Zhen Chen, F. Geng, A. Zeng

Biotechnology Journal • 2015

Protein engineering to expand the substrate spectrum of native enzymes opens new possibilities for bioproduction of valuable chemicals from non-natural pathways. No natural microorganism can directly use sugars to produce 1,3-propanediol (PDO). Here, we present a de novo route for the biosynthesis of PDO from sugar, which may overcome the mentioned limitations by expanding the homoserine synthesis pathway. The accomplishment of pathway from homoserine to PDO is achieved by protein engineering of glutamate dehydrogenase (GDH) and pyruvate decarboxylase to sequentially convert homoserine to 4-hydroxy-2-ketobutyrate and 3-hydroxypropionaldehyde. The latter is finally converted to PDO by using a native alcohol dehydrogenase. In this work, we report on experimental accomplishment of this non-natural pathway, especially by protein engineering of GDH for the key step of converting homoserine to 4-hydroxy-2-ketobutyrate. These results show the feasibility and significance of protein engineering for de novo pathway design and overproduction of desired industrial products.

Khaled Elbanna, Fatimah S Alsulami, L. Neyaz et al.

Frontiers in Microbiology • 2024

Microbial biopolymers have emerged as promising solutions for environmental pollution-related human health issues. Poly-γ-glutamic acid (γ-PGA), a natural anionic polymeric compound, is composed of highly viscous homo-polyamide of D and L-glutamic acid units. The extracellular water solubility of PGA biopolymer facilitates its complete biodegradation and makes it safe for humans. The unique properties have enabled its applications in healthcare, pharmaceuticals, water treatment, foods, and other domains. It is applied as a thickener, taste-masking agent, stabilizer, texture modifier, moisturizer, bitterness-reducing agent, probiotics cryoprotectant, and protein crystallization agent in food industries. γ-PGA is employed as a biological adhesive, drug carrier, and non-viral vector for safe gene delivery in tissue engineering, pharmaceuticals, and medicine. It is also used as a moisturizer to improve the quality of hair care and skincare cosmetic products. In agriculture, it serves as an ideal stabilizer, environment-friendly fertilizer synergist, plant-growth promoter, metal biosorbent in soil washing, and animal feed additive to reduce body fat and enhance egg-shell strength.

John Löfblom, R. Rosenstein, M. Nguyen et al.

Applied Microbiology and Biotechnology • 2017

Since the 1950s, Staphylococcus carnosus is used as a starter culture for sausage fermentation where it contributes to food safety, flavor, and a controlled fermentation process. The long experience with S. carnosus has shown that it is a harmless and “food grade” species. This was confirmed by the genome sequence of S. carnosus TM300 that lacks genes involved in pathogenicity. Since the development of a cloning system in TM300, numerous genes have been cloned, expressed, and characterized and in particular, virulence genes that could be functionally validated in this non-pathogenic strain. A secretion system was developed for production and secretion of industrially important proteins and later modified to also enable display of heterologous proteins on the surface. The display system has been employed for various purposes, such as development of live bacterial delivery vehicles as well as microbial biocatalysts or bioadsorbents for potential environmental or biosensor applications. Recently, this surface display system has been utilized for display of peptide and protein libraries for profiling of protease substrates and for generation of various affinity proteins, e.g., Affibody molecules and scFv antibodies. In addition, by display of fragmented antigen-encoding genes, the surface expression system has been successfully used for epitope mapping of antibodies. Reviews on specific applications of S. carnosus have been published earlier, but here we provide a more extensive overview, covering a broad range of areas from food fermentation to sophisticated methods for protein-based drug discovery, which are all based on S. carnosus.

Anna M. Duraj-Thatte, Avinash Manjula-Basavanna, J. Rutledge et al.

Nature Communications • 2021

Living cells have the capability to synthesize molecular components and precisely assemble them from the nanoscale to build macroscopic living functional architectures under ambient conditions. The emerging field of living materials has leveraged microbial engineering to produce materials for various applications but building 3D structures in arbitrary patterns and shapes has been a major challenge. Here we set out to develop a bioink, termed as “microbial ink” that is produced entirely from genetically engineered microbial cells, programmed to perform a bottom-up, hierarchical self-assembly of protein monomers into nanofibers, and further into nanofiber networks that comprise extrudable hydrogels. We further demonstrate the 3D printing of functional living materials by embedding programmed Escherichia coli (E. coli) cells and nanofibers into microbial ink, which can sequester toxic moieties, release biologics, and regulate its own cell growth through the chemical induction of rationally designed genetic circuits. In this work, we present the advanced capabilities of nanobiotechnology and living materials technology to 3D-print functional living architectures. Living cells can precisely assemble to build 3D functional architectures. Here the authors produce an extrudable microbial ink entirely from the engineered cells, which can be further programmed to 3D print functional living materials.

Xing Kai Chia, Tony Hadibarata, R. A. Kristanti et al.

Bioprocess and Biosystems Engineering • 2024

The use of pesticides and the subsequent accumulation of residues in the soil has become a worldwide problem. Organochlorine (OC) pesticides have spread widely in the environment and caused contamination from past agricultural activities. This article reviews the bioremediation of pesticide compounds in soil using microbial enzymes, including the enzymatic degradation pathway and the recent development of enzyme-mediated bioremediation. Enzyme-mediated bioremediation is divided into phase I and phase II, where the former increases the solubility of pesticide compounds through oxidation–reduction and hydrolysis reactions, while the latter transforms toxic pollutants into less toxic or nontoxic products through conjugation reactions. The identified enzymes that can degrade OC insecticides include dehalogenases, phenol hydroxylase, and laccases. Recent developments to improve enzyme-mediated bioremediation include immobilization, encapsulation, and protein engineering, which ensure its stability, recyclability, handling and storage, and better control of the reaction.

