Biotechnological Production Processes Research Articles

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.

Saccharomyces cerevisiae as a Host for Chondroitin Production

Chondroitin is a glycosaminoglycan that has gained widespread use in nutraceuticals and pharmaceuticals, mainly for treating osteoarthritis. Traditionally, it has been extracted from animal cartilage but recently, biotechnological processes have emerged as a commercial alternative to avoid the risk of viral or prion contamination and offer a vegan-friendly source. Typically, these methods involve producing the chondroitin backbone using pathogenic bacteria and then modifying it enzymatically through the action of sulfotransferases. Despite the challenges of expressing active sulfotransferases in bacteria, the use of eukaryotic microorganisms is still limited to a few works using Pichia pastoris. To create a safer and efficient biotechnological platform, we constructed a biosynthetic pathway for chondroitin production in S. cerevisiae as a proof-of-concept. Up to 125 mg/L and 200 mg/L of intracellular and extracellular chondroitin were produced, respectively. Furthermore, as genome-scale models are valuable tools for identifying novel targets for metabolic engineering, a stoichiometric model of chondroitin-producing S. cerevisiae was developed and used in optimization algorithms. Our research yielded several novel targets, such as uridine diphosphate (UDP)-N-acetylglucosamine pyrophosphorylase (QRI1), glucosamine-6-phosphate acetyltransferase (GNA1), or N-acetylglucosamine-phosphate mutase (PCM1) overexpression, that might enhance chondroitin production and guide future experimental research to develop more efficient host organisms for the biotechnological production process.

Enhanced biotechnological process for antivenom production using Quality by Design methodology

Snakebites envenoming are a global public health problem that largely affects economically vulnerable populations. The primary recognized treatment involves hyperimmune serum, consisting of sterile and heterologous immunoglobulin solutions targeting specific antigens. One established production method in Vital Brazil Institute involves antibody digestion by pepsin, fractionation with ammonium sulfate salt, and thermocoagulation, yielding 30-40% and requiring a 5-day duration. This study aims to assess a novel production method centered on recovering the (Fab’)2 fraction using caprylic acid, intending to implement a more efficient technological process. A new methodology was developed, defining digestion conditions (37 °C, pH 3.2; 2.5 g L-1 of pepsin over 120 minutes at 150 rpm) and fractionation parameters (37 °C, pH 5.8; 2% caprylic acid over 60 minutes at 250 rpm). These conditions were established through the application of Quality by Design, supported by a critical risk analysis. This method reduced the usage of raw materials, process time, and labor cost per hour while maintaining yield with greater purity. These modifications were promising, resulting in higher purity and lower manufacturing costs, thereby expanding the prospect of achieving a more robust production process with a safer, optimized product. This allows meeting production goals and exploring new opportunities in foreign markets.

Streptomycetes as Microbial Cell Factories for the Biotechnological Production of Melanin.

Melanins are complex, polymeric pigments with interesting properties like UV-light absorbance ability, metal ion chelation capacity, antimicrobial action, redox behaviors, and scavenging properties. Based on these characteristics, melanins might be applied in different industrial fields like food packaging, environmental bioremediation, and bioelectronic fields. The actual melanin manufacturing process is not environmentally friendly as it is based on extraction and purification from cuttlefish. Synthetic melanin is available on the market, but it is more expensive than animal-sourced pigment and it requires long chemical procedures. The biotechnological production of microbial melanin, instead, might be a valid alternative. Streptomycetes synthesize melanins as pigments and as extracellular products. In this review, the melanin biotechnological production processes by different Streptomyces strains have been revised according to papers in the literature. The different fermentation strategies to increase melanin production such as the optimization of growth conditions and medium composition or the use of raw sources as growth substrates are here described. Diverse downstream purification processes are also reported as well as all the different analytical methods used to characterize the melanin produced by Streptomyces strains before its application in different fields.

