Creating Single-Copy Genetic Circuits.

Synthetic biology is increasingly used to develop sophisticated living devices for basic and applied research. Many of these genetic devices are engineered using multi-copy plasmids, but as the field progresses from proof-of-principle demonstrations to practical applications, it is important to develop single-copy synthetic modules that minimize consumption of cellular resources and can be stably maintained as genomic integrants. Here we use empirical design, mathematical modeling, and iterative construction and testing to build single-copy, bistable toggle switches with improved performance and reduced metabolic load that can be stably integrated into the host genome. Deterministic and stochastic models led us to focus on basal transcription to optimize circuit performance and helped to explain the resulting circuit robustness across a large range of component expression levels. The design parameters developed here provide important guidance for future efforts to convert functional multi-copy gene circuits into optimized single-copy circuits for practical, real-world use.

Can microbes be harnessed to reduce atmospheric loads of greenhouse gases?

Reducing atmospheric loads of greenhouse gases (GHGs), especially CO2 and CH4 , has been considered the key to alleviating global crises we are facing, such as climate change, sea level elevation and ocean acidification. To this end, development of strategies and technologies for carbon capture, sequestration and utilization (CCSU) is urgently needed. Although physicochemical methods have been the most actively studied in the early stages of developing CCSU technologies, there have recently been growing interests in developing microbe-based CCSU processes. In this article, we discuss advantages of microbe-based CCSU technologies over physicochemical approaches and even plant-based approaches. Next, various parts of the global carbon cycle where microorganisms can contribute, such as sequestering atmospheric GHGs, facilitating the carbon cycle, and slowing down the depletion of carbon reservoirs are described, emphasizing the impacts of microbes on the carbon cycle. Strategies to upgrade microbes and increase their performance in assimilating GHGs or converting GHGs to value-added chemicals are also provided. Moreover, several examples of exploiting microbes to address environmental crises are discussed. Finally, we discuss things to overcome in microbe-based CCSU technologies and provide future perspectives.

Improved terephthalic acid production from p-xylene using metabolically engineered Pseudomonas putida.

Terephthalic acid (TPA) is an important commodity chemical used as a monomer of polyethylene terephthalate (PET). Since a large quantity of PET is routinely manufactured and consumed worldwide, the development of sustainable biomanufacturing processes for its monomers (i.e. TPA and ethylene glycol) has recently gained much attention. In a previous study, we reported the development of a metabolically engineered Escherichia coli strain producing 6.7 g/L of TPA from p-xylene (pX) with a productivity and molar conversion yield of 0.278 g/L/h and 96.7 mol%, respectively. Here, we report metabolic engineering of Pseudomonas putida KT2440, a microbial chassis particularly suitable for the synthesis of aromatic compounds, for improved biocatalytic conversion of pX to TPA. To develop a plasmid-free, antibiotic-free, and inducer-free biocatalytic process for cost-competitive TPA production, all heterologous genes required for the synthetic pX-to-TPA bioconversion pathway were integrated into the chromosome of P. putida KT2440 by RecET-based markerless recombineering and overexpressed under the control of constitutive promoters. Next, TPA production was enhanced by integrating multiple copies of the heterologous genes to the ribosomal RNA genes through iteration of recombineering-based random integration and subsequent screening of high-performance strains. Finally, fed-batch fermentation process was optimized to further improve the performance of the engineered P. putida strain. As a result, 38.25 ± 0.11 g/L of TPA was produced from pX with a molar conversion yield of 99.6 ± 0.6%, which is equivalent to conversion of 99.3 ± 0.8 g pX to 154.6 ± 0.5 g TPA. This superior pX-to-TPA biotransformation process based on the engineered P. putida strain will pave the way to the commercial biomanufacturing of TPA in an industrial scale.

Improved production of heme using metabolically engineered Escherichia coli.