Shannon M. Hoffman, Allison Tang, J. Avalos

Annual Review of Chemical and Biomolecular Engineering • 2022

Optogenetics has been used in a variety of microbial engineering applications, such as chemical and protein production, studies of cell physiology, and engineered microbe-host interactions. These diverse applications benefit from the precise spatiotemporal control that light affords, as well as its tunability, reversibility, and orthogonality. This combination of unique capabilities has enabled a surge of studies in recent years investigating complex biological systems with completely new approaches. We briefly describe the optogenetic tools that have been developed for microbial engineering, emphasizing the scientific advancements that they have enabled. In particular, we focus on the unique benefits and applications of implementing optogenetic control, from bacterial therapeutics to cybergenetics. Finally, we discuss future research directions, with special attention given to the development of orthogonal multichromatic controls. With an abundance of advantages offered by optogenetics, the future is bright in microbial engineering. Expected final online publication date for the Annual Review of Chemical and Biomolecular Engineering, Volume 13 is October 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Oliver P. F. Windram, Rui Rodrigues, Sangjin Lee et al.

Nucleic Acids Research • 2017

Abstract The ability to program cellular behaviour is a major goal of synthetic biology, with applications in health, agriculture and chemicals production. Despite efforts to build ‘orthogonal’ systems, interactions between engineered genetic circuits and the endogenous regulatory network of a host cell can have a significant impact on desired functionality. We have developed a strategy to rewire the endogenous cellular regulatory network of yeast to enhance compatibility with synthetic protein and metabolite production. We found that introducing novel connections in the cellular regulatory network enabled us to increase the production of heterologous proteins and metabolites. This strategy is demonstrated in yeast strains that show significantly enhanced heterologous protein expression and higher titers of terpenoid production. Specifically, we found that the addition of transcriptional regulation between free radical induced signalling and nitrogen regulation provided robust improvement of protein production. Assessment of rewired networks revealed the importance of key topological features such as high betweenness centrality. The generation of rewired transcriptional networks, selection for specific phenotypes, and analysis of resulting library members is a powerful tool for engineering cellular behavior and may enable improved integration of heterologous protein and metabolite pathways.

Nicole M. Gaudelli, Alexis C. Komor, H. Rees et al.

Nature • 2017

The spontaneous deamination of cytosine is a major source of transitions from C•G to T•A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted A•T base pairs to G•C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR–Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted A•T base pairs efficiently to G•C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.

J. Gu, N. Isozumi, Shouli Yuan et al.

Molecular Biology and Evolution • 2021

Abstract Antimicrobial peptides (AMPs) have been considered as the alternatives to antibiotics because of their less susceptibility to microbial resistance. However, compared with conventional antibiotics they show relatively low activity and the consequent high cost and nonspecific cytotoxicity, hindering their clinical application. What’s more, engineering of AMPs is a great challenge due to the inherent complexity in their sequence, structure, and function relationships. Here, we report an evolution-based strategy for improving the antifungal activity of a nematode-sourced defensin (Cremycin-5). This strategy utilizes a sequence-activity comparison between Cremycin-5 and its functionally diverged paralogs to identify sites associated with antifungal activity for screening of enhanceable activity-modulating sites for subsequent saturation mutagenesis. Using this strategy, we identified a site (Glu-15) whose mutations with nearly all other types of amino acids resulted in a universally enhanced activity against multiple fungal species, which is thereby defined as a Universally Enhanceable Activity-Modulating Site (UEAMS). Especially, Glu15Lys even exhibited >9-fold increased fungicidal potency against several clinical isolates of Candida albicans through inhibiting cytokinesis. This mutant showed high thermal and serum stability and quicker killing kinetics than clotrimazole without detectable hemolysis. Molecular dynamic simulations suggest that the mutations at the UEAMS likely limit the conformational flexibility of a distant functional residue via allostery, enabling a better peptide–fungus interaction. Further sequence, structural, and mutational analyses of the Cremycin-5 ortholog uncover an epistatic interaction between the UEAMS and another site that may constrain its evolution. Our work lights one new road to success of engineering AMP drug leads.

Wenliang Hao, Wenjing Cui, Zhongmei Liu et al.

Advanced Science • 2024

Base editors (BEs) are widely used as revolutionary genome manipulation tools for cell evolution. To screen the targeted individuals, it is often necessary to expand the editing window to ensure highly diverse variant library. However, current BEs suffer from a limited editing window of 5–6 bases, corresponding to only 2–3 amino acids. Here, by engineering the CRISPR‒Cas12b, the study develops dCas12b‐based CRISPRi system, which can efficiently repress gene expression by blocking the initiation and elongation of gene transcription. Further, based on dCas12b, a new‐generation of BEs with an expanded editing window is established, covering the entire protospacer or more. The expanded editing window results from the smaller steric hindrance compared with other Cas proteins. The universality of the new BE is successfully validated in Bacillus subtilis and Escherichia coli. As a proof of concept, a spectinomycin‐resistant E. coli strain (BL21) and a 6.49‐fold increased protein secretion efficiency in E. coli JM109 are successfully obtained by using the new BE. The study, by tremendously expanding the editing window of BEs, increased the capacity of the variant library exponentially, greatly increasing the screening efficiency for microbial cell evolution.

Daniel C. Zielinski, Arjun Patel, B. Palsson

Microorganisms • 2020

Microbial strains are being engineered for an increasingly diverse array of applications, from chemical production to human health. While traditional engineering disciplines are driven by predictive design tools, these tools have been difficult to build for biological design due to the complexity of biological systems and many unknowns of their quantitative behavior. However, due to many recent advances, the gap between design in biology and other engineering fields is closing. In this work, we discuss promising areas of development of computational tools for engineering microbial strains. We define five frontiers of active research: (1) Constraint-based modeling and metabolic network reconstruction, (2) Kinetics and thermodynamic modeling, (3) Protein structure analysis, (4) Genome sequence analysis, and (5) Regulatory network analysis. Experimental and machine learning drivers have enabled these methods to improve by leaps and bounds in both scope and accuracy. Modern strain design projects will require these tools to be comprehensively applied to the entire cell and efficiently integrated within a single workflow. We expect that these frontiers, enabled by the ongoing revolution of big data science, will drive forward more advanced and powerful strain engineering strategies.

