insecticides, with different modes of action, also have a 14-d preharvest interval when used on peach, making it imperative to detect these pests well before harvest. Chlorantraniliprole and the microbial-based products had no effect on adult beetles regardless of the exposure method.

Neoepitope based assays to detect C5a – Pitfalls and interpretations

Published literature regarding calf nutrition was reviewed to create an information base for the implementation of proper nutritional management to maximize health and productivity.The main source of data and information for this review was peer-reviewed literature.Feeding a sufficient volume of colostrum during the first hours of life is crucial to calf health and survival; however, less is known about transition milk feeding and the potential benefits of the myriad of bioactive compounds it contains. After feeding colostrum and transition milk, calves are susceptible to diarrhea when moved onto high amounts of milk, and antibiotic use is often necessary to decrease disease. Feeding an elevated plane of milk nutrition results in increased ADG and, in some studies, increased future milk production. Thus, this nutritional strategy is recommended; however, weaning calves from high volumes of milk represents massive changes in the structure and microbiology of the gastrointestinal tract.Colostrum and transition milk contain an abundance of bioactive molecules that can positively affect gut development and microbiota. There is significant potential for the use of novel feeding strategies and microbial-based products as alternatives to antibiotics. Calves fed an elevated plane of nutrition in the first month of life have greater productivity and growth. However, weaning should take place later in life. Moreover, applying a proper step-down feeding protocol is recommended, as it allows calves to intake and digest sufficient solid feed for growth and minimize distress at weaning.

De novo biosynthesis of trans-cinnamic acid derivatives in Saccharomyces cerevisiae.

The production of natural aroma compounds is an expanding field within the branch of white biotechnology. Three aromatic compounds of interest are cinnamaldehyde, the typical cinnamon aroma that has applications in agriculture and medical sciences, as well as cinnamyl alcohol and hydrocinnamyl alcohol, which have applications in the cosmetic industry. Current production methods, which rely on extraction from plant materials or chemical synthesis, are associated with drawbacks regarding scalability, production time, and environmental impact. These considerations make the development of a sustainable microbial-based production highly desirable. Through steps of rational metabolic engineering, we engineered the yeast Saccharomyces cerevisiae as a microbial host to produce trans-cinnamic acid derivatives cinnamaldehyde, cinnamyl alcohol, and hydrocinnamyl alcohol, from externally added trans-cinnamic acid or de novo from glucose as a carbon source. We show that the desired products can be de novo synthesized in S. cerevisiae via the heterologous overexpression of the genes encoding phenylalanine ammonia lyase 2 from Arabidopsis thaliana (AtPAL2), aryl carboxylic acid reductase (acar) from Nocardia sp., and phosphopantetheinyl transferase (entD) from Escherichia coli, together with endogenous alcohol dehydrogenases. This study provides a proof of concept and a strain that can be further optimized for production of high-value aromatic compounds.

Optimized Production of Tannase from Cashew Testa using Aspergillus niger MTCC 5898

ABSTRACTThe production of enzymes by bioprocessing is a good alternative to add value to agro-industry residues. Production cost of enzymes, the major constraint in bioprocessing, may be brought down by multifaceted approaches which include the use of cheap agro-residues for the microbial-based production of the enzyme, and the use of cost-efficient aerobic cell growth strategies and anaerobic-combined strategies like solid state fermentation (SSF). The current study investigated the production of tannase by Aspergillus niger CEPC 11 (MTCC 5898) on an abundantly available waste byproduct cashew testa under solid state A. niger growth. The optimum values of parameters obtained through response surface methodology (RSM) were cashew testa (23%), K2HPO4 (3.40 mM), sodium chloride (0.47 mM) and temperature (32–35°C). Optimization of the amount of cashew testa, content of the medium and incubation temperature resulted in a 3.02-fold increase from 97.32–301.70 U/g DS of tannase.