Metabolic Engineering Approaches Research Articles

Enhancing the biosynthesis of taxadien-5α-yl-acetate in Escherichia coli by combinatorial metabolic engineering approaches

Biosynthesis of paclitaxel (Taxol™) is a hot topic with extensive and durable interests for decades. However, it is severely hindered due to the very low titers of intermediates. In this study, Escherichia coli was employed to de novo synthesize a key intermediate of paclitaxel, taxadien-5α-yl-acetate (T5OAc). Plasmid-based pathway reconstruction and optimization were conducted for T5OAc production. The endogenous methylerythritol phosphate pathway was enhanced to increase the precursor supply. Three taxadien-5α-ol O-acetyltransferases were tested to obtain the best enzyme for the acetylation step. Metabolic burden was relieved to restore cell growth and promote production through optimizing the plasmid production system. In order to achieve metabolic balance, the biosynthesis pathway was regulated precisely by multivariate-modular metabolic engineering. Finally, in a 5-L bioreactor, the T5OAc titer was enhanced to reach 10.9 mg/L. This represents an approximately 272-fold increase in production compared to the original strain, marking the highest yield of T5OAc ever documented in E. coli, which is believed to be helpful for promoting the progress of paclitaxel biosynthesis.

Use of acetate as substrate for sustainable production of homoserine and threonine by Escherichia coli W3110: a modular metabolic engineering approach

Acetate, a promising yet underutilized carbon source for biological production, was explored for the efficient production of homoserine and threonine in Escherichia coli W. A modular metabolic engineering approach revealed the crucial roles of both acetate assimilation pathways (AckA/Pta and Acs), optimized TCA cycle flux and glyoxylate shunt activity, and enhanced CoA availability, mediated by increased pantothenate kinase activity, for efficient homoserine production. The engineered strain W-H22/pM2/pR1P exhibited a high acetate assimilation rate (5.47 mmol/g cell/h) and produced 44.1 g/L homoserine in 52 h with a 53% theoretical yield (0.18 mol/mol) in fed-batch fermentation. Similarly, strain W-H31/pM2/pR1P achieved 45.8 g/L threonine in 52 h with a 65% yield (0.22 mol/mol). These results represent the highest reported levels of amino acid production using acetate, highlighting its potential as a valuable and sustainable feedstock for biomanufacturing.

Enhancing erythromycin production in Saccharopolyspora erythraea through rational engineering and fermentation refinement: A Design-Build-Test-Learn approach.

Industrial production of bioactive compounds from actinobacteria, such as erythromycin and its derivatives, faces challenges in achieving optimal yields. To this end, the Design-Build-Test-Learn (DBTL) framework, a systematic metabolic engineering approach, was employed to enhance erythromycin production in Saccharopolyspora erythraea (S. erythraea) E3 strain. A genetically modified strain, S. erythraea E3-CymRP21-dcas9-sucC (S. erythraea CS), was developed by suppressing the sucC gene using an inducible promoter and dcas9 protein. The strain exhibited improved erythromycin synthesis, attributed to enhanced precursor synthesis and increased NADPH availability. Transcriptomic and metabolomic analyses revealed altered central carbon metabolism, amino acid metabolism, energy metabolism, and co-factor/vitamin metabolism in CS. Augmented amino acid metabolism led to nitrogen depletion, potentially causing cellular autolysis during later fermentation stages. By refining the fermentation process through ammonium sulfate supplementation, erythromycin yield reached 1125.66mg L-1, a 43.5% increase. The results demonstrate the power of the DBTL methodology in optimizing erythromycin production, shedding light on its potential for revolutionizing antibiotic manufacturing in response to the global challenge of antibiotic resistance.

Tailoring regulatory components for metabolic engineering in cyanobacteria.

The looming climate crisis has prompted an ever-growing interest in cyanobacteria due to their potential as sustainable production platforms for the synthesis of energy carriers and value-added chemicals from CO2 and sunlight. Nonetheless, cyanobacteria are yet to compete with heterotrophic systems in terms of space-time yields and consequently production costs. One major drawback leading to the low production performance observed in cyanobacteria is the limited ability to utilize the full capacity of the photosynthetic apparatus and its associated systems, i.e. CO2 fixation and the directly connected metabolism. In this review, novel insights into various levels of metabolic regulation of cyanobacteria are discussed, including the potential of targeting these regulatory mechanisms to create a chassis with a phenotype favorable for photoautotrophic production. Compared to conventional metabolic engineering approaches, minor perturbations of regulatory mechanisms can have wide-ranging effects.