Biotechnological Utilization of Agro-Industrial Residues and By-Products-Sustainable Production of Biosurfactants.

The importance and interest in the efficient use and valorization of agro-industrial residues and by-products have grown due to environmental problems associated with improper disposal. Biotechnological production processes, including microbial biosurfactant production, represent a sustainable way to utilize agro-industrial residues and by-products, which are applied as substrates in these processes. Biosurfactants produced by microorganisms using renewable resources are a viable alternative to traditional petrochemical surfactants and have several potential uses in a wide range of industrial sectors due to their minimal ecotoxicity, easy biodegradability, and moderate production conditions. The common applications of biosurfactants, besides in food industry as food additives and preservatives, are in agriculture, environmental protection, the cosmetics and pharmaceutical industry, wastewater treatment, the petroleum industry, etc. This review aims to summarize the comprehensive scientific research related to the use of various agro-industrial residues and by-products in the microbial production of biosurfactants, as well as to emphasize the present state and the importance of their sustainable production. Additionally, based on the available biosurfactant market analysis datasets and research studies, the current situation in science and industry and the future perspectives of microbial biosurfactant production have been discussed.

Disposable Bioreactors Used in Process Development and Production Processes with Plant Cell and Tissue Cultures.

The bioreactor is the centerpiece of the upstream processing in any biotechnological production process. Its design, the cultivation parameters, the production cell line, and the culture medium all have a major influence on the efficiency of the process and the result of the cultivation. Disposable bioreactors have been used for the past 20 years, playing a major role in process development and commercial production of high-value substances at medium scales.Our review deals with scalable, disposable bioreactors that have proven to be useful for the cultivation of plant cell and tissue cultures. Based on the definitions of terms and a categorization approach, the most commonly used, commercially available, disposable bioreactor types are presented below. The focus is on wave-mixed, stirred, and orbitally shaken bioreactors. In addition to their instrumentation and bioengineering characteristics, cultivation results are discussed, and emerging trends for the development of disposable bioreactors for plant cell and tissue cultures are also addressed.

Biotechnological Advancements in Streptokinase Production

This article offers a deep analysis of activating streptokinase which looks into the biotechnological challenges and future perspectives. One of the main problems associated with streptokinase is its short half-life, complex production process, and immunogenicity which hinders patients from getting a successful reperfusion. Yet, the development of biotechnology methods that would not face these drawbacks, such as the strategy of recombinant streptokinase production and enzyme engineering theories, is a promise. This paper aims to provide an overview of biotechnological breakthroughs in the production of streptokinase with a focus on discovering long-lasting formulations exhibiting less immunogenicity and in addition the economic viability and scalability of biotechnological production processes. This article also gives the comping image of known purification methods like chromatography, ultrafiltration, and I-A chromatography for the separation and purification of streptokinase. In addition, the author presents the advantage of such suitable production technologies as cell-free production systems and E. Coli system optimization, such as recombinant streptokinase synthesis in Yeast systems. Biotechnology is turning out to be a new area, having a future boom due to the rising innovation and demand that are associated with the prospects revealed by the market trends.

Diamide-based screening method for the isolation of improved oxidative stress tolerance phenotypes in Bacillus mutant libraries.

