Engineering Cellular Metabolism.

Metabolic engineering is the science of rewiring the metabolism of cells to enhance production of native metabolites or to endow cells with the ability to produce new products. The potential applications of such efforts are wide ranging, including the generation of fuels, chemicals, foods, feeds, and pharmaceuticals. However, making cells into efficient factories is challenging because cells have evolved robust metabolic networks with hard-wired, tightly regulated lines of communication between molecular pathways that resist efforts to divert resources. Here, we will review the current status and challenges of metabolic engineering and will discuss how new technologies can enable metabolic engineering to be scaled up to the industrial level, either by cutting off the lines of control for endogenous metabolism or by infiltrating the system with disruptive, heterologous pathways that overcome cellular regulation.

Flux optimization using multiple promoters in Halomonas bluephagenesis as a model chassis of the next generation industrial biotechnology.

Predictability and robustness are challenges for bioproduction because of the unstable intracellular synthetic activities. With the deeper understanding of the gene expression process, fine-tuning has become a meaningful tool for biosynthesis optimization. This study characterized several gene expression elements and constructed a multiple inducible system that responds to ten different small chemical inducers in halophile bacterium Halomonas bluephagenesis. Genome insertion of regulators was conducted for the purpose of gene cluster stabilization and regulatory plasmid simplification. Additionally, dynamic ranges of the multiple inducible systems were tuned by promoter sequence mutations to achieve diverse scopes for high-resolution gene expression control. The multiple inducible system was successfully employed to precisely control chromoprotein expression, lycopene and poly-3-hydroxybutyrate (PHB) biosynthesis, resulting in colorful bacterial pictures, optimized cell growth, lycopene and PHB accumulation. This study demonstrates a desirable approach for fine-tuning of rational and efficient gene expressions, displaying the significance for metabolic pathway optimization.

Ectoine hyperproduction by engineered Halomonas bluephagenesis.

Ectoine, a crucial osmoprotectant for salt adaptation in halophiles, has gained growing interest in cosmetics and medical industries. However, its production remains challenged by stringent fermentation process in model microorganisms and low production level in its native producers. Here, we systematically engineered the native ectoine producer Halomonas bluephagenesis for ectoine production by overexpressing ectABC operon, increasing precursors availability, enhancing product transport system and optimizing its growth medium. The final engineered H. bluephagenesis produced 85 g/L ectoine in 52 h under open unsterile incubation in a 7 L bioreactor in the absence of plasmid, antibiotic or inducer. Furthermore, it was successfully demonstrated the feasibility of decoupling salt concentration with ectoine synthesis and co-production with bioplastic P(3HB-co-4HB) by the engineered H. bluephagenesis. The unsterile fermentation process and significantly increased ectoine titer indicate that H. bluephagenesis as the chassis of Next-Generation Industrial Biotechnology (NGIB), is promising for the biomanufacturing of not only intracellular bioplastic PHA but also small molecular compound such as ectoine.

A long-term growth stable Halomonas sp. deleted with multiple transposases guided by its metabolic network model Halo-ecGEM.

Microbial instability is a common problem during bio-production based on microbial hosts. Halomonas bluephagenesis has been developed as a chassis for next generation industrial biotechnology (NGIB) under open and unsterile conditions. However, the hidden genomic information and peculiar metabolism have significantly hampered its deep exploitation for cell-factory engineering. Based on the freshly completed genome sequence of H. bluephagenesis TD01, which reveals 1889 biological process-associated genes grouped into 84 GO-slim terms. An enzyme constrained genome-scale metabolic model Halo-ecGEM was constructed, which showed strong ability to simulate fed-batch fermentations. A visible salt-stress responsive landscape was achieved by combining GO-slim term enrichment and CVT-based omics profiling, demonstrating that cells deploy most of the protein resources by force to support the essential activity of translation and protein metabolism when exposed to salt stress. Under the guidance of Halo-ecGEM, eight transposases were deleted, leading to a significantly enhanced stability for its growth and bioproduction of various polyhydroxyalkanoates (PHA) including 3-hydroxybutyrate (3HB) homopolymer PHB, 3HB and 3-hydroxyvalerate (3HV) copolymer PHBV, as well as 3HB and 4-hydroxyvalerate (4HB) copolymer P34HB. This study sheds new light on the metabolic characteristics and stress-response landscape of H. bluephagenesis, achieving for the first time to construct a long-term growth stable chassis for industrial applications. For the first time, it was demonstrated that genome encoded transposons are the reason for microbial instability during growth in flasks and fermentors.