Characterization of anthranilic acid produced by Virgibacillus salarius MML1918 and its bio-imaging application.

Anthranilic acid (AA) holds significant importance in the chemical industry. It serves as a crucial building block for the amino acid tryptophan by manipulating the tryptophan biosynthesis pathway, it is possible to increase the production of anthranilic acid. In this study, we utilized metabolic engineering approaches to produce anthranilic acid from the halophilic bacterium Virgibacillus salarius MML1918. The halophilic bacteria were grown in an optimized production medium, and mass production of secondary metabolites was made in ATCC medium 1097 Proteose peptone-for halophilic bacteria and subjected to column chromatography followed by sub-column chromatography the single band for the purified compound was confirmed. Further, various spectral analyses were made for the partially purified compounds, and fluorescence microscopy for fungal cell observation was performed. The purified compound was confirmed by single crystal X-ray diffraction (XRD) analysis, and it was identified as 2-amino benzoic acid. The Fourier transform infrared Spectroscopy (FT-IR) spectrum and nuclear magnetic resonance (NMR) spectrum also confirm the structural characteristic of 2-amino benzoic acid. The UV-Vis absorption spectrum of AA shows the maximum absorption at 337.86nm. The emission spectrum of 2-amino benzoic acid showed the maximum emission at 453nm. The bio-imaging application of 2-amino benzoic acid was examined with fungal mycelium of Rhizoctonia solani. It was effectively bound and emitted the blue color at the concentration of 200 and 300µg/mL. The halophilic bacterium (V. salarius), may have unique metabolic pathways and requirements compared to non-halophilic organisms, to produce AA effectively. This could have implications for industrial biotechnology, particularly in manufacturing environments where high salt concentrations are present and also it can be used as bio-imaging agent.

Efficient Plant Triterpenoids Synthesis in Saccharomyces cerevisiae: from Mechanisms to Engineering Strategies.

Triterpenoids possess a range of biological activities and are extensively utilized in the pharmaceutical, food, cosmetic, and chemical industries. Traditionally, they are acquired through chemical synthesis and plant extraction. However, these methods have drawbacks, including high energy consumption, environmental pollution, and being time-consuming. Recently, the de novo synthesis of triterpenoids in microbial cell factories has been achieved. This represents a promising and environmentally friendly alternative to traditional supply methods. Saccharomyces cerevisiae, known for its robustness, safety, and ample precursor supply, stands out as an ideal candidate for triterpenoid biosynthesis. However, challenges persist in industrial production and economic feasibility of triterpenoid biosynthesis. Consequently, metabolic engineering approaches have been applied to improve the triterpenoid yield, leading to substantial progress. This review explores triterpenoids biosynthesis mechanisms in S.cerevisiae and strategies for efficient production. Finally, the review also discusses current challenges and proposes potential solutions, offering insights for future engineering.

Morphological Engineering of Filamentous Fungi: Research Progress and Perspectives.

Filamentous fungi are important cell factories for the production of high-value enzymes and chemicals for the food, chemical, and pharmaceutical industries. Under submerged fermentation, filamentous fungi exhibit diverse fungal morphologies that are influenced by environmental factors, which in turn affect the rheological properties and mass transfer of the fermentation system, and ultimately the synthesis of products. In this review, we first summarize the mechanisms of mycelial morphogenesis and then provide an overview of current developments in methods and strategies for morphological regulation, including physicochemical and metabolic engineering approaches. We also anticipate that rapid developments in synthetic biology and genetic manipulation tools will accelerate morphological engineering in the future.