Heme has recently attracted much attention due to its promising applications in the food and healthcare industries. However, the current titers and productivities of heme produced by recombinant microorganisms are not high enough for a wide range of applications. In this study, the process for the fermentation of the metabolically engineered Escherichia coli HAEM7 strain was optimized for the high-level production of heme. To improve the production of heme, different carbon sources, iron concentration in the medium, pH control strategies, induction points, and iron content in the feeding solution were examined. Moreover, strategies of increasing cell density, regular iron supplementation, and supply of excess feeding solution were developed to further improve the production of heme. In the optimized fermentation process, the HAEM7 strain produced 1.03 g/L heme with productivity of 21.5 mg/L/h. The fermentation process and strategies reported here will expedite establishing industry-level production of heme.

Production of zinc protoporphyrin IX by metabolically engineered Escherichia coli.

Zinc protoporphyrin IX (ZnPPIX) has been considered as a promising red colorant for food industries as well as an anticancer drug. However, bio-based production of ZnPPIX from a renewable carbon source has not been reported yet. In this study, a fermentation process of the metabolically engineered Escherichia coli HAEM7 strain was optimized for the high-level production of ZnPPIX. To repurpose the HAEM7 strain that was originally developed for the production of heme into a producer of ZnPPIX, the concentrations of iron and zinc in the culture medium were rebalanced. Next, the concentration of zinc in the feeding solution was optimized to improve ZnPPIX production. Moreover, the pH control strategy, induction point, and the strategy of increasing the cell density, which were optimized in the accompanying paper for the high-level production of heme, were applied together. In the optimized fermentation process, the HAEM7 strain produced 2.2 g/L ZnPPIX with a productivity of 39.9 mg/L/h. The fermentation process and strategies reported here will expedite establishing industry-level production of ZnPPIX.

Application of Lactic Acid Bacteria (LAB) in Sustainable Agriculture: Advantages and Limitations.

Lactic acid bacteria (LAB) are significant groups of probiotic organisms in fermented food and are generally considered safe. LAB regulate soil organic matter and the biochemical cycle, detoxify hazardous chemicals, and enhance plant health. They are found in decomposing plants, traditional fermented milk products, and normal human gastrointestinal and vaginal flora. Exploring LAB identified in unknown niches may lead to isolating unique species. However, their classification is quite complex, and they are adapted to high sugar concentrations and acidic environments. LAB strains are considered promising candidates for sustainable agriculture, and they promote soil health and fertility. Therefore, they have received much attention regarding sustainable agriculture. LAB metabolites promote plant growth and stimulate shoot and root growth. As fertilizers, LAB can promote biodegradation, accelerate the soil organic content, and produce organic acid and bacteriocin metabolites. However, LAB show an antagonistic effect against phytopathogens, inhibiting fungal and bacterial populations in the rhizosphere and phyllosphere. Several studies have proposed the LAB bioremediation efficiency and detoxification of heavy metals and mycotoxins. However, LAB genetic manipulation and metabolic engineered tools provide efficient cell factories tailor-made to produce beneficial industrial and agro-products. This review discusses lactic acid bacteria advantages and limitations in sustainable agricultural development.

Revisiting Statistical Design and Analysis in Scientific Research.

Statistics is essential to design experiments and interpret experimental results. Inappropriate use of the statistical analysis, however, often leads to a wrong conclusion. This concept article revisits basic concepts of statistics and provides a brief guideline of applying the statistical analysis for scientific research from designing experiments to analyzing and presenting the data.

Markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida.