The bacterium Bacillus subtilis is of high importance both as a model organism for Gram-positive bacteria and as an industrial workhorse in the production of biomolecules. In recent years, advancements have been made to engineer the bacterium even further toward industrial applications. In this study, we present a novel screening method for mutant libraries using diamide, an oxidizing agent that binds free thiols and creates disulfide bonds between them, thereby causing a so-called "disulfide stress" in bacteria. The method shows promise to selectively identify phenotypes in B. subtilis with improved tolerance toward oxidative and disulfide-associated stress. Phenotypes initially identified by transposon mutagenesis were recreated through targeted gene deletions. Among the resulting deletion mutants, the largest difference in diamide tolerance compared to the parental strain was observed for pfkA and ribT deletion strains. A proteomics analysis showed that diamide tolerance can be achieved through different routes involving increased expression of stress management proteins and reduced availability or activity of the RNA degradosome. We conclude that our screening method allows the facile identification of Bacillus strains with improved oxidative stress tolerance phenotypes. IMPORTANCE During their life cycle, bacteria are exposed to a range of different stresses that need to be managed appropriately in order to ensure their growth and viability. This applies not only to bacteria in their natural habitats but also to bacteria employed in biotechnological production processes. Oxidative stress is one of these stresses that may originate either from bacterial metabolism or external factors. In biotechnological settings, it is of critical importance that production strains are resistant to oxidative stresses. Accordingly, this also applies to the major industrial cell factory Bacillus subtilis. In the present study, we, therefore, developed a screen for B. subtilis strains with enhanced oxidative stress tolerance. The results show that our approach is feasible and time-, space-, and resource-efficient. We, therefore, anticipate that it will enhance the development of more robust industrial production strains with improved robustness under conditions of oxidative stress.

High-cell-density cultivation of Vibrio natriegens in a low-chloride chemically defined medium

Vibrio natriegens is a halophilic bacterium with the fastest generation time of non-pathogenic bacteria reported so far. It therefore has high potential as a production strain for biotechnological production processes or other applications in biotechnology. Culture media for V. natriegens typically contain high sodium chloride concentrations. The corresponding high chloride concentrations can lead to corrosion processes on metal surfaces in bioreactors. Here we report the development of a low-chloride chemically defined medium for V. natriegens. Sodium chloride was completely replaced by the sodium salts disodium hydrogen phosphate, disodium sulfate, and sodium citrate, while keeping the total concentration of sodium ions constant. The use of citrate prevents the occurrence of precipitates, especially of ammonium magnesium phosphate. With this defined medium, high-cell-density fed-batch cultivations in laboratory-scale bioreactors using exponential feeding yielded biomass concentrations of more than 60 g L−1.Key pointsA defined medium for V. natriegens that only contains traces of chloride was developedCorrosion processes on metal surfaces in industrial bioreactors can thus be preventedHigh yields of biomass can be achieved in fed-batch cultivation with this medium

Biotechnological Production of Fumaric Acid by Rhizopus arrhizus—Reaching Industrially Relevant Final Titers

Fumaric acid is used in various areas of the chemical industry due to its functional groups. For example, it is used in the polymer industry to produce unsaturated polyester resins, which are nowadays mostly produced from fossil raw materials. With regard to sustainable biotechnological fumaric acid production, the main challenge is to develop a cost-effective and robust fermentation process with industrially relevant final titers, productivities and yields. For biotechnological fumaric acid production, mainly fungi of the genus Rhizopus are used, which require very complex and challenging morphology control. The aim of this work is the development of an effective biotechnological fumaric acid production process with R. arrhizus NRRL 1526. Significant insights into the morphology control of the fungus and optimization of production characteristics were obtained, and a final titer of 86.3 g/L fumaric acid was achieved in a batch cultivation, with a yield of 0.67 g/g and a productivity of 0.60 g/(L∙h). In addition, a fed-batch process was developed, in which the production phase was extended, and a maximum final titer of 195.4 g/L fumaric acid was achieved. According to current knowledge, this value is the highest final concentration of fumaric acid produced using biotechnology.