Cyclodepsipeptides: Isolation, Bioactivities, Biosynthesis and Total Synthesis

Abstract: Cyclodepsipeptides, mainly derived from marine organisms and soil microorganisms, are amphiphilic molecules consisting of short oligopeptides with fatty acid tails attached to form a macrocyclic structure. Studies on the activity of cyclodepsipeptides have shown that they have cytotoxicity, antibacterial and anthelmintic effects, and are widely used in biological control, drug development, environmental remediation and disease treatment. Cyclodepsipeptides play a prominent role in the development of new drugs and drug lead compounds, especially as antibiotics with great medicinal potentiall, and are slowly seeping into the public consciousness. The biosynthesis of cyclodepsipeptides is mainly based on the synthesis of non-ribosomal peptide synthases, and selection of key regulatory enzymes for homologue regulation and biosynthetic strategies using genetic engineering and metabolic engineering approaches. The biosynthesis method is miniaturised, recyclable, and safer. The total synthesis methods of cyclodepsipeptides are mainly combined solid-liquid phase methods, which synthesise cyclodepsipeptides faster and are easy to purify. This paper reviews the biological activities of cyclodepsipeptides, their biosynthesis, and total synthesis.

Biosynthesis of melatonin from l-tryptophan by an engineered microbial cell factory

BackgroundThe demand for melatonin is increasing due to its health-promoting bioactivities such as antioxidant and sleep benefits. Although melatonin is present in various organisms, its low content and high extraction cost make it unsustainable. Biosynthesis is a promising alternative method for melatonin production. However, the ectopic production of melatonin in microorganisms is very difficult due to the low or insoluble expression of melatonin synthesis genes. Hence, we aim to explore the biosynthesis of melatonin using Escherichia coli as a cell factory and ways to simultaneously coordinated express genes from different melatonin synthesis pathways.ResultsIn this study, the mXcP4H gene from Xanthomonas campestris, as well as the HsAADC, HsAANAT and HIOMT genes from human melatonin synthesis pathway were optimized and introduced into E. coli via a multi-monocistronic vector. The obtained strain BL7992 successfully synthesized 1.13 mg/L melatonin by utilizing L-tryptophan (l-Trp) as a substrate in a shake flask. It was determined that the rate-limiting enzyme for melatonin synthesis is the arylalkylamine N-acetyltransferase, which is encoded by the HsAANAT gene. Targeted metabolomics analysis of l-Trp revealed that the majority of l-Trp flowed to the indole pathway in BL7992, and knockout of the tnaA gene may be beneficial for increasing melatonin production.ConclusionsA metabolic engineering approach was adopted and melatonin was successfully synthesized from low-cost l-Trp in E. coli. This study provides a rapid and economical strategy for the synthesis of melatonin.

Differential gene expression and potential regulatory network of fatty acid biosynthesis during fruit and leaf development in yellowhorn (Xanthoceras sorbifolium), an oil-producing tree with significant deployment values.

Xanthoceras sorbifolium (yellowhorn) is a woody oil plant with super stress resistance and excellent oil characteristics. The yellowhorn oil can be used as biofuel and edible oil with high nutritional and medicinal value. However, genetic studies on yellowhorn are just in the beginning, and fundamental biological questions regarding its very long-chain fatty acid (VLCFA) biosynthesis pathway remain largely unknown. In this study, we reconstructed the VLCFA biosynthesis pathway and annotated 137 genes encoding relevant enzymes. We identified four oleosin genes that package triacylglycerols (TAGs) and are specifically expressed in fruits, likely playing key roles in yellowhorn oil production. Especially, by examining time-ordered gene co-expression network (TO-GCN) constructed from fruit and leaf developments, we identified key enzymatic genes and potential regulatory transcription factors involved in VLCFA synthesis. In fruits, we further inferred a hierarchical regulatory network with MYB-related (XS03G0296800) and B3 (XS02G0057600) transcription factors as top-tier regulators, providing clues into factors controlling carbon flux into fatty acids. Our results offer new insights into key genes and transcriptional regulators governing fatty acid production in yellowhorn, laying the foundation for efforts to optimize oil content and fatty acid composition. Moreover, the gene expression patterns and putative regulatory relationships identified here will inform metabolic engineering and molecular breeding approaches tailored to meet biofuel and bioproduct demands.

A versatile method for conjugating lipid nanoparticles on T cells through combination of click chemistry and metabolic glycoengineering.