Rong Chen, Yuheng Liu, Shu Chen et al.

Frontiers in Plant Science • 2022

Germacrene A (GA) is a key intermediate for the synthesis of medicinal active compounds, especially for β-elemene, which is a broad-spectrum anticancer drug. The production of sufficient GA in the microbial platform is vital for the precursors supply of active compounds. In this study, Escherichia coli BL21 Star (DE3) was used as the host and cultivated in SBMSN medium, obtaining a highest yield of FPP. The GA synthase from Lactuca sativa (LTC2) exhibited the highest level of GA production. Secondly, two residues involved in product release (T410 and T392) were substituted with Ser and Ala, respectively, responsible for relatively higher activities. Next, substitution of selected residues S243 with Asn caused an increase in activity. Furthermore, I364K-T410S and T392A-T410S were created by combination with the beneficial mutation, and they demonstrated dramatically enhanced titers with 1.90-fold and per-cell productivity with 5.44-fold, respectively. Finally, the production titer of GA reached 126.4 mg/L, and the highest productivity was 7.02 mg/L.h by the I364K-T410S mutant in a shake-flask batch culture after fermentation for 18 h. To our knowledge, the productivity of the I364K-T410S mutant is the highest level ever reported. These results highlight a promising method for the industrial production of GA in E. coli, and lay a foundation for pathway reconstruction and the production of valuable natural sesquiterpenes.

Linxia Liu, Jinlong Li, Yuanming Gai et al.

Nature Communications • 2023

Vitamin B6 is an essential nutrient with extensive applications in the medicine, food, animal feed, and cosmetics industries. Pyridoxine (PN), the most common commercial form of vitamin B6, is currently chemically synthesized using expensive and toxic chemicals. However, the low catalytic efficiencies of natural enzymes and the tight regulation of the metabolic pathway have hindered PN production by the microbial fermentation process. Here, we report an engineered Escherichia coli strain for PN production. Parallel pathway engineering is performed to decouple PN production and cell growth. Further, protein engineering is rationally designed including the inefficient enzymes PdxA, PdxJ, and the initial enzymes Epd and Dxs. By the iterative multimodule optimization strategy, the final strain produces 1.4 g/L of PN with productivity of 29.16 mg/L/h by fed-batch fermentation. The strategies reported here will be useful for developing microbial strains for the production of vitamins and other bioproducts having inherently low metabolic fluxes.

Melodie M. Machovina, S.J.B. Mallinson, B. Knott et al.

Proceedings of the National Academy of Sciences • 2019

Significance Lignin is an abundant but underutilized heterogeneous polymer found in terrestrial plants. In current lignocellulosic biorefinery paradigms, lignin is primarily slated for incineration, but for a nonfood plant-based bioeconomy to be successful, lignin valorization is critical. An emerging concept to valorize lignin uses aromatic–catabolic pathways and microbes to funnel heterogeneous lignin-derived aromatic compounds to single high-value products. For this approach to be viable, the discovery and engineering of enzymes to conduct key reactions is critical. In this work, we have engineered a two-component cytochrome P450 enzyme system to conduct one of the most important reactions in biological lignin conversion: aromatic O-demethylation of syringol, the base aromatic unit of S-lignin, which is highly abundant in hardwoods and grasses. Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism is O-aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringol O-demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover in Pseudomonas putida KT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromatic O-demethylases in the biological conversion of lignin-derived aromatic compounds.

Giles Obinna Ndochinwa, Qing-Yan Wang, N. O. Okoro et al.

Open Life Sciences • 2024

Abstract Recent advancements in protein/enzyme engineering have enabled the production of a diverse array of high-value compounds in microbial systems with the potential for industrial applications. The goal of this review is to articulate some of the most recent protein engineering advances in bacteria, yeast, and other microbial systems to produce valuable substances. These high-value substances include α-farnesene, vitamin B12, fumaric acid, linalool, glucaric acid, carminic acid, mycosporine-like amino acids, patchoulol, orcinol glucoside, d-lactic acid, keratinase, α-glucanotransferases, β-glucosidase, seleno-methylselenocysteine, fatty acids, high-efficiency β-glucosidase enzymes, cellulase, β-carotene, physcion, and glucoamylase. Additionally, recent advances in enzyme engineering for enhancing thermostability will be discussed. These findings have the potential to revolutionize various industries, including biotechnology, food, pharmaceuticals, and biofuels.

Kristof Verbeeck, Jo De Vrieze, Ilje Pikaar et al.

Microbial Biotechnology • 2021

Summary Anaerobic digesters produce biogas, a mixture of predominantly CH 4 and CO 2 , which is typically incinerated to recover electrical and/or thermal energy. In a context of circular economy, the CH 4 and CO 2 could be used as chemical feedstock in combination with ammonium from the digestate. Their combination into protein‐rich bacterial, used as animal feed additive, could contribute to the ever growing global demand for nutritive protein sources and improve the overall nitrogen efficiency of the current agro‐ feed/food chain. In this concept, renewable CH 4 and H 2 can serve as carbon‐neutral energy sources for the production of protein‐rich cellular biomass, while assimilating and upgrading recovered ammonia from the digestate. This study evaluated the potential of producing sustainable high‐quality protein additives in a decentralized way through coupling anaerobic digestion and microbial protein production using methanotrophic and hydrogenotrophic bacteria in an on‐farm bioreactor. We show that a practical case digester handling liquid piggery manure, of which the energy content is supplemented for 30% with co‐substrates, provides sufficient biogas to allow the subsequent microbial protein as feed production for about 37% of the number of pigs from which the manure was derived. Overall, producing microbial protein on the farm from available methane and ammonia liberated by anaerobic digesters treating manure appears economically and technically feasible within the current range of market prices existing for high‐quality protein. The case of producing biomethane for grid injection and upgrading the CO 2 with electrolytic hydrogen to microbial protein by means of hydrogen‐oxidizing bacteria was also examined but found less attractive at the current production prices of renewable hydrogen. Our calculations show that this route is only of commercial interest if the protein value equals the value of high‐value protein additives like fishmeal and if the avoided costs for nutrient removal from the digestate are taken into consideration.