Pseudomonas putida has gained much interest among metabolic engineers as a workhorse for producing valuable natural products. While a few gene knockout tools for P. putida have been reported, integration of heterologous genes into the chromosome of P. putida, an essential strategy to develop stable industrial strains producing heterologous bioproducts, requires development of a more efficient method. Current methods rely on time-consuming homologous recombination techniques and transposon-mediated random insertions. Here we report a RecET recombineering system for markerless integration of heterologous genes into the P. putida chromosome. The efficiency and capacity of the recombineering system were first demonstrated by knocking out various genetic loci on the P. putida chromosome with knockout lengths widely spanning 0.6-101.7 kb. The RecET recombineering system developed here allowed successful integration of biosynthetic gene clusters for four proof-of-concept bioproducts, including protein, polyketide, isoprenoid, and amino acid derivative, into the target genetic locus of P. putida chromosome. The markerless recombineering system was completed by combining Cre/lox system and developing efficient plasmid curing systems, generating final strains free of antibiotic markers and plasmids. This markerless recombineering system for efficient gene knockout and integration will expedite metabolic engineering of P. putida, a bacterial host strain of increasing academic and industrial interest.

Metabolomics for industrial fermentation.

Metabolomics is essential to understand the metabolism and identify engineering targets to improve the performances of strains and bioprocesses. Although numerous metabolomics techniques have been developed and applied to various organisms, the metabolome of Saccharopolyspora erythraea, a native producer of erythromycin, had never been studied. The 2017 best paper of Bioprocess and Biosystems Engineering reports examination of three methods for quenching and extraction to analyze the intracellular metabolome of S. erythraea, and identified the most reliable methods for studying different groups of the metabolites. Subsequent studies on the dynamics of the intracellular metabolome of S. erythraea during the fed-batch fermentation identified a positive correlation between the specific erythromycin production rate and the pool size of intracellular propionyl-CoA and other precursors of erythromycin. A series of follow-up studies, such as demonstrating the applicability of the quenching/extraction methods in other related antibiotic producers, demonstrating the generality of the best matches between the quenching/extraction methods and the metabolite groups, and combining metabolomics approaches with the fluxomics and systems metabolic engineering approaches, will facilitate the metabolomics studies on important antibiotic producers, enable standardization of the quenching/extraction protocols, and improve the performance of the antibiotic production with deeper insight into their metabolism.

CRISPR/Cas9-coupled recombineering for metabolic engineering of Corynebacterium glutamicum.

Genome engineering of Corynebacterium glutamicum, an important industrial microorganism for amino acids production, currently relies on random mutagenesis and inefficient double crossover events. Here we report a rapid genome engineering strategy to scarlessly knock out one or more genes in C. glutamicum in sequential and iterative manner. Recombinase RecT is used to incorporate synthetic single-stranded oligodeoxyribonucleotides into the genome and CRISPR/Cas9 to counter-select negative mutants. We completed the system by engineering the respective plasmids harboring CRISPR/Cas9 and RecT for efficient curing such that multiple gene targets can be done iteratively and final strains will be free of plasmids. To demonstrate the system, seven different mutants were constructed within two weeks to study the combinatorial deletion effects of three different genes on the production of γ-aminobutyric acid, an industrially relevant chemical of much interest. This genome engineering strategy will expedite metabolic engineering of C. glutamicum.

Rapsyn mediates subsynaptic anchoring of PKA type I and stabilisation of acetylcholine receptor in vivo.

The stabilisation of acetylcholine receptors (AChRs) at the neuromuscular junction depends on muscle activity and the cooperative action of myosin Va and protein kinase A (PKA) type I. To execute its function, PKA has to be present in a subsynaptic microdomain where it is enriched by anchoring proteins. Here, we show that the AChR-associated protein, rapsyn, interacts with PKA type I in C2C12 and T-REx293 cells as well as in live mouse muscle beneath the neuromuscular junction. Molecular modelling, immunoprecipitation and bimolecular fluorescence complementation approaches identify an α-helical stretch of rapsyn to be crucial for binding to the dimerisation and docking domain of PKA type I. When expressed in live mouse muscle, a peptide encompassing the rapsyn α-helical sequence efficiently delocalises PKA type I from the neuromuscular junction. The same peptide, as well as a rapsyn construct lacking the α-helical domain, induces severe alteration of acetylcholine receptor turnover as well as fragmentation of synapses. This shows that rapsyn anchors PKA type I in close proximity to the postsynaptic membrane and suggests that this function is essential for synapse maintenance.