Biomimetic CO2 Capture Unlocked through Enzyme Mining: Discovery of a Highly Thermo- and Alkali-Stable Carbonic Anhydrase.

Taking immediate action to combat the urgent threat of CO2-driven global warming is crucial for ensuring a habitable planet. Decarbonizing the industrial sector requires implementing sustainable carbon-capture technologies, such as biomimetic hot potassium carbonate capture (BioHPC). BioHPC is superior to traditional amine-based strategies due to its eco-friendly nature. This innovative technology relies on robust carbonic anhydrases (CAs), enzymes that accelerate CO2 hydration and endure harsh industrial conditions like high temperature and alkalinity. Thus, the discovery of highly stable CAs is crucial for the BioHPC technology advancement. Through high-throughput bioinformatics analysis, we identified a highly thermo- and alkali-stable CA, termed CA-KR1, originating from a metagenomic sample collected at a hot spring in Kirishima, Japan. CA-KR1 demonstrates remarkable stability at high temperatures and pH, with a half-life of 24 h at 80 °C and retains activity and solubility even after 30 d in a 20% (w/v) K2CO3/pH 11.5 solution─a standard medium for HPC. In pressurized batch reactions, CA-KR1 enhanced CO2 absorption by >90% at 90 °C, 20% K2CO3, and 7 bar. To our knowledge, CA-KR1 constitutes the most resilient CA biocatalyst for efficient CO2 capture under HPC-relevant conditions, reported to date. CA-KR1 integration into industrial settings holds great promise in promoting efficient BioHPC, a potentially game-changing development for enhancing carbon-capture capacity toward industrial decarbonization.

Synthetic biology toolkit of Ralstonia eutropha (Cupriavidus necator).

Synthetic biology encompasses many kinds of ideas and techniques with the common theme of creating something novel. The industrially relevant microorganism, Ralstonia eutropha (also known as Cupriavidus necator), has long been a subject of metabolic engineering efforts to either enhance a product it naturally makes (polyhydroxyalkanoate) or produce novel bioproducts (e.g., biofuels and other small molecule compounds). Given the metabolic versatility of R. eutropha and the existence of multiple molecular genetic tools and techniques for the organism, development of a synthetic biology toolkit is underway. This toolkit will allow for novel, user-friendly design that can impart new capabilities to R. eutropha strains to be used for novel application. This article reviews the different synthetic biology techniques currently available for modifying and enhancing bioproduction in R. eutropha. KEY POINTS: • R. eutropha (C. necator) is a versatile organism that has been examined for many applications. • Synthetic biology is being used to design more powerful strains for bioproduction. • A diverse synthetic biology toolkit is being developed to enhance R. eutropha's capabilities.

Carbon efficient production of chemicals with yeasts.

Microbial metabolism offers a wide variety of opportunities to produce chemicals from renewable resources. Employing such processes of industrial biotechnology provides valuable means to fight climate change by replacing fossil feedstocks by renewable substrate to reduce or even revert carbon emission. Several yeast species are well suited chassis organisms for this purpose, illustrated by the fact that the still largest microbial production of a chemical, namely bioethanol is based on yeast. Although production of ethanol and some other chemicals is highly efficient, this is not the case for many desired bulk chemicals. One reason for low efficiency is carbon loss, which decreases the product yield and increases the share of total production costs that is taken by substrate costs. Here we discuss the causes for carbon loss in metabolic processes, approaches to avoid carbon loss, as well as opportunities to incorporate carbon from CO2 , based on the electron balance of pathways. These aspects of carbon efficiency are illustrated for the production of succinic acid from a diversity of substrates using different pathways.

PHB production from food waste hydrolysates by Halomonas bluephagenesis Harboring PHB operon linked with an essential gene.

