Rationally Engineered Synthetic Coculture for Improved Biomass and Product Formation

In microbial ecosystems, bacteria are dependent on dynamic interspecific interactions related to carbon and energy flow. Substrates and end-metabolites are rapidly converted to other compounds, which protects the community from high concentrations of inhibitory molecules. In biotechnological applications, pure cultures are preferred because of the more straight-forward metabolic engineering and bioprocess control. However, the accumulation of unwanted side products can limit the cell growth and process efficiency. In this study, a rationally engineered coculture with a carbon channeling system was constructed using two well-characterized model strains Escherichia coli K12 and Acinetobacter baylyi ADP1. The directed carbon flow resulted in efficient acetate removal, and the coculture showed symbiotic nature in terms of substrate utilization and growth. Recombinant protein production was used as a proof-of-principle example to demonstrate the coculture utility and the effects on product formation. As a result, the biomass and recombinant protein titers of E. coli were enhanced in both minimal and rich medium simple batch cocultures. Finally, harnessing both the strains to the production resulted in enhanced recombinant protein titers. The study demonstrates the potential of rationally engineered cocultures for synthetic biology applications.     

De novo phenol bioproduction from glucose using biosensor-assisted microbial coculture engineering

Microbial biosynthesis has been extensively adapted for the production of commodity chemicals using renewable feedstocks. This study integrated metabolite biosensors into rationally designed microbial cocultures to achieve high-efficiency bioproduction of phenol from simple carbon substrate glucose. Specifically, two sets of E. coli–E. coli cocultures were first constructed for accommodation of two independent phenol biosynthesis pathways via 4-hydroxybenzoate (4HB) and tyrosine (TYR), respectively. Biosensor-assisted microbial cell selection mechanisms were subsequently incorporated into the coculture systems to address the insufficient pathway intermediate provision that limited the overall bioproduction. For the 4HB- and TYR-dependent pathways, this approach improved the phenol production by 2.3- and 3.9-fold, respectively, compared to the monoculture controls. Notably, the use of biosensor-assisted cell selection strategy in monocultures resulted in reduced phenol production, highlighting the advantage of coculture engineering for coupling with biosensing. After stepwise optimization, the phenol bioproduction yield of the engineered coculture's reached 0.057 g/g glucose. Furthermore, the coculture biosynthesis was successfully scaled up at both shake flask and bioreactor levels. Overall, the findings of this study demonstrate the outstanding potential of coupling biosensing and modular coculture engineering for advancing microbial biosynthesis of valuable molecules from renewable carbon substrates.     

Reactive oxygen species (ROS) generated by cyanobacteria act as an electron acceptor in the biocathode of a bio-electrochemical system

The enhanced electricity generation in a biocathode bio-electrochemical system (BES) with Microcystis aeruginosa IPP as the cathodic microorganism under illumination is investigated. The results show that this cyanobacterium is able to act as a potential cathodic microorganism under illumination. In addition, M. aeruginosa IPP is found to produce reactive oxygen species (ROS) in its growth in the BES. ROS, as more competitive electron acceptors than oxygen, are utilized prior to oxygen. The BES current is substantially reduced when the ROS production is inhibited by mannitol, indicating that the ROS secreted by the cyanobacterium play an important role in the electricity generation of such a biocathode BES. This work demonstrates that the ROS released by cyanobacteria benefit for an enhanced electricity generation of BES.     

Integration of biocatalyst and process engineering for sustainable and efficient n‐butanol production

Recent environmental economic developments generate a need for sustainable and cost‐effective (microbial) processes for the production of high‐volume, low‐priced bulk chemicals. As an example, n‐butanol has, as a second‐generation biofuel, beneficial characteristics compared to ethanol in liquid transportation fuel applications. The industrial revival of the classic n‐butanol (ABE) fermentation requires process and strain engineering solutions for overcoming the main process limitations: product toxicity and low space–time yield. Reaction intensification on the biocatalyst, fermentation, and bioprocess level can be based on economic and ecologic evaluations using quantifiable constraints. This review describes the means of process intensification for biotechnological processes. A quantitative approach is then used for the comparison of the massive literature on n‐butanol fermentation. A comprehensive literature study—including key fermentation performance parameters—is presented and the results are visualized using the window of operation methodology. The comparison allowed the identification of the key constraints, high cell densities, high strain stability, high specific production rate, cheap in situ product removal, high n‐butanol tolerance, to operate in situ product removal efficiently, and cheap carbon source. It can thus be used as a guideline for the bioengineer during the combined biocatalyst, fermentation, and bioprocess development and intensification.     