Engineering Corynebacterium glutamicum for de novo production of 2-phenylethanol from lignocellulosic biomass hydrolysate

Background2-Phenylethanol is a specific aromatic alcohol with a rose-like smell, which has been widely used in the cosmetic and food industries. At present, 2-phenylethanol is mainly produced by chemical synthesis. The preference of consumers for “natural” products and the demand for environmental-friendly processes have promoted biotechnological processes for 2-phenylethanol production. Yet, high 2-phenylethanol cytotoxicity remains an issue during the bioproduction process.ResultsCorynebacterium glutamicum with inherent tolerance to aromatic compounds was modified for the production of 2-phenylethanol from glucose and xylose. The sensitivity of C. glutamicum to 2-phenylethanol toxicity revealed that this host was more tolerant than Escherichia coli. Introduction of a heterologous Ehrlich pathway into the evolved phenylalanine-producing C. glutamicum CALE1 achieved 2-phenylethanol production, while combined expression of the aro10. Encoding 2-ketoisovalerate decarboxylase originating from Saccharomyces cerevisiae and the yahK encoding alcohol dehydrogenase originating from E. coli was shown to be the most efficient. Furthermore, overexpression of key genes (aroGfbr, pheAfbr, aroA, ppsA and tkt) involved in the phenylpyruvate pathway increased 2-phenylethanol titer to 3.23 g/L with a yield of 0.05 g/g glucose. After introducing a xylose assimilation pathway from Xanthomonas campestris and a xylose transporter from E. coli, 3.55 g/L 2-phenylethanol was produced by the engineered strain CGPE15 with a yield of 0.06 g/g xylose, which was 10% higher than that with glucose. This engineered strain CGPE15 also accumulated 3.28 g/L 2-phenylethanol from stalk hydrolysate.ConclusionsIn this study, we established and validated an efficient C. glutamicum strain for the de novo production of 2-phenylethanol from corn stalk hydrolysate. This work supplied a promising route for commodity 2-phenylethanol bioproduction from nonfood lignocellulosic feedstock.Graphical

Assessment of Photosynthetic Carbon Capture versus Carbon Footprint of an Industrial Microalgal Process

It is often read that industrial microalgal biotechnology could contribute to carbon capture through photosynthesis. While technically accurate, this claim is rarely supported by sound figures nor put in regard to the carbon emissions associated with said processes. In this view, this work provides a quantitative assessment of the extent microalgal processes compensation for their carbon dioxide emissions. To do so, microalgae were cultivated under photolimited conditions. Their growth dynamic and photosynthetic apparatus status were monitored by daily cell density measurement and fluorescence assays. Ultimate analyses were used to determine microalgal carbon content. Simultaneously, the power consumption of the process was recorded, and the associated carbon dioxide emissions were computed using European electrical production carbon intensity. All in all, the recorded values confirmed microalgae growth under good physiological conditions and allowed computing the carbon capture rate, the energy storing rate, and the carbon dioxide emissions of the process. The process captured 0.72 ± 0.19 gCO2/day while emitting 182 gCO2/day, on average (over 15 days). The photoconversion efficiency was 4.34 ± 0.68%. Even if it were highly optimized (red/blue LED instead of white, for example), the process could only capture 1.02 ± 0.40% of its emissions. From these figures, the claim stating that a biotechnological microalgal production process could partly compensate for its emission seems rather bold. Authors should, therefore, emphasize other ecosystemic benefits of microalgal cultivation, such as phosphorous intake. Finally, we were also able to evaluate Chlorella vulgaris light and dark respiration (0.0377 ± 0.042 day−1 and 7.42 × 10−3 ± 3.33 × 10−3 day−1), which could help to assess carbon emission by biomass respiratory activity.

Engineering Strategies for Efficient Bioconversion of Glycerol to Value-Added Products by Yarrowia lipolytica

Yarrowia lipolytica has been a valuable biotechnological workhorse for the production of commercially important biochemicals for over 70 years. The knowledge gained so far on the native biosynthetic pathways, as well as the availability of numerous systems and synthetic biology tools, enabled not only the regulation and the redesign of the existing metabolic pathways, but also the introduction of novel synthetic ones; further consolidating the position of the yeast in industrial biotechnology. However, for the development of competitive and sustainable biotechnological production processes, bioengineering should be reinforced by bioprocess optimization strategies. Although there are many published reviews on the bioconversion of various carbon sources to value-added products by Yarrowia lipolytica, fewer works have focused on reviewing up-to-date strain, medium, and process engineering strategies with an aim to emphasize the significance of integrated engineering approaches. The ultimate goal of this work is to summarize the necessary knowledge and inspire novel routes to manipulate at a systems level the yeast biosynthetic machineries by combining strain and bioprocess engineering. Due to the increasing surplus of biodiesel-derived waste glycerol and the favored glycerol-utilization metabolic pathways of Y. lipolytica over other carbon sources, the present review focuses on pure and crude glycerol-based biomanufacturing.