From sustainable feedstocks to microbial foods.

Climate change-induced alterations in weather patterns, such as frequent and severe heatwaves, cold waves, droughts, floods, heavy rain and storms, are reducing crop yields and agricultural productivity. At the same time, greenhouse gases arising from food production and supply account for almost 30% of anthropogenic emissions. This vicious circle is producing a global food crisis. Sustainable food resources and production systems are needed now, and microbial foods are one possible solution. In this Perspective, we highlight the most promising technologies, and carbon and energy sources, for microbial food production.

Microbial lysates repurposed as liquid egg substitutes.

Microbial lysates, rich in protein and essential nutrients, demonstrate remarkable capabilities in forming gels and stable foams when heated and whisked, similar to liquid eggs. These characteristics make them an excellent alternative to animal-derived liquid eggs, contributing to sustainable food production and consumption while maintaining high nutritional value. Their versatility positions microbial lysates as promising ingredients in culinary applications, offering a sustainable and nutritious substitute.

Enhanced production of difficult-to-express proteins through knocking down rnpA gene expression.

Escherichia coli has been employed as a workhorse for the efficient production of recombinant proteins. However, some proteins were found to be difficult to produce in E. coli. The stability of mRNA has been considered as one of the important factors affecting recombinant protein production. Here we report a generally applicable and simple strategy for enhancing mRNA stability, and consequently improving recombinant protein production in E. coli. RNase P, a ribozyme comprising an RNA subunit (RnpB) and a protein subunit (RnpA), is involved in tRNA maturation. Based on the finding that purified RnpA can digest rRNA and mRNA in vitro, it was reasoned that knocking down the level of RnpA might enhance recombinant protein production. For this, the synthetic small regulatory RNA-based knockdown system was applied to reduce the expression level of RnpA. The developed RnpA knockdown system allowed successful overexpression of 23 different recombinant proteins of various origins and sizes, including Cas9 protein, antibody fragment, and spider silk protein. Notably, a 284.9-kDa ultra-high molecular weight, highly repetitive glycine-rich spider silk protein, which is one of the most difficult proteins to produce, could be produced to 1.38 g L-1 , about two-fold higher than the highest value previously achieved, by a fed-batch culture of recombinant E. coli strain employing the RnpA knockdown system. The RnpA-knockdown strategy reported here will be generally useful for the production of recombinant proteins including those that have been difficult to produce.

Targeted and high-throughput gene knockdown in diverse bacteria using synthetic sRNAs.

Synthetic sRNAs allow knockdown of target genes at translational level, but have been restricted to a limited number of bacteria. Here, we report the development of a broad-host-range synthetic sRNA (BHR-sRNA) platform employing the RoxS scaffold and the Hfq chaperone from Bacillus subtilis. BHR-sRNA is tested in 16 bacterial species including commensal, probiotic, pathogenic, and industrial bacteria, with >50% of target gene knockdown achieved in 12 bacterial species. For medical applications, virulence factors in Staphylococcus epidermidis and Klebsiella pneumoniae are knocked down to mitigate their virulence-associated phenotypes. For metabolic engineering applications, high performance Corynebacterium glutamicum strains capable of producing valerolactam (bulk chemical) and methyl anthranilate (fine chemical) are developed by combinatorial knockdown of target genes. A genome-scale sRNA library covering 2959 C. glutamicum genes is constructed for high-throughput colorimetric screening of indigoidine (natural colorant) overproducers. The BHR-sRNA platform will expedite engineering of diverse bacteria of both industrial and medical interest.

Microbial food: microorganisms repurposed for our food.