Volatiles produced by soil‐borne endophytic bacteria increase plant pathogen resistance and affect tritrophic interactions

Volatile organic compounds (VOCs) released by soil microorganisms influence plant growth and pathogen resistance. Yet, very little is known about their influence on herbivores and higher trophic levels. We studied the origin and role of a major bacterial VOC, 2,3‐butanediol (2,3‐BD), on plant growth, pathogen and herbivore resistance, and the attraction of natural enemies in maize. One of the major contributors to 2,3‐BD in the headspace of soil‐grown maize seedlings was identified as Enterobacter aerogenes, an endophytic bacterium that colonizes the plants. The production of 2,3‐BD by E. aerogenes rendered maize plants more resistant against the Northern corn leaf blight fungus Setosphaeria turcica. On the contrary, E. aerogenes‐inoculated plants were less resistant against the caterpillar Spodoptera littoralis. The effect of 2,3‐BD on the attraction of the parasitoid Cotesia marginiventris was more variable: 2,3‐BD application to the headspace of the plants had no effect on the parasitoids, but application to the soil increased parasitoid attraction. Furthermore, inoculation of seeds with E. aerogenes decreased plant attractiveness, whereas inoculation of soil with a total extract of soil microbes increased parasitoid attraction, suggesting that the effect of 2,3‐BD on the parasitoid is indirect and depends on the composition of the microbial community.     

The biology of habitat dominance; can microbes behave as weeds?

Competition between microbial species is a product of, yet can lead to a reduction in, the microbial diversity of specific habitats. Microbial habitats can resemble ecological battlefields where microbial cells struggle to dominate and/or annihilate each other and we explore the hypothesis that (like plant weeds) some microbes are genetically hard‐wired to behave in a vigorous and ecologically aggressive manner. These ‘microbial weeds’ are able to dominate the communities that develop in fertile but uncolonized – or at least partially vacant – habitats via traits enabling them to out‐grow competitors; robust tolerances to habitat‐relevant stress parameters and highly efficient energy‐generation systems; avoidance of or resistance to viral infection, predation and grazers; potent antimicrobial systems; and exceptional abilities to sequester and store resources. In addition, those associated with nutritionally complex habitats are extraordinarily versatile in their utilization of diverse substrates. Weed species typically deploy multiple types of antimicrobial including toxins; volatile organic compounds that act as either hydrophobic or highly chaotropic stressors; biosurfactants; organic acids; and moderately chaotropic solutes that are produced in bulk quantities (e.g. acetone, ethanol). Whereas ability to dominate communities is habitat‐specific we suggest that some microbial species are archetypal weeds including generalists such as: Pichia anomala, Acinetobacter spp. and Pseudomonas putida; specialists such as Dunaliella salina, Saccharomyces cerevisiae, Lactobacillus spp. and other lactic acid bacteria; freshwater autotrophs Gonyostomum semen and Microcystis aeruginosa; obligate anaerobes such as Clostridium acetobutylicum; facultative pathogens such as Rhodotorula mucilaginosa, Pantoea ananatis and Pseudomonas aeruginosa; and other extremotolerant and extremophilic microbes such as Aspergillus spp., Salinibacter ruber and Haloquadratum walsbyi. Some microbes, such as Escherichia coli, Mycobacterium smegmatis and Pseudoxylaria spp., exhibit characteristics of both weed and non‐weed species. We propose that the concept of nonweeds represents a ‘dustbin’ group that includes species such as Synodropsis spp., Polypaecilum pisce, Metschnikowia orientalis, Salmonella spp., and Caulobacter crescentus. We show that microbial weeds are conceptually distinct from plant weeds, microbial copiotrophs, r‐strategists, and other ecophysiological groups of microorganism. Microbial weed species are unlikely to emerge from stationary‐phase or other types of closed communities; it is open habitats that select for weed phenotypes. Specific characteristics that are common to diverse types of open habitat are identified, and implications of weed biology and open‐habitat ecology are discussed in the context of further studies needed in the fields of environmental and applied microbiology.     