Hongyu Zhao, Zhenyu Yao, Xiangbin Chen et al.

Microbial Biotechnology • 2017

Summary Phasins are unusual amphiphilic proteins that bind to microbial polyhydroxyalkanoate ( PHA ) granules in nature and show great potential for various applications in biotechnology and medicine. Despite their remarkable diversity, only the crystal structure of Pha P A h from Aeromonas hydrophila has been solved to date. Based on the structure of Pha P A h , homology models of Pha P A z from Azotobacter sp . FA ‐8 and Pha P TD from Halomonas bluephagenesis TD were successfully established, allowing rational mutagenesis to be conducted to enhance the stability and surfactant properties of these proteins. Pha P A z mutants, including Pha P A z Q38L and Pha P A z Q78L, as well as Pha P TD mutants, including Pha P TD Q 38M and Pha P TD Q 72M, showed better emulsification properties and improved thermostability (6‐10°C higher melting temperatures) compared with their wild‐type homologues under the same conditions. Importantly, the established PhaP homology‐modelling approach, based on the high‐resolution structure of Pha P A h , can be generalized to facilitate the study of other PhaP members.

Jitendra Kumar

Advances in Poultry Nutrition Research • 2021

Feathers are hard waste products, mainly composed of hard β-keratin, and are produced in large quantities in commercial poultry processing plants. Therefore, their industrial utilization is important economically as well as environmentally. Feathers degradation through keratinolytic microorganisms has been considered as an important method for efficient bioconversion, nutritional enhancement and eco-friendliness. The use of crude keratinase significantly increased the amino acid digestibility of raw feathers and commercial feather meal. This enzyme increased the digestibility of commercial feather meal and could replace as much as 7% of the dietary protein for growing chicks. However, feathers are currently utilized on a limited basis as a dietary protein supplement for animal feed because feather meal production is an expensive process, requiring significant amounts of energy. This review paper explains the nutritive value of feathers which makes suitable and inexpensive animal and poultry feed.

Jackson Z. Lee, Andrew Logan, Seth Terry et al.

Microbial Biotechnology • 2015

Summary As global fisheries decline, microbial single‐cell protein ( SCP ) produced from brewery process water has been highlighted as a potential source of protein for sustainable animal feed. However, biotechnological investigation of SCP is difficult because of the natural variation and complexity of microbial ecology in wastewater bioreactors. In this study, we investigate microbial response across a full‐scale brewery wastewater treatment plant and a parallel pilot bioreactor modified to produce an SCP product. A pyrosequencing survey of the brewery treatment plant showed that each unit process selected for a unique microbial community. Notably, flow equalization basins were dominated by P revotella , methanogenesis effluent had the highest levels of diversity, and clarifier wet‐well samples were sources of sequences for the candidate bacterial phyla of TM 7 and BD 1‐5. Next, the microbial response of a pilot bioreactor producing SCP was tracked over 1 year, showing that two different production trials produced two different communities originating from the same starting influent. However, SCP production resulted generally in enrichment of several clades of rhizospheric diazotrophs of A lphaproteobacteria and B etaproteobacteria in the bioreactor and even more so in the final product. These diazotrophs are potentially useful as the basis of a SCP product for commercial feed production.

Joseph A. Gredell, Christopher S. Frei, Patrick C. Cirino

Biotechnology Journal • 2012

Abstract Nature takes advantage of the malleability of protein and RNA sequence and structure to employ these macromolecules as molecular reporters whose conformation and functional roles depend on the presence of a specific ligand (an “effector” molecule). By following nature's example, ligand‐responsive proteins and RNA molecules are now routinely engineered and incorporated into customized molecular reporting systems (biosensors). Microbial small‐molecule biosensors and endogenous molecular reporters based on these sensing components find a variety of applications that include high‐throughput screening of biosynthesis libraries, environmental monitoring, and novel gene regulation in synthetic biology. Here, we review recent advances in engineering small‐molecule recognition by proteins and RNA and in coupling in vivo ligand binding to reporter‐gene expression or to allosteric activation of a protein conferring a detectable phenotype. Emphasis is placed on microbial screening systems that serve as molecular reporters and facilitate engineering the ligand‐binding component to recognize new molecules.

Silvio Matassa, Nico Boon, Ilje Pikaar et al.

Microbial Biotechnology • 2016

Summary Microbial biotechnology has a long history of producing feeds and foods. The key feature of today's market economy is that protein production by conventional agriculture based food supply chains is becoming a major issue in terms of global environmental pollution such as diffuse nutrient and greenhouse gas emissions, land use and water footprint. Time has come to re‐assess the current potentials of producing protein‐rich feed or food additives in the form of algae, yeasts, fungi and plain bacterial cellular biomass, producible with a lower environmental footprint compared with other plant or animal‐based alternatives. A major driver is the need to no longer disintegrate but rather upgrade a variety of low‐value organic and inorganic side streams in our current non‐cyclic economy. In this context, microbial bioconversions of such valuable matters to nutritive microbial cells and cell components are a powerful asset. The worldwide market of animal protein is of the order of several hundred million tons per year, that of plant protein several billion tons of protein per year; hence, the expansion of the production of microbial protein does not pose disruptive challenges towards the process of the latter. Besides protein as nutritive compounds, also other cellular components such as lipids (single cell oil), polyhydroxybuthyrate, exopolymeric saccharides, carotenoids, ectorines, (pro)vitamins and essential amino acids can be of value for the growing domain of novel nutrition. In order for microbial protein as feed or food to become a major and sustainable alternative, addressing the challenges of creating awareness and achieving public and broader regulatory acceptance are real and need to be addressed with care and expedience.

Anping Su, Qijun Yu, Ying Luo et al.