Sustainable food production is a key to solve complicated and intertwined issues of overpopulation, climate change, environment and sustainability. Microorganisms, which have been routinely consumed as a part of fermented foods and more recently as probiotic dietary supplements, can be repurposed for our food to present a sustainable solution to current food production system. This paper begins with three snapshots of our future life with microbial foods. Next, the importance, possible forms, and raw materials (i.e. microorganisms and their carbon and energy sources) of microbial foods are discussed. In addition, the production strategies, further applications and current limitations of microbial foods are discussed.

Metabolic engineering strategies toward production of biofuels.

Exacerbation of climate change and air pollution around the world have emphasized the necessity of replacing fossil fuels with clean and sustainable energy. Metabolic engineering has provided strategies to engineer diverse organisms for the production of biofuels from renewable carbon sources. Although some of the processes are commercialized, there has been continued effort to produce advanced biofuels with higher efficiencies. In this article, metabolic engineering strategies recently exploited to enhance biofuel production and facilitate utilization of non-edible low-value carbon sources are reviewed. The strategies include engineering enzymes, exploiting new pathways, and systematically optimizing metabolism and fermentation processes, among others. In addition, metabolic and bioprocess engineering strategies to achieve competitiveness of current biofuel production systems compared with fossil fuels are discussed.

Protocols for RecET-based markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida.

Pseudomonas putida has emerged as a promising host for the production of chemicals and materials thanks to its metabolic versatility and cellular robustness. In particular, P. putida KT2440 has been officially classified as a generally recognized as safe (GRAS) strain, which makes it suitable for the production of compounds that humans directly consume, including secondary metabolites of high importance. Although various tools and strategies have been developed to facilitate metabolic engineering of P. putida, modification of large genes/clusters essential for heterologous expression of natural products with large biosynthetic gene clusters (BGCs) has not been straightforward. Recently, we reported a RecET-based markerless recombineering system for engineering P. putida and demonstrated deletion of multiple regions as large as 101.7 kb throughout the chromosome by single rounds of recombineering. In addition, development of a donor plasmid system allowed successful markerless integration of heterologous BGCs to P. putida chromosome using the recombineering system with examples of - but not limited to - integrating multiple heterologous BGCs as large as 7.4 kb to the chromosome of P. putida KT2440. In response to the increasing interest in our markerless recombineering system, here we provide detailed protocols for markerless gene knockout and integration for the genome engineering of P. putida and related species of high industrial importance.

Microbial production of fatty acids and derivative chemicals.

Microbial production of fatty acids and derivatives from non-edible biomass has attracted much attention as an alternative to their production from plant oils and animal fats. Fatty acids and some of their derivatives are ubiquitous metabolites synthesized for membrane biosynthesis and other metabolic purposes in microorganisms. These compounds, however, are rarely produced beyond cellular demands, frequently resulting in low titers even after metabolic engineering. Recently, more advanced metabolic engineering strategies including systems metabolic engineering allowed improved production of fatty acids and their derivatives by employing non-oleaginous and oleaginous microorganisms. Here, we review metabolic engineering strategies developed for the production of fatty acids and derivative chemicals by non-oleaginous and oleaginous microorganisms in recent years.

Systems Metabolic Engineering Strategies: Integrating Systems and Synthetic Biology with Metabolic Engineering.

Metabolic engineering allows development of microbial strains efficiently producing chemicals and materials, but it requires much time, effort, and cost to make the strains industrially competitive. Systems metabolic engineering, which integrates tools and strategies of systems biology, synthetic biology, and evolutionary engineering with traditional metabolic engineering, has recently been used to facilitate development of high-performance strains. The past decade has witnessed this interdisciplinary strategy continuously being improved toward the development of industrially competitive overproducer strains. In this article, current trends in systems metabolic engineering including tools and strategies are reviewed, focusing on recent developments in selection of host strains, metabolic pathway reconstruction, tolerance enhancement, and metabolic flux optimization. Also, future challenges and prospects are discussed.