Optogenetic closed-loop feedback control of the unfolded protein response optimizes protein production

In biotechnological protein production processes, the onset of protein unfolding at high gene expression levels leads to diminishing production yields and reduced efficiency. Here we show that in silico closed-loop optogenetic feedback control of the unfolded protein response (UPR) in S. cerevisiae clamps gene expression rates at intermediate near-optimal values, leading to significantly improved product titers. Specifically, in a fully-automated custom-built 1L-photobioreactor, we used a cybergenetic control system to steer the level of UPR in yeast to a desired set-point by optogenetically modulating the expression of α-amylase, a hard-to-fold protein, based on real-time feedback measurements of the UPR, resulting in 60% higher product titers. This proof-of-concept study paves the way for advanced optimal biotechnology production strategies that diverge from and complement current strategies employing constitutive overexpression or genetically hardwired circuits.

Machine learning for metabolic pathway optimization: A review

Optimizing the metabolic pathways of microbial cell factories is essential for establishing viable biotechnological production processes. However, due to the limited understanding of the complex setup of cellular machinery, building efficient microbial cell factories remains tedious and time-consuming. Machine learning (ML), a powerful tool capable of identifying patterns within large datasets, has been used to analyze biological datasets generated using various high-throughput technologies to build data-driven models for complex bioprocesses. In addition, ML can also be integrated with Design–Build–Test–Learn to accelerate development. This review focuses on recent ML applications in genome-scale metabolic model construction, multistep pathway optimization, rate-limiting enzyme engineering, and gene regulatory element designing. In addition, we have discussed some limitations of these methods as well as potential solutions.

Leveraging substrate flexibility and product selectivity of acetogens in two-stage systems for chemical production.

Carbon dioxide (CO2 ) stands out as sustainable feedstock for developing a circular carbon economy whose energy supply could be obtained by boosting the production of clean hydrogen from renewable electricity. H2 -dependent CO2 gas fermentation using acetogenic microorganisms offers a viable solution of increasingly demonstrated value. While gas fermentation advances to achieve commercial process scalability, which is currently limited to a few products such as acetate and ethanol, it is worth taking the best of the current state-of-the-art technology by its integration within innovative bioconversion schemes. This review presents multiple scenarios where gas fermentation by acetogens integrate into double-stage biotechnological production processes that use CO2 as sole carbon feedstock and H2 as energy carrier for products' synthesis. In the integration schemes here reviewed, the first stage can be biotic or abiotic while the second stage is biotic. When the first stage is biotic, acetogens act as a biological platform to generate chemical intermediates such as acetate, formate and ethanol that become substrates for a second fermentation stage. This approach holds the potential to enhance process titre/rate/yield metrics and products' spectrum. Alternatively, when the first stage is abiotic, the integrated two-stage scheme foresees, in the first stage, the catalytic transformation of CO2 into C1 products that, in the second stage, can be metabolized by acetogens. This latter scheme leverages the metabolic flexibility of acetogens in efficient utilization of the products of CO2 abiotic hydrogenation, namely formate and methanol, to synthesize multicarbon compounds but also to act as flexible catalysts for hydrogen storage or production.