Microbial Biotechnology • 2021

Summary Gamma‐aminobutyric acid (GABA) and delta‐aminolevulinic acid (ALA), playing important roles in agriculture, medicine and other fields, are multifunctional non‐protein amino acids with similar and comparable properties and biosynthesis pathways. Recently, microbial synthesis has become an inevitable trend to produce GABA and ALA due to its green and sustainable characteristics. In addition, the development of metabolic engineering and synthetic biology has continuously accelerated and increased the GABA and ALA yield in microorganisms. Here, focusing on the current trends in metabolic engineering strategies for microbial synthesis of GABA and ALA, we analysed and compared the efficiency of various metabolic strategies in detail. Moreover, we provide the insights to meet challenges of realizing industrially competitive strains and highlight the future perspectives of GABA and ALA production.

Meirong Zhao, Jianfan Ma, Lei Zhang et al.

Microbial Cell Factories • 2024

Abstract Microbial proteins are promising substitutes for animal- and plant-based proteins. S. cerevisiae , a generally recognized as safe (GRAS) microorganism, has been frequently employed to generate heterologous proteins. However, constructing a universal yeast chassis for efficient protein production is still a challenge due to the varying properties of different proteins. With progress in synthetic biology, a multitude of molecular biology tools and metabolic engineering strategies have been employed to alleviate these issues. This review first analyses the advantages of protein production by S. cerevisiae . The most recent advances in improving heterologous protein yield are summarized and discussed in terms of protein hyperexpression systems, protein secretion engineering, glycosylation pathway engineering and systems metabolic engineering. Furthermore, the prospects for efficient and sustainable heterologous protein production by S. cerevisiae are also provided.

Marianna Villano, Federico Aulenta, Mauro Majone

Asia-Pacific Journal of Chemical Engineering • 2012

ABSTRACT Bioelectrochemical systems (BESs) are an emerging technology that uses solid‐state electrodes to stimulate microbial metabolism (either for substrate degradation or for products formation). Because of their versatility and unmatched level of control over the biological reactions, BESs hold a great potential for application in industrial and environmental bioprocesses. Among them, the bioelectrochemical production of renewable and carbon‐neutral energy carriers, such as hydrogen and methane, at the cathode of bioelectrochemical systems is recently attracting considerable attention. While exciting as a concept, the performance of the process seems to be still primarily limited by the low kinetics and efficiencies of the cathodic reactions. In this review, key opportunities for gaseous biofuels production with bioelectrochemical systems are addressed and compared with existing biotechnological approaches such as anaerobic digestion and dark fermentation. The major bottlenecks and challenges that still need to be faced to make this novel technology practical are presented and critically discussed. © 2012 Curtin University of Technology and John Wiley & Sons, Ltd.

Alice Boo, Harman Mehta, Rodrigo Ledesma Amaro et al.

bioRxiv (Cold Spring Harbor Laboratory) • 2023

Abstract Microbial consortia have been utilised for centuries to produce fermented foods and have great potential in applications such as therapeutics, biomaterials, fertilisers, and biobased production. Working together, microbes become specialized and perform complex tasks more efficiently, strengthening both cooperation and stability of the microbial community. However, imbalanced proportions of microbial community members can lead to unoptimized and diminished yields in biotechnology. To address this, we developed a burden-aware RNA-based multicellular feedback control system that stabilises and tunes coculture compositions. The system consists of three modules: a quorum sensing-based communication module to provide information about the densities of cocultured strains, an RNA-based comparator module to compare the ratio of densities of both strains to a pre-set desired ratio, and a customisable growth module that relies either on heterologous gene expression or on CRISPRi knockdowns to tune growth rates. We demonstrated that heterologous expression burden could be used to stabilise composition in a two-member E. coli coculture. This is the first coculture composition controller that does not rely on toxins or syntrophy for growth regulation and uses RNA sequestration to stabilise and control coculture composition. This work provides a fundamental basis to explore burden-aware multicellular feedback control strategies for robust stabilisation of synthetic community compositions.

Jong-Won Lee, Cong T. Trinh

bioRxiv (Cold Spring Harbor Laboratory) • 2018

ABSTRACT Background Green organic solvents such as lactate esters have broad industrial applications and favorable environmental profiles. Thus, manufacturing and use of these biodegradable solvents from renewable feedstocks help benefit the environment. However, to date, the direct microbial biosynthesis of lactate esters from fermentable sugars has not yet been demonstrated. Results In this study, we present a microbial conversion platform for direct biosynthesis of lactate esters from fermentable sugars. First, we designed a pyruvate-to-lactate ester module, consisting of a lactate dehydrogenase ( ldhA ) to convert pyruvate to lactate, a propionate CoA-transferase ( pct ) to convert lactate to lactyl-CoA, and an alcohol acyltransferase ( AAT ) to condense lactyl-CoA and alcohol(s) to make lactate ester(s). By generating a library of five pyruvate-to-lactate ester modules with divergent AATs, we screened for the best module(s) capable of producing a wide range of linear, branched, and aromatic lactate esters with an external alcohol supply. By co-introducing a pyruvate-to-lactate ester module and an alcohol (i.e., ethanol, isobutanol) module into a modular Escherichia coli (chassis) cell, we demonstrated for the first time the microbial biosynthesis of ethyl and isobutyl lactate esters directly from glucose. In an attempt to enhance ethyl lactate production as a proof-of-study, we re-modularized the pathway into 1) the upstream module to generate the ethanol and lactate precursors and 2) the downstream module to generate lactyl-CoA and condense it with ethanol to produce the target ethyl lactate. By manipulating the metabolic fluxes of the upstream and downstream modules through plasmid copy numbers, promoters, ribosome binding sites, and environmental perturbation, we were able to probe and alleviate the metabolic bottlenecks by improving ethyl lactate production by 4.96-fold. We found that AAT is the most rate limiting step in biosynthesis of lactate esters likely due to its low activity and specificity towards the non-natural substrate lactyl-CoA and alcohols. Conclusions We have successfully established the biosynthesis pathway of lactate esters from fermentable sugars and demonstrated for the first time the direct fermentative production of lactate esters from glucose using an E. coli modular cell. This study defines a cornerstone for the microbial production of lactate esters as green solvents from renewable resources with novel industrial applications.