Food wastes can be hydrolyzed into soluble microbial substrates, contributing to sustainability. Halomonas spp.-based Next Generation Industrial Biotechnology (NGIB) allows open, unsterile fermentation, eliminating the need for sterilization to avoid the Maillard reaction that negatively affects cell growth. This is especially important for food waste hydrolysates, which have a high nutrient content but are unstable due to batch, sources, or storage conditions. These make them unsuitable for polyhydroxyalkanoate (PHA) production, which usually requires limitation on either nitrogen, phosphorous, or sulfur. In this study, H. bluephagenesis was constructed by overexpressing the PHA synthesis operon phaCABCn (cloned from Cupriavidus necator) controlled by the essential gene ompW (encoding outer membrane protein W) promoter and the constitutive porin promoter that are continuously expressed at high levels throughout the cell growth process, allowing poly(3-hydroxybutyrate) (PHB) production to proceed in nutrient-rich (also nitrogen-rich) food waste hydrolysates of various sources. The recombinant H. bluephagenesis termed WZY278 generated 22 g L-1 cell dry weight (CDW) containing 80 wt% PHB when cultured in food waste hydrolysates in shake flasks, and it was grown to 70 g L-1 CDW containing 80 wt% PHB in a 7-L bioreactor via fed-batch cultivation. Thus, unsterilizable food waste hydrolysates can become nutrient-rich substrates for PHB production by H. bluephagenesis able to be grown contamination-free under open conditions.

Cambial meristematic cell culture: a sustainable technology toward in vitro specialized metabolites production.

Cambial meristematic cells (CMCs) culture has received a fair share of scientific and industrial attention among the trending topics of plant cell culture, especially their potential toward secondary metabolites production. However, the conventional plant cell culture is often not commercially feasible because of difficulties associated with culture dedifferentiated cells. Several reports have been published to culture CMCs and bypass the dedifferentiation process in plant cell culture. Numerous mitochondria, multiple vacuoles, genetic stability, self-renewal, higher biomass, and stable metabolites accumulation are the characteristics features of CMCs compared with dedifferentiated cells (DDCs) culture. The CMCs culture has a broader application to produce large-scale natural compounds for: pharmaceuticals, food, and cosmetic industries. Cutting-edge progress in plant cellular and molecular biology has allowed unprecedented insights into cambial stem cell culture and its fundamental processes. Therefore, regarding sustainability and natural compound production, cambial cell culture ranks among the most vital biotechnological interventions for industrial and economic perspectives. This review highlights the recent advances in plant stem cell culture and understands the cambial cells induction and culture mechanisms that affect the growth and natural compounds production.

Recent advances in biosynthesis mechanisms and yield enhancement strategies of erythritol.

Erythritol is a four-carbon sugar alcohol naturally produced by microorganisms as an osmoprotectant. As a new sugar substitute, erythritol has recently been popular on the ingredient market because of its unique nutritional characteristics. Even though the history of erythritol biosynthesis dates from the turn of the twentieth century, scientific advancement has lagged behind other polyols due to the relative complexity of making it. In recent years, biosynthetic methods for erythritol have been rapidly developed due to an increase in market demand, a better understanding of metabolic pathways, and the rapid development of genetic engineering tools. This paper reviews the history of industrial strain development and focuses on the underlying mechanism of high erythritol production by strains gained through screening or mutagenesis. Meanwhile, we highlight the metabolic pathway knowledge of erythritol biosynthesis in microorganisms and summarize the metabolic engineering and research progress on critical genes involved in different stages of the synthetic pathway. Lastly, we talk about the still-contentious issues and promising future research directions that will help break the erythritol production bottleneck and make erythritol production greener and more sustainable.

Exploring the Impact of Tensions in Stakeholder Norms on Designing for Value Change: The Case of Biosafety in Industrial Biotechnology.