Pseudomonas aeruginosa-derived rhamnolipids subvert the host innate immune response through manipulation of the human beta-defensin-2 expression

Pseudomonas aeruginosa is a well‐known cause of infections especially in compromised patients. To neutralize this pathogen, the expression of antimicrobial factors in epithelial cells is crucial. In particular the human beta‐defensin hBD‐2 is especially active against P. aeruginosa. In this study, we identified rhamnolipids in P. aeruginosa culture supernatants that are able to prevent the pathogen‐induced hBD‐2 response in keratinocytes. The presence of rhamnolipids within the host cells and inhibition assays suggest that calcium‐regulated pathways and protein kinase C activation are impaired by rhamnolipids. In consequence, the induction of hBD‐2 in keratinocytes by P. aeruginosa‐derived flagellin as well as the host's own hBD‐2 mediator interleukin IL‐1β is inhibited. Strikingly, rhamnolipids did not affect the release of the proinflammatory mediator interleukin IL‐8 by flagellin. Thus, in addition to their function in establishment and persistence of P. aeruginosa infections, rhamnolipids can be engaged by P. aeruginosa for a targeted attenuation of the innate immunity to manage its survival and colonization on compromised epithelia.     

The mechanism of competition between two bloom‐forming Microcystis species

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Alkaline phosphatase dual‐binding sites for collagen dictate cell migration and microvessel assembly in vitro

Interactions between cell types, growth factors, and extracellular matrix components involved in angiogenesis are crucial for new vessel formation leading to tissue regeneration. This study investigated whether cocultures of fibroblasts and endothelial cells (ECs; from macro‐ or microvasculature) play a role in the formation of microvessel‐like structures by ECs, as well as modulate fibroblast differentiation and growth factors production (vascular endothelial cell growth factor, basic fibroblast growth factor, active transforming growth factor‐β1, and interleukin‐8), which are important for vessel sprouting and maturation. Data obtained revealed that in vitro coculture systems of fibroblasts and human ECs stimulate collagen synthesis and growth factors production by fibroblasts that ultimately affect the formation and distribution of microvessel‐like structures in cell cultures. In this study, areas with activated fibroblasts and high alkaline phosphatase (ALP) activity were also observed in cocultures. Molecular docking assays revealed that ALP has two binding positions for collagen, suggesting its impact in collagen proteins’ aggregation, cell migration, and microvessel assembly. These findings indicate that bioinformatics and coculture systems are complementary tools for investigating the participation of proteins, like collagen and ALP in angiogenesis.     

Large-scale production of diesel-like biofuels – process design as an inherent part of microorganism development

Industrial biotechnology is playing an important role in the transition to a bio-based economy. Currently, however, industrial implementation is still modest, despite the advances made in microorganism development. Given that the fuels and commodity chemicals sectors are characterized by tight economic margins, we propose to address overall process design and efficiency at the start of bioprocess development. While current microorganism development is targeted at product formation and product yield, addressing process design at the start of bioprocess development means that microorganism selection can also be extended to other critical targets for process technology and process scale implementation, such as enhancing cell separation or increasing cell robustness at operating conditions that favor the overall process. In this paper we follow this approach for the microbial production of diesel-like biofuels. We review current microbial routes with both oleaginous and engineered microorganisms. For the routes leading to extracellular production, we identify the process conditions for large scale operation. The process conditions identified are finally translated to microorganism development targets. We show that microorganism development should be directed at anaerobic production, increasing robustness at extreme process conditions and tailoring cell surface properties. All the same time, novel process configurations integrating fermentation and product recovery, cell reuse and low-cost technologies for product separation are mandatory. This review provides a state-of-the-art summary of the latest challenges in large-scale production of diesel-like biofuels.     

Streptomyces sp. TEM 33 possesses high lipolytic activity in solid-state fermentation in comparison with submerged fermentation

Solid-state fermentation (SSF) is a bioprocess that doesn’t need an excess of free water, and it offers potential benefits for microbial cultivation for bioprocesses and product development. In comparing the antibiotic production, few detailed reports could be found with lipolytic enzyme production by Streptomycetes in SSF. Taking this knowledge into consideration, we prefer to purify Actinomycetes species as a new source for lipase production. The lipase-producing strain Streptomyces sp. TEM 33 was isolated from soil and lipase production was managed by solid-state fermentation (SSF) in comparison with submerged fermentation (SmF). Bioprocess-affecting factors like initial moisture content, incubation time, and various carbon and nitrogen additives and the other enzymes secreted into the media were optimized. Lipase activity was measured as 1.74 ± 0.0005 U/g dry substrate (gds) by the p-nitrophenylpalmitate (pNPP) method on day 6 of fermentation with 71.43% final substrate moisture content. In order to understand the metabolic priority in SSF, cellulase and xylanase activity of Streptomyces sp. TEM33 was also measured. The microorganism degrades the wheat bran to its usable form by excreting cellulases and xylanases; then it secretes the lipase that is necessary for degrading the oil in the medium.     