Kenneth T. Walker, Ivy S Li, Jennifer Keane et al.

Nature Biotechnology • 2024

Environmental concerns are driving interest in postpetroleum synthetic textiles produced from microbial and fungal sources. Bacterial cellulose (BC) is a promising sustainable leather alternative, on account of its material properties, low infrastructure needs and biodegradability. However, for alternative textiles like BC to be fully sustainable, alternative ways to dye textiles need to be developed alongside alternative production methods. To address this, we genetically engineer Komagataeibacter rhaeticus to create a bacterial strain that grows self-pigmenting BC. Melanin biosynthesis in the bacteria from recombinant tyrosinase expression achieves dark black coloration robust to material use. Melanated BC production can be scaled up for the construction of prototype fashion products, and we illustrate the potential of combining engineered self-pigmentation with tools from synthetic biology, through the optogenetic patterning of gene expression in cellulose-producing bacteria. With this study, we demonstrate that combining genetic engineering with current and future methods of textile biofabrication has the potential to create a new class of textiles.

Ying-Ying Chen, Jia-Cong Huang, Cai-Yun Wu et al.

Critical Reviews in Biotechnology • 2024

Abstract 5-Aminolevulinic acid (5-ALA) is a non-proteinogenic amino acid essential for synthesizing tetrapyrrole compounds, including heme, chlorophyll, cytochrome, and vitamin B12. As a plant growth regulator, 5-ALA is extensively used in agriculture to enhance crop yield and quality. The complexity and low yield of chemical synthesis methods have led to significant interest in the microbial synthesis of 5-ALA. Advanced strategies, including the: enhancement of precursor and cofactor supply, compartmentalization of key enzymes, product transporters engineering, by-product formation reduction, and biosensor-based dynamic regulation, have been implemented in bacteria for 5-ALA production, significantly advancing its industrialization. This article offers a comprehensive review of recent developments in 5-ALA production using engineered bacteria and presents new insights to propel the field forward. Graphical Abstract

Yaofeng Zhou, Qianying Li, Yuhao Wu et al.

Advanced Materials • 2024

Engineered bacteria are widely used in cancer treatment because live facultative/obligate anaerobes can selectively proliferate at tumor sites and reach hypoxic regions, thereby causing nutritional competition, enhancing immune responses, and producing anticancer microbial agents in situ to suppress tumor growth. Despite the unique advantages of bacteria‐based cancer biotherapy, the insufficient treatment efficiency limits its application in the complete ablation of malignant tumors. The combination of nanomedicine and engineered bacteria has attracted increasing attention owing to their striking synergistic effects in cancer treatment. Engineered bacteria that function as natural vehicles can effectively deliver nanomedicines to tumor sites. Moreover, bacteria provide an opportunity to enhance nanomedicines by modulating the TME and producing substrates to support nanomedicine‐mediated anticancer reactions. Nanomedicine exhibits excellent optical, magnetic, acoustic, and catalytic properties, and plays an important role in promoting bacteria‐mediated biotherapies. The synergistic anticancer effects of engineered bacteria and nanomedicines in cancer therapy are comprehensively summarized in this review. Attention is paid not only to the fabrication of nanobiohybrid composites, but also to the interpromotion mechanism between engineered bacteria and nanomedicine in cancer therapy. Additionally, recent advances in engineered bacteria‐synergized multimodal cancer therapies are highlighted.

Laure Lapinsonnière, Matthieu Picot, F. Barrière

ChemSusChem • 2012

Catalyses of electrode reactions by oxidoreductases or living electroactive bacteria are compared and recent advances reviewed. The relation between the biological and nevertheless inert nature of enzymes and the living machinery of electroactive microbes is discussed. The way these biocatalysts may be electrically contacted to anodes or cathodes is considered with a focus on their immobilization at electrodes and on the issue of time stability of these assemblies. Recent improvements in power output of biofuel cells are reviewed together with applications that have appeared in the literature. This account also reviews new approaches for combining enzymes and living microbes in bioelectrochemical systems such as reproducing microbial metabolisms with enzyme cascades and expressing oxidoreductases on genetically engineered microbes. Finally, the use of surface chemistry for studying the microbe-electrode interface and bioelectrodes with cell organelles, such as mitochondria, or with higher organisms, such as yeasts, are discussed. Some perspectives for future research to extend this field are offered as conclusions.

N. Sekar, Jian Wang, Yan Zhou et al.

Biotechnology and Bioengineering • 2018

Cyanobacteria are used as anode catalysts in photo-bioelectrochemical cells to generate electricity in a sustainable, economic, and environmental friendly manner using only water and sunlight. Though cyanobacteria (CB) possess unique advantage for solar energy conversion by virtue of its robust photosynthesis, they cannot efficiently perform extracellular electron transfer (EET). The reasons being, unlike dissimilatory metal reducing bacteria (that are usually exploited in microbial fuel cells to generate electricity), (1) CB do not possess any special features on their outer membrane to carry out EET and, (2) the electrons generated in photosynthetic electron transport chain are channeled into competing respiratory pathways rather than to the anode. CB, genetically engineered to express outer membrane cytochrome S (OmcS), was found to generate ∼nine-fold higher photocurrent compared to that of wild-type cyanobacterium in our previous work. In this study, each of the three respiratory terminal oxidases in Synechococcus elongatus PCC7942 namely bd-type quinol oxidase, aa3 -type cytochrome oxidase, and cbb3 -type cytochrome oxidase was knocked-out one at a time (cyd- , cox- , and cco- respectively) and its contribution for extracellular ferricyanide reduction and photocurrent generation was investigated. The knock-out mutant lacking functional bd-type quinol oxidase (cyd- ) exhibited greater EET by reducing more ferricyanide compared to other single knock-out mutants as well as the wild type. Further, cyd- omcs (the cyd- mutant expressing OmcS) was found to generate more photocurrent than the corresponding single knock out controls and the wild-type. This study clearly demonstrates that the bd-quinol oxidase diverted more electrons from the photosynthetic electron transport chain towards respiratory oxygen reduction and knocking it out had certainly enhanced the cyanobacterial EET.