Cell-mediated drug delivery by conjugating nanomedicine to the surface of living cells is a promising strategy for enhancing the efficacy of both drug delivery and cell therapy. It exploits the tissue homing properties of the specific cell types to overcome in vivo barriers and forms a drug depot by directly putting the therapeutic payload in target cells. An important concern of developing this system is the method to conjugate nanoparticles on cells. Herein, we developed a bioorthogonal T cell conjugation strategy using SPAAC click chemistry, which allows controllable and highly efficient conjugation without affecting the viability and functions of the cytotoxic T lymphocytes. Azide groups were incorporated on the surface of T cells through metabolic glycoengineering, followed by reacting with dibenzylcyclooctyne (DBCO) modified lipid nanoparticles (LNPs). LNPs can be conjugated to T cells, allowing for the loading of different drug molecules on the cells. The metabolic engineering and click reaction approach provides a simple and versatile strategy to conjugate NPs to living cells and enable the development of sophisticated therapeutic cell products.

Recent advances in keratinase production via protein engineering, breeding, and fermentation

The gene editing and synthetic biological tools have led to the implementation of diverse metabolic engineering approaches to enhance the production of specific enzymes. Microbial keratinases can convert keratin wastes into valuable compounds for mankind. Since the market for keratinases cannot be satisfied by production from wild hosts, it is obligatory to develop hosts with high keratinase yields. The intention of this review is to evaluate microbial keratinase advancement through protein engineering, breeding techniques, and fermentation optimization. The main aim of protein engineering is to improve the heat resistance ability and catalytic activity of keratinases by employing mutagenesis methods. Moreover, modifying the expression elements and host engineering are also two unique ways to augment the keratinase yield. Intending to accelerate the production of modified keratinase, this review attempts to highlight the optimization of expression elements, such as promoter engineering, UTR, signal peptide, and codon optimization. Moreover, the approaches of host engineering including strengthening precursor supply, membrane surface engineering, and optimization of secretion pathways were also explained here. Furthermore, it is also essential to optimize the medium composition and fermentation condition for high keratinase yield. This review also addressed the present advancements, difficulties, and tendencies in the field of microbial keratinase production, along with its potential.

Improved production of Taxol® precursors in S. cerevisiae using combinatorial in silico design and metabolic engineering

BackgroundIntegrated metabolic engineering approaches that combine system and synthetic biology tools enable the efficient design of microbial cell factories for synthesizing high-value products. In this study, we utilized in silico design algorithms on the yeast genome-scale model to predict genomic modifications that could enhance the production of early-step Taxol® in engineered Saccharomyces cerevisiae cells.ResultsUsing constraint-based reconstruction and analysis (COBRA) methods, we narrowed down the solution set of genomic modification candidates. We screened 17 genomic modifications, including nine gene deletions and eight gene overexpressions, through wet-lab studies to determine their impact on taxadiene production, the first metabolite in the Taxol® biosynthetic pathway. Under different cultivation conditions, most single genomic modifications resulted in increased taxadiene production. The strain named KM32, which contained four overexpressed genes (ILV2, TRR1, ADE13, and ECM31) involved in branched-chain amino acid biosynthesis, the thioredoxin system, de novo purine synthesis, and the pantothenate pathway, respectively, exhibited the best performance. KM32 achieved a 50% increase in taxadiene production, reaching 215 mg/L. Furthermore, KM32 produced the highest reported yields of taxa-4(20),11-dien-5α-ol (T5α-ol) at 43.65 mg/L and taxa-4(20),11-dien-5-α-yl acetate (T5αAc) at 26.2 mg/L among early-step Taxol® metabolites in S. cerevisiae.ConclusionsThis study highlights the effectiveness of computational and integrated approaches in identifying promising genomic modifications that can enhance the performance of yeast cell factories. By employing in silico design algorithms and wet-lab screening, we successfully improved taxadiene production in engineered S. cerevisiae strains. The best-performing strain, KM32, achieved substantial increases in taxadiene as well as production of T5α-ol and T5αAc. These findings emphasize the importance of using systematic and integrated strategies to develop efficient yeast cell factories, providing potential implications for the industrial production of high-value isoprenoids like Taxol®.

Gene identification and semisynthesis of the anti-inflammatory oleanane-type triterpenoid wilforlide A.