Economic analysis of pilot‐scale production of B‐phycoerythrin

β‐Phycoerythrin is a color protein with several applications, from food coloring to molecular labeling. Depending on the application, different purity is required, affecting production cost and price. Different production and purification strategies for B‐phycoerythrin have been developed, the most studied are based on the production using Porphyridium cruentum and purified using chromatographic techniques or aqueous two‐phase systems. The use of the latter can result in a less expensive and intensive recovery of the protein, but there is lack of a proper economic analysis to study the effect of using aqueous two‐phase systems in a scaled‐up process. This study analyzed the production of B‐Phycoerythrin using real data obtained during the scale‐up of a bioprocess using specialized software (BioSolve, Biopharm Services, UK). First, a sensitivity analysis was performed to identify critical parameters for the production cost, then a Monte Carlo analysis to emulate real processes by adding uncertainty to the identified parameters. Next, the bioprocess was analyzed to determine its financial attractiveness and possible optimization strategies were tested and discussed. Results show that aqueous two‐phase systems retain their advantages of low cost and intensive recovery (54.56%); the costs of production per gram calculated (before titer optimization: US$15,709 and after optimization: US$2,374) allowed to obtain profit (in the range of US$millions in a 10‐year period) for a potential company taking this production method by comparing the production cost against commercial prices. The bioprocess analyzed is a promising and profitable method for the generation of a highly purified B‐phycoerythrin. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1472–1479, 2016     

Characterization of the hypersensitive response‐like cell death phenomenon induced by targeting antiviral lectin griffithsin to the secretory pathway

Griffithsin (GRFT) is an antiviral lectin, originally derived from a red alga, which is currently being investigated as a topical microbicide to prevent transmission of human immunodeficiency virus (HIV). Targeting GRFT to the apoplast for production in Nicotiana benthamiana resulted in necrotic symptoms associated with a hypersensitive response (HR)‐like cell death, accompanied by H₂O₂ generation and increased PR1 expression. Mannose‐binding lectins surfactant protein D (SP‐D), cyanovirin‐N (CV‐N) and human mannose‐binding lectin (hMBL) also induce salicylic acid (SA)‐dependent HR‐like cell death in N. benthamiana, and this effect is mediated by the lectin's glycan binding activity. We found that secreted GRFT interacts with an endogenous glycoprotein, α‐xylosidase (XYL1), which is involved in cell wall organization. The necrotic effect could be mitigated by overexpression of Arabidopsis XYL1, and by co‐expression of SA‐degrading enzyme NahG, providing strategies for enhancing expression of oligomannose‐binding lectins in plants.     

Stratified microbial structure and activity within anode biofilm during electrochemically assisted brewery wastewater treatment

In a bioelectrochemical system (BES), microbial community of anode biofilm is crucial to BES performance. In this study, the stratified pattern of community structure and activity of an anode-respiring biofilm in a BES fueled with brewery wastewater was investigated over time. The anode biofilm exhibited a superior performance in the removal of ethanol to that of an open-circuit system. The electrical current density reached a high level of 0.55mA/cm² with a Coulombic efficiency of 71.4%, but decreased to 0.18mA/cm² in the late stage of operation. A mature biofilm developed a more active outer layer covering a less active inner core, although the activities of the outer and inner layers of biofilm were similar in the early stage. More Geobacter spp., typical exoelectrogens, were enriched in the outer layer than in the inner layer of biofilm in the early stage, while more Geobacter spp. were distributed in the inner layer than in the outer layer in the late stage. The inactive and Geobacter-occupied inner layer of biofilm might be responsible for the decreased electricity generation from wastewater in the late stage of operation. This study provides better understanding of the effect of anode biofilm structure on BES performance.     

Enhanced electron transfer of different mediators for strictly opposite shifting of metabolism in Clostridium pasteurianum grown on glycerol in a new electrochemical bioreactor

Microbial electrosynthesis or electro-fermentation in bioelectrochemical systems (BES) have recently received much attention. Here, we demonstrate with the glycerol metabolism by Clostridium pasteurianum that H ₂ from in situ water electrolysis, especially in combination with a redox mediator, provides a simple and flexible way for shifting product selectivity and enhancing product yield in the fermentation process. In particular, we report and quantify for the first time strictly different effects of Neutral Red (NR) and the barely studied redox mediator Brilliant Blue (BB) on the growth and product formation of C. pasteurianum grown on glycerol in a newly developed BES. We were able to switch the product formation pattern of C. pasteurianum with a concentration-dependent addition of NR and BB under varied iron availability. Interestingly, NR and BB influenced the glycerol metabolism in a strictly opposite manner concerning the formation of the major products 1,3-propanediol (1,3-PDO) and n-butanol (BuOH). Whereas, NR and iron generally enhance the formation of BuOH, BB favors the formation of 1,3-PDO. In BES the metabolic shifts were enhanced, leading to a further increased yield by as high as 33% for BuOH in NR fermentations and 21% for 1,3-PDO in BB fermentations compared with the respective controls. For the first time, the electron transfer mediated by these mediators and their recycle (recharge) were unambiguously quantified by excluding the overlapping effect of iron. BB has a higher capacity than NR and iron. The extra electron transfer by BB can account for as high as 30–75% of the total NAD ⁺ regeneration under certain conditions, contributing significantly to the product formation.     