Gen Nakagawa, A. Kouzuma, Atsumi Hirose et al.

PLOS ONE • 2015

In bioelectrochemical systems, the electrode potential is an important parameter affecting the electron flow between electrodes and microbes and microbial metabolic activities. Here, we investigated the metabolic characteristics of a glucose-utilizing strain of engineered Shewanella oneidensis under electrode-respiring conditions in electrochemical reactors for gaining insight into how metabolic pathways in electrochemically active bacteria are affected by the electrode potential. When an electrochemical reactor was operated with its working electrode poised at +0.4 V (vs. an Ag/AgCl reference electrode), the engineered S. oneidensis strain, carrying a plasmid encoding a sugar permease and glucose kinase of Escherichia coli, generated current by oxidizing glucose to acetate and produced D-lactate as an intermediate metabolite. However, D-lactate accumulation was not observed when the engineered strain was grown with a working electrode poised at 0 V. We also found that transcription of genes involved in pyruvate and D-lactate metabolisms was upregulated at a high electrode potential compared with their transcription at a low electrode potential. These results suggest that the carbon catabolic pathway of S. oneidensis can be modified by controlling the potential of a working electrode in an electrochemical bioreactor.

Lei Cheng, Di Min, Ru-Li He et al.

Biotechnology and Bioengineering • 2020

Shewanella oneidensis MR‐1, a model strain of exoelectrogenic bacteria (EEB), plays a key role in environmental bioremediation and bioelectrochemical systems because of its unique respiration capacity. However, only a narrow range of substrates can be utilized by S. oneidensis MR‐1 as carbon sources, resulting in its limited applications. In this study, a rapid, highly efficient, and easily manipulated base‐editing system pCBEso was developed by fusing a Cas9 nickase (Cas9n (D10A)) with the cytidine deaminase rAPOBEC1 in S. oneidensis MR‐1. The C‐to‐T conversion of suitable C within the base‐editing window could be readily and efficiently achieved by the pCBEso system without requiring double‐strand break or repair templates. Moreover, double‐locus simultaneous editing was successfully accomplished with an efficiency of 87.5%. With this tool, the key genes involving in N‐acetylglucosamine (GlcNAc) or glucose metabolism in S. oneidensis MR‐1 were identified. Furthermore, an engineered strain with expanded carbon source utilization spectra was constructed and exhibited a higher degradation rate for multiple organic pollutants (i.e., azo dyes and organoarsenic compounds) than the wild‐type when glucose or GlcNAc was used as the sole carbon source. Such a base‐editing system could be readily applied to other EEB. This study not only enhances the substrate utilization and pollutant degradation capacities of S. oneidensis MR‐1 but also accelerates the robust construction of engineered strains for environmental bioremediation.

Sandipan Banerjee, Nitu Gupta, Krishnendu Pramanik et al.

Environmental Science and Pollution Research • 2023

Degradation, detoxification, or removal of the omnipresent polycyclic aromatic hydrocarbons (PAHs) from the ecosphere as well as their prevention from entering into food chain has never appeared simple. In this context, cost-effective, eco-friendly, and sustainable solutions like microbe-mediated strategies have been adopted worldwide. With this connection, measures have been taken by multifarious modes of microbial remedial strategies, i.e., enzymatic degradation, biofilm and biosurfactant production, application of biochar-immobilized microbes, lactic acid bacteria, rhizospheric-phyllospheric-endophytic microorganisms, genetically engineered microorganisms, and bioelectrochemical techniques like microbial fuel cell. In this review, a nine-way directional approach which is based on the microbial resources reported over the last couple of decades has been described. Fungi were found to be the most dominant taxa among the CPAH-degrading microbial community constituting 52.2%, while bacteria, algae, and yeasts occupied 37.4%, 9.1%, and 1.3%, respectively. In addition to these, category-wise CPAH degrading efficiencies of each microbial taxon, consortium-based applications, CPAH degradation–related molecular tools, and factors affecting CPAH degradation are the other important aspects of this review in light of their appropriate selection and application in the PAH-contaminated environment for better human-health management in order to achieve a sustainable ecosystem. Graphical Abstract

Sara Díaz-Rullo Edreira, I. Vasiliadou, Amanda Prado et al.

Communications Biology • 2024

Reducing greenhouse gas emissions is critical for humanity nowadays, but it can be beneficial by developing engineered systems that valorize CO2 into commodities, thus mimicking nature’s wisdom. Purple phototrophic bacteria (PPB) naturally accept CO2 into their metabolism as a primary redox sink system in photo-heterotrophy. Dedicated use of this feature for developing sustainable processes (e.g., through negative-emissions photo-bioelectrosynthesis) requires a deep knowledge of the inherent metabolic mechanisms. This work provides evidence of tuning the PPB metabolic mechanisms upon redox stressing through negative polarization (−0.4 and −0.8 V vs. Ag/AgCl) in photo-bioelectrochemical devices. A mixed PPB-culture upregulates its ability to capture CO2 from organics oxidation through the Calvin-Besson-Bassam cycle and anaplerotic pathways, and the redox imbalance is promoted to polyhydroxyalkanoates production. The ecological relationship of PPB with mutualist bacteria stabilizes the system and opens the door for future development of photo-bioelectrochemical devices focused on CO up-cycling.

Yu‐Jing Jiang, Su Hui, Liping Jiang et al.

Chemistry – A European Journal • 2022

Microbial fuel cell (MFC) is a promising approach that could utilize microorganisms to oxidize biodegradable pollutants in wastewater and generate electrical power simultaneously. Introducing advanced anode nanomaterials is generally considered as an effective way to enhance MFC performance by increasing bacterial adhesion and facilitating bacteria extracellular electron transfer (EET). This review focuses on the key advances of recent anode modification materials, as well as the current understanding of the microbial EET process occurring at the bacteria-electrode interface. Based on the difference in combination mode of the exoelectrogens and nanomaterials, anode surface modification, hybrid biofilm construction and single-bacterial surface modification strategies are elucidated exhaustively. The inherent mechanisms may help to break through the performance output bottleneck of MFCs by rational design of EET-related nanomaterials, and lead to the widespread application of microbial electrochemical systems.