Synthetic biologists design and engineer organisms for a better and more sustainable future. While the manifold prospects are encouraging, concerns about the uncertain risks of genome editing affect public opinion as well as local regulations. As a consequence, biosafety and associated concepts, such as the Safe-by-design framework and genetic safeguard technologies, have gained notoriety and occupy a central position in the conversation about genetically modified organisms. Yet, as regulatory interest and academic research in genetic safeguard technologies advance, the implementation in industrial biotechnology, a sector that is already employing engineered microorganisms, lags behind. The main goal of this work is to explore the utilization of genetic safeguard technologies for designing biosafety in industrial biotechnology. Based on our results, we posit that biosafety is a case of a changing value, by means of further specification of how to realize biosafety. Our investigation is inspired by the Value Sensitive Design framework, to investigate scientific and technological choices in their appropriate social context. Our findings discuss stakeholder norms for biosafety, reasonings about genetic safeguards, and how these impact the practice of designing for biosafety. We show that tensions between stakeholders occur at the level of norms, and that prior stakeholder alignment is crucial for value specification to happen in practice. Finally, we elaborate in different reasonings about genetic safeguards for biosafety and conclude that, in absence of a common multi-stakeholder effort, the differences in informal biosafety norms and the disparity in biosafety thinking could end up leading to design requirements for compliance instead of for safety.

Identification of a novel gene required for competitive growth at high temperature in the thermotolerant yeast Kluyveromyces marxianus.

It is important to understand the basis of thermotolerance in yeasts to broaden their application in industrial biotechnology. The capacity to run bioprocesses at temperatures above 40 °C is of great interest but this is beyond the growth range of most of the commonly used yeast species. In contrast, some industrial yeasts such as Kluyveromyces marxianus can grow at temperatures of 45 °C or higher. Such species are valuable for direct use in industrial biotechnology and as a vehicle to study the genetic and physiological basis of yeast thermotolerance. In previous work, we reported that evolutionarily young genes disproportionately changed expression when yeast were growing under stressful conditions and postulated that such genes could be important for long-term adaptation to stress. Here, we tested this hypothesis in K. marxianus by identifying and studying species-specific genes that showed increased expression during high-temperature growth. Twelve such genes were identified and 11 were successfully inactivated using CRISPR-mediated mutagenesis. One gene, KLMX_70384, is required for competitive growth at high temperature, supporting the hypothesis that evolutionary young genes could play roles in adaptation to harsh environments. KLMX_70384 is predicted to encode an 83 aa peptide, and RNA sequencing and ribo-sequencing were used to confirm transcription and translation of the gene. The precise function of KLMX_70384 remains unknown but some features are suggestive of RNA-binding activity. The gene is located in what was previously considered an intergenic region of the genome, which lacks homologues in other yeasts or in databases. Overall, the data support the hypothesis that genes that arose de novo in K. marxianus after the speciation event that separated K. marxianus and K. lactis contribute to some of its unique traits.

Development of a ribosome profiling protocol to study translation in Kluyveromyces marxianus.

Kluyveromyces marxianus is an interesting and important yeast because of particular traits such as thermotolerance and rapid growth, and for applications in food and industrial biotechnology. For both understanding its biology and developing bioprocesses, it is important to understand how K. marxianus responds and adapts to changing environments. For this, a full suite of omics tools to measure and compare global patterns of gene expression and protein synthesis is needed. We report here the development of a ribosome profiling method for K. marxianus, which allows codon resolution of translation on a genome-wide scale by deep sequencing of ribosome locations on mRNAs. To aid in the analysis and sharing of ribosome profiling data, we added the K. marxianus genome as well as transcriptome and ribosome profiling data to the publicly accessible GWIPS-viz and Trips-Viz browsers. Users are able to upload custom ribosome profiling and RNA-Seq data to both browsers, therefore allowing easy analysis and sharing of data. We also provide a set of step-by-step protocols for the experimental and bioinformatic methods that we developed.

Protocols for marker-free gene knock-out and knock-down in Kluyveromyces marxianus using CRISPR/Cas9.

There is increased interest in strain engineering in the food and industrial yeast Kluyveromyces marxianus and a number of CRISPR/Cas9 systems have been described and used by different groups. The methods that we developed allow for very rapid and efficient inactivation of target genes using the endogenous DNA repair mechanisms of the cell. The strains and plasmids that we use are freely available, and here we provide a set of integrated protocols to easily inactivate genes and to precisely integrate DNA fragments into the genome, for example for promoter replacement, allelic swaps or introduction of point mutations. The protocols use the Cas9/gRNA expression plasmid pUCC001 and Golden Gate assembly for molecular cloning of targeting sequences. A genome-wide set of target sequences is provided. Using these plasmids in wild-type strains or in strains lacking non-homologous end-joining (NHEJ) DNA repair, the first set of protocols explain how to introduce indels (NHEJ-mediated) or precise deletions (homology-dependent repair (HDR)-mediated) at precise targets. The second set of protocols describe how to swap a promoter or coding sequence to yield a reprogrammed gene. The methods do not require the use of dominant or auxotrophic marker genes and thus the strains generated are marker-free. The protocols have been tested in multiple K. marxianus strains, are straightforward and can be carried out in any molecular biology laboratory without specialized equipment.