Effect of Different Carbon and Nitrogen Sources on Clostridium argentinense Toxigenicity in Coculture withPseudomonas mendocina

The effect of different carbon and nitrogen sources on the production of toxin by Clostridium argentinense was examined. The toxin production by C. argentinense in coculture with Pseudomonas mendocina increased in all the cases in relation to that produced by monocultures independent of the nature of the source. Using dextrin as carbon source C. argentinense produced the highest levels of toxin both in monocultures (300 LD₅₀/mL) and in cocultures with P. mendocina (5000 LD₅₀/mL). Experiments run in a microfermenter showed that the slow growth of cocultures associated with the assimilation of dextrin and the pH and Eh profiles favoured the production of toxin. Of the nitrogen sources assayed, corn steep liquor sustained the highest levels of toxin in both monocultures and cocultures with 3 and 2.8 fold increases with respect to that obtained using proteose peptone. The toxin production by C. argentinense cultures and C. argentinense–P. mendocina cocultures was highly dependent on the nature of the carbon and nitrogen sources used in the culture media. Growth of C. argentinense on substrates slowly assimilated stimulated the production of toxin.     

Relevance of microbial coculture fermentations in biotechnology

The purpose of this article is to review coculture fermentations in industrial biotechnology. Examples for the advantageous utilization of cocultures instead of single cultivations include the production of bulk chemicals, enzymes, food additives, antimicrobial substances and microbial fuel cells. Coculture fermentations may result in increased yield, improved control of product qualities and the possibility of utilizing cheaper substrates. Cocultivation of different micro‐organisms may also help to identify and develop new biotechnological substances. The relevance of coculture fermentations and the potential of improving existing processes as well as the production of new chemical compounds in industrial biotechnology are pointed out here by means of more than 35 examples.     

Exploring characteristics of bioelectricity generation and dye decolorization of mixed and pure bacterial cultures from wine-bearing wastewater treatment

This study uncovered microbial characteristics of bioelectricity generation and dye decolorization in single-chamber microbial fuel cells (MFCs) using activated sludge for wine-containing wastewater treatment. Phylogenetic tree analysis on 16S rRNA gene fragments indicated that the predominant strains on anodic biofilm in acclimatized MFCs were Gamma-Proteobacteria Aeromonas punctata NIU-P9, Pseudomonas plecoglossicida NIU-Y3, Pseudomonas koreensis NIU-X8, Acinetobacter junii NIU-Y8, Stenotrophomonas maltophila NIU-X2. Our findings showed that the current production capabilities of these pure strains were only ca. 10% of those of their mother activated sludge, indicating that synergistic interactions among microbes might be the most influential factor to maximize power generation in MFCs. Plus, these electrochemically active strains also performed reductive decolorization of C.I. reactive blue 160, suggesting that bioelectricity generation might be directly associated to azo dye decolorization to deal with electron transfer on anodic biofilm in MFCs.     

Prospects for an HIV Vaccine: Conventional Approaches and DNA Immunization

Abstract

Microbial transglutaminase is an important enzyme in food processing for improving protein properties by catalyzing the cross-linking of proteins. Recently, this enzyme has been shown to exhibit wider potential application in tissue engineering, textiles and leather processing, site-specific protein conjugation and wheat gluten allergy reduction. The production of microbial transglutaminase has been significantly improved thanks to advances in bioprocess engineering and genetic engineering during the last three decades. More recently, studies on the biological mechanism of transglutaminase synthesis have further contributed towards the understanding of microbial transglutaminase production by Streptomyces. This will further facilitate improving the production of recombinant microbial transglutaminase. In this paper, we will review the progress in bioprocess engineering and genetic engineering in microbial transglutaminase production. We will highlight our understanding of the biological mechanisms of microbial transglutaminase synthesis, including biotechnological approaches used based on these biological mechanisms as a way of improving transglutaminase production.We address in addition the future research needs for microbial transglutaminase production.