Wilforlide A is one of the main active constituents produced in trace amounts in Tripterygium wilfordii Hook F, which has excellent anti-inflammatory and immune suppressive effects. Despite the seeming structural simplicity of the compound, the biosynthetic pathway of wilforlide A remains unknown. Gene-specific expression analysis and genome mining were used to identify the gene candidates, and their functions were studied in vitro and in vivo. A modularized two-step (M2S) technique and CRISPR-Cas9 methods were used to construct engineering yeast. Here, we identified a cytochrome P450, TwCYP82AS1, that catalyses C-22 hydroxylation during wilforlide A biosynthesis. We also found that TwCYP712K1 to K3 can further oxidize the C-29 carboxylation of oleanane-type triterpenes in addition to friedelane-type triterpenes. Reconstitution of the biosynthetic pathway in engineered yeast increased the precursor supply, and combining TwCYP82AS1 and TwCYP712Ks produced abrusgenic acid, which was briefly acidified to achieve the semisynthesis of wilforlide A. Our work presents an alternative metabolic engineering approach for obtaining wilforlide A without relying on extraction from plants.

The oxygen-tolerant reductive glycine pathway assimilates methanol, formate and CO2 in the yeast Komagataella phaffii

The current climatic change is predominantly driven by excessive anthropogenic CO2 emissions. As industrial bioprocesses primarily depend on food-competing organic feedstocks or fossil raw materials, CO2 co-assimilation or the use of CO2-derived methanol or formate as carbon sources are considered pathbreaking contributions to solving this global problem. The number of industrially-relevant microorganisms that can use these two carbon sources is limited, and even fewer can concurrently co-assimilate CO2. Here, we search for alternative native methanol and formate assimilation pathways that co-assimilate CO2 in the industrially-relevant methylotrophic yeast Komagataella phaffii (Pichia pastoris). Using 13C-tracer-based metabolomic techniques and metabolic engineering approaches, we discover and confirm a growth supporting pathway based on native enzymes that can perform all three assimilations: namely, the oxygen-tolerant reductive glycine pathway. This finding paves the way towards metabolic engineering of formate and CO2 utilisation to produce proteins, biomass, or chemicals in yeast.

Perspectives for Using CO2 as a Feedstock for Biomanufacturing of Fuels and Chemicals.

Microbial cell factories offer an eco-friendly alternative for transforming raw materials into commercially valuable products because of their reduced carbon impact compared to conventional industrial procedures. These systems often depend on lignocellulosic feedstocks, mainly pentose and hexose sugars. One major hurdle when utilizing these sugars, especially glucose, is balancing carbon allocation to satisfy energy, cofactor, and other essential component needs for cellular proliferation while maintaining a robust yield. Nearly half or more of this carbon is inevitably lost as CO2 during the biosynthesis of regular metabolic necessities. This loss lowers the production yield and compromises the benefit of reducing greenhouse gas emissions-a fundamental advantage of biomanufacturing. This review paper posits the perspectives of using CO2 from the atmosphere, industrial wastes, or the exhausted gases generated in microbial fermentation as a feedstock for biomanufacturing. Achieving the carbon-neutral or -negative goals is addressed under two main strategies. The one-step strategy uses novel metabolic pathway design and engineering approaches to directly fix the CO2 toward the synthesis of the desired products. Due to the limitation of the yield and efficiency in one-step fixation, the two-step strategy aims to integrate firstly the electrochemical conversion of the exhausted CO2 into C1/C2 products such as formate, methanol, acetate, and ethanol, and a second fermentation process to utilize the CO2-derived C1/C2 chemicals or co-utilize C5/C6 sugars and C1/C2 chemicals for product formation. The potential and challenges of using CO2 as a feedstock for future biomanufacturing of fuels and chemicals are also discussed.

Production of poly(3-hydroxybutyrate)/poly(lactic acid) from industrial wastewater by wild-type Cupriavidus necator H16

The massive production of urban and industrial wastes has created a clear need for alternative waste management processes. One of the more promising strategies is to use waste as raw material for the production of biopolymers such as polyhydroxyalkanoates (PHAs). In this work, a lactate-enriched stream obtained by anaerobic digestion (AD) of wastewater (WW) from a candy production plant was used as a feedstock for PHA production in wild-type Cupriavidus necator H16. Unexpectedly, we observed the accumulation of poly(3-hydroxybutyrate)/poly(lactic acid) (P(3HB)/PLA), suggesting that the non-engineered strain already possesses the metabolic potential to produce these polymers of interest. The systematic study of factors, such as incubation time, nitrogen and lactate concentration, influencing the synthesis of P(3HB)/PLA allowed the production of a panel of polymers in a resting cell system with tailored lactic acid (LA) content according to the GC–MS of the biomass. Further biomass extraction suggested the presence of methanol soluble low molecular weight molecules containing LA, while 1 % LA could be detected in the purified polymer fraction. These results suggested that the cells are producing a blend of polymers. A proteomic analysis of C. necator resting cells under P(3HB)/PLA production conditions provides new insights into the latent pathways involved in this process. This study is a proof of concept demonstrating that LA can polymerize in a non-modified organism and paves the way for new metabolic engineering approaches for lactic acid polymer production in the model bacterium C. necator H16.