J. D'aes, Marie-Alice Fraiture, Bert Bogaerts et al.

Life • 2022

Genetically modified microorganisms (GMM) are frequently employed for manufacturing microbial fermentation products such as food enzymes or vitamins. Although the fermentation product is required to be pure, GMM contaminations have repeatedly been reported in numerous commercial microbial fermentation produce types, leading to several rapid alerts at the European level. The aim of this study was to investigate the added value of shotgun metagenomic high-throughput sequencing to confirm and extend the results of classical analysis methods for the genomic characterization of unauthorized GMM. By combining short- and long-read metagenomic sequencing, two transgenic constructs were characterized, with insertions of alpha-amylase genes originating from B. amyloliquefaciens and B. licheniformis, respectively, and a transgenic construct with a protease gene insertion originating from B. velezensis, which were all present in all four investigated samples. Additionally, the samples were contaminated with up to three unculturable Bacillus strains, carrying genetic modifications that may hamper their ability to sporulate. Moreover, several samples contained viable Bacillus strains. Altogether these contaminations constitute a considerable load of antimicrobial resistance genes, that may represent a potential public health risk. In conclusion, our study showcases the added value of metagenomics to investigate the quality and safety of complex commercial microbial fermentation products.

D. Nosek, O. Samsel, T. Pokój et al.

International Journal of Environmental Science and Technology • 2023

The commercialization of microbial fuel cell technology is limited by high operating costs and low electricity production due to poor electron transfer to the anode. Operational costs can be lowered by utilizing waste materials, and cell performance can be improved by anode modification. This study investigated how anode modification with iron compounds changed the efficiency of energy generation and the microbiome of microbial fuel cells fueled with waste volatile fatty acids from a full-scale anaerobic digestion. Anode modification with 2.5 g Fe_2O_3/m^2 increased the power density, current density, and voltage by 3.6-fold, 1.8-fold, and 1.4-fold, respectively. In the microbial fuel cell influent, propionic, enanthic, and iso-caproic acids predominated (60, 15, and 13% of all volatile fatty acids, respectively); in the outflow, propionic (71%) and valeric acids (17%) predominated. In anodic biofilms, Acidovorax sp. were most abundant; they have a great capacity for volatile fatty acids decomposition, and their abundance doubled in the microbial fuel cell with an iron-modified anode. The presence of iron significantly increased the abundance of the genera Pseudomonas and Geothrix , which were mainly responsible for electricity production. These results indicate that anode modification with iron changes the anode microbiome, favoring efficient volatile fatty acids metabolism and a greater abundance of electrogens in the biofilm, which ensures better electricity generation.

Yangkai Duan, Zhi Zhu, Ke Cai et al.

PLoS ONE • 2011

Biodiesel is a renewable alternative to petroleum diesel fuel that can contribute to carbon dioxide emission reduction and energy supply. Biodiesel is composed of fatty acid alkyl esters, including fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs), and is currently produced through the transesterification reaction of methanol (or ethanol) and triacylglycerols (TAGs). TAGs are mainly obtained from oilseed plants and microalgae. A sustainable supply of TAGs is a major bottleneck for current biodiesel production. Here we report the de novo biosynthesis of FAEEs from glucose, which can be derived from lignocellulosic biomass, in genetically engineered Escherichia coli by introduction of the ethanol-producing pathway from Zymomonas mobilis, genetic manipulation to increase the pool of fatty acyl-CoA, and heterologous expression of acyl-coenzyme A: diacylglycerol acyltransferase from Acinetobacter baylyi. An optimized fed-batch microbial fermentation of the modified E. coli strain yielded a titer of 922 mg L−1 FAEEs that consisted primarily of ethyl palmitate, -oleate, -myristate and -palmitoleate.

S. A. Hussain, Alexis García, Md Ahsanul Kabir Khan et al.

Genes • 2020

Concerns about global warming, fossil-fuel depletion, food security, and human health have promoted metabolic engineers to develop tools/strategies to overproduce microbial functional oils directly from renewable resources. Medium-chain fatty acids (MCFAs, C8–C12) have been shown to be important sources due to their diverse biotechnological importance, providing benefits ranging from functional lipids to uses in bio-fuel production. However, oleaginous microbes do not carry native pathways for the production of MCFAs, and therefore, diverse approaches have been adapted to compensate for the requirements of industrial demand. Mucor circinelloides is a promising organism for lipid production (15–36% cell dry weight; CDW) and the investigation of mechanisms of lipid accumulation; however, it mostly produces long-chain fatty acids (LCFAs). To address this challenge, we genetically modified strain M. circinelloides MU758, first by integrating heterologous acyl-ACP thioesterase (TE) into fatty acid synthase (FAS) complex and subsequently by modifying the β-oxidation pathway by disrupting the acyl-CoA oxidase (ACOX) and/or acyl-CoA thioesterase (ACOT) genes with a preference for medium-chain acyl-CoAs, to elevate the yield of MCFAs. The resultant mutant strains (M-1, M-2, and M-3, respectively) showed a significant increase in lipid production in comparison to the wild-type strain (WT). MCFAs in M-1 (47.45%) was sharply increased compared to the wild type strain (2.25%), and it was further increased in M-2 (60.09%) suggesting a negative role of ACOX in MCFAs production. However, MCFAs in M-3 were much decreased compared to M-1,suggesting a positive role of ACOT in MCFAs production. The M-2 strain showed maximum lipid productivity (~1800 milligram per liter per day or mg/L.d) and MCFAs productivity (~1100 mg/L.d). Taken together, this study elaborates on how the combination of two multidimensional approaches, TE gene over-expression and modification of the β-oxidation pathway via substantial knockout of specific ACOX gene, significantly increased the production of MCFAs. This synergistic approach ultimately offers a novel opportunity for synthetic/industrial biologists to increase the content of MCFAs.