Efficient production of biliverdin through whole-cell biocatalysis using recombinant Escherichia coli

Biliverdin is an important cellular antioxidant. Traditionally, biliverdin is produced by chemical oxidation of bilirubin, which is a complex process and the final product is of low purity. Here we report an efficient, green and safe process for biotechnological production of biliverdin. A heme oxygenase (HO) gene from Clostridium tetani was screened, and a recombinant strain Escherichia coli BL21/pETDuet-hoCt with the ability of transforming heme into biliverdin was constructed. A biliverdin yield of 32.9 mg/L from 100 mg/L substrate was achieved under pH 7.0 and 35 ℃. In order to improve the supply of reducing power, an NADPH regeneration system using glutamate dehydrogenase (GdhA) was constructed, resulting in a recombinant strain E. coli BL21/pETDuet-gdhAEc-hoCt which was capable of producing 71.5 mg/L biliverdin. Moreover, through introduction of a membrane surface display system, a recombinant strain E. coli BL21/pETDuet-gdhAEc-blc/hoCt was constructed to shorten the transformation time, and the production of biliverdin was further increased to 76.3 mg/L, this is the highest titer of biosynthesized biliverdin reported to date, and the research may thus facilitate the green production of biliverdin.

Artificial microbial consortia for bioproduction processes.

The application of artificial microbial consortia for biotechnological production processes is an emerging field in research as it offers great potential for the improvement of established as well as the development of novel processes. In this review, we summarize recent highlights in the usage of various microbial consortia for the production of, for example, platform chemicals, biofuels, or pharmaceutical compounds. It aims to demonstrate the great potential of co-cultures by employing different organisms and interaction mechanisms and exploiting their respective advantages. Bacteria and yeasts often offer a broad spectrum of possible products, fungi enable the utilization of complex lignocellulosic substrates via enzyme secretion and hydrolysis, and microalgae can feature their abilities to fixate CO2 through photosynthesis for other organisms as well as to form lipids as potential fuelstocks. However, the complexity of interactions between microbes require methods for observing population dynamics within the process and modern approaches such as modeling or automation for process development. After shortly discussing these interaction mechanisms, we aim to present a broad variety of successfully established co-culture processes to display the potential of artificial microbial consortia for the production of biotechnological products.

Applications of Coarse-Grained Models in Metabolic Engineering.

Mathematical modeling is a promising tool for better understanding of cellular processes. In recent years, the development of coarse-grained models has gained attraction since these simple models are able to capture and describe a broad range of growth conditions. Coarse-grained models often comprise only two cellular components, a low molecular component as representative for central metabolism and energy generation and a macromolecular component, representing the entire proteome. A framework is presented that presents a strict mass conservative model for bacterial growth during a biotechnological production process. After providing interesting properties for the steady-state solution, applications are presented 1) for a production process of an amino acid and 2) production of a metabolite from central metabolism.

Application of an Escherichia coli triple reporter strain for at-line monitoring of single-cell physiology during L-phenylalanine production.

Biotechnological production processes are sustainable approaches for the production of biobased components such as amino acids for food and feed industry. Scale-up from ideal lab-scale bioreactors to large-scale processes is often accompanied by loss in productivity. This may be related to population heterogeneities of cells originating from isogenic cultures that arise due to dynamic non-ideal conditions in the bioreactor. To better understand this phenomenon, deeper insights into single-cell physiologies in bioprocesses are mandatory before scale-up. Here, a triple reporter strain (3RP) was developed by chromosomally integrating the fluorescent proteins mEmerald, CyOFP1, and mTagBFP2 into the L-phenylalanine producing Escherichia coli strain FUS4 (pF81kan) to allow monitoring of growth, oxygen availability, and general stress response of the single cells. Functionality of the 3RP was confirmed in well-mixed lab-scale fed-batch processes with glycerol as carbon source in comparison to the strain without fluorescent proteins, leading to no difference in process performance. Fluorescence levels could successfully reflect the course of related process state variables, revealed population heterogeneities during the transition between different process phases and potentially subpopulations that exhibit superior process performance. Furthermore, indications were found for noise in gene expression as regulation strategy against environmental perturbation.