Recent Challenges and Trends of Polyhydroxyalkanoate Production by Extremophilic Bacteria Using Renewable Feedstocks.

Polyhydroxyalkanoates (PHAs) are biodegradable polymers with immense potential in addressing the global plastic pollution crisis and advancing sustainable bioplastics production. Among the various microbes known for PHA production, extremophilic bacteria possess unique capabilities to thrive under extreme conditions, making them attractive candidates for PHA synthesis. Furthermore, the utilization of renewable feedstocks for PHA production aligns with the growing demand for sustainable bioplastic alternatives. A diverse range of extremophilic bacteria, especially halophiles and thermophiles, has provided cost-competitive platforms for producing customized PHA polymers. Extremophilic bacteria offer unique advantages over mesophiles due to their contamination resistance, high cell density growth, and unique culture conditions. The current status of Halomonas spp. as a chassis further allows exploration of metabolic engineering approaches to overcome the challenges associated with current industrial biotechnology. This article especially focuses on extremophilic bacteria and explores recent advances in utilizing renewable feedstocks such as lignocellulosic biomass, agro-industrial residues, and waste streams for PHA production. The integration of biorefinery concepts and circular economy principles in PHA manufacturing is also examined. This review is an attempt to provide an understanding of renewable substrates as feedstocks and emerging trends in PHA production by extremophilic bacteria. It underscores the pivotal role of extremophiles and sustainable feedstock sources in advancing the feasibility and eco-friendliness of PHAs as a promising biopolymer alternative.

Biofuels from cyanobacteria -a metabolic engineering approach

The concern about the limited availability of petroleum-based fuels and their role in increasing CO2 levels in the atmosphere has sparked significant attention toward biofuel and bioenergy production. The global pursuit of sustainable energy sources has catalyzed innovative research into alternative biofuel production strategies. Transforming CO2 into usable fuels and chemicals is gaining even more prominence. Cyanobacteria, renowned for their photosynthetic ability, have emerged as promising candidates for biofuel synthesis. Their ability to convert solar energy and carbon dioxide into valuable biofuels makes them a compelling avenue for sustainable energy solutions. Using metabolic engineering principles, researchers have endeavored to optimize cyanobacterial metabolic pathways, enhance photosynthetic efficiency, and redirect carbon flux toward biofuel precursors. Numerous species of cyanobacteria offer genetic and metabolic traits that facilitate manipulation, and their photosynthetic characteristics imply that carbohydrates, fatty acids, and even alcohol could serve as potential renewable sources for biofuels. This review showcases cyanobacteria's ability as a biofuel source and emphasizes the transformative influence of metabolic engineering employed in the creation and production of "cyanofuels”

Recent advances in methanol production from methanotrophs.

Methanol, the simplest aliphatic molecule of the alcohol family, finds diverse range of applications as an industrial solvent, a precursor for producing other chemicals (e.g., dimethyl ether, acetic acid and formaldehyde), and a potential fuel. There are conventional chemical routes for methanol production such as, steam reforming of natural gas to form syngas, followed by catalytic conversion into methanol; direct catalytic oxidation of methane, or hydrogenation of carbon dioxide. However, these chemical routes are limited by the requirement for expensive catalysts and extreme process conditions, and plausible environmental implications. Alternatively, methanotrophic microorganisms are being explored as biological alternative for methanol production, under milder process conditions, bypassing the requirement for chemical catalysts, and without imposing any adverse environmental impact. Methanotrophs possess inherent metabolic pathways for methanol production via biological methane oxidation or carbon dioxide reduction, thus offering a surplus advantage pertaining to the sequestration of two major greenhouse gases. This review sheds light on the recent advances in methanotrophic methanol production including metabolic pathways, feedstocks, metabolic engineering, and bioprocess engineering approaches. Furthermore, various reactor configurations are discussed in view of the challenges associated with solubility and mass transfer limitations in methanotrophic gas fermentation systems.