Substantial gradient mitigation in simulated large-scale bioreactors by optimally placed multiple feed points.

The performance of large-scale stirred tank and bubble column bioreactors is often hindered by insufficient macromixing of feeds, leading to heterogeneities in pH, substrate, and oxygen, which complicates process scale-up. Appropriate feed placement or the use of multiple feed points could improve mixing. Here, theoretically optimal placement of feed points was derived using one-dimensional diffusion equations. The utility of optimal multipoint feeds was evaluated with mixing, pH control, and bioreaction simulations using three-dimensional compartment models of four industrially relevant bioreactors with working volumes ranging from 8 to 237 m3 . Dividing the vessel axially in equal-sized compartments and locating a feed point or multiple feed points symmetrically in each compartment reduced the mixing time substantially by more than a minute and mitigated gradients of pH, substrate, and oxygen. Performance of the large-scale bioreactors was consequently restored to ideal, homogeneous reactor performance: oxygen consumption and biomass yield were recovered and the phenotypical heterogeneity of the biomass population was diminished.

Dynamic feedback regulation for efficient membrane protein production using a small RNA-based genetic circuit in Escherichia coli.

BACKGROUND: Membrane proteins (MPs) are an important class of molecules with a wide array of cellular functions and are part of many metabolic pathways. Despite their great potential-as therapeutic drug targets or in microbial cell factory optimization-many challenges remain for efficient and functional expression in a host such as Escherichia coli. RESULTS: A dynamically regulated small RNA-based circuit was developed to counter membrane stress caused by overexpression of different MPs. The best performing small RNAs were able to enhance the maximum specific growth rate with 123%. On culture level, the total MP production was increased two-to three-fold compared to a system without dynamic control. This strategy not only improved cell growth and production of the studied MPs, it also suggested the potential use for countering metabolic burden in general. CONCLUSIONS: A dynamically regulated feedback circuit was developed that can sense metabolic stress caused by, in casu, the overexpression of an MP and responds to it by balancing the metabolic state of the cell and more specifically by downregulating the expression of the MP of interest. This negative feedback mechanism was established by implementing and optimizing simple-to-use genetic control elements based on post-transcriptional regulation: small non-coding RNAs. In addition to membrane-related stress when the MP accumulated in the cytoplasm as aggregates, the sRNA-based feedback control system was still effective for improving cell growth but resulted in a decreased total protein production. This result suggests promiscuity of the MP sensor for more than solely membrane stress.

Towards an understanding of oleate hydratases and their application in industrial processes.

Fatty acid hydratases are unique to microorganisms. Their native function is the oxidation of unsaturated C-C bonds to enable detoxification of environmental toxins. Within this enzyme family, the oleate hydratases (Ohys), which catalyze the hydroxylation of oleic acid to 10-(R)-hydroxy stearic acid (10-HSA) have recently gained particular industrial interest. 10-HSA is considered to be a replacement for 12-(R)-hydroxy stearic acid (12-HSA), which has a broad application in the chemical and pharmaceutical industry. As 12-HSA is obtained through an energy consuming synthesis process, the biotechnological route for sustainable 10-HSA production is of significant industrial interest. All Ohys identified to date have a non-redox active FAD bound in their active site. Ohys can be divided in several subfamilies, that differ in their oligomerization state and the decoration with amino acids in their active sites. The latter observation indicates a different reaction mechanism across those subfamilies. Despite intensive biotechnological, biochemical and structural investigations, surprising little is known about substrate binding and the reaction mechanism of this enzyme family. This review, summarizes our current understanding of Ohys with a focus on sustainable biotransformation.

The soil bacterium, Corynebacterium glutamicum, from biosynthesis of value-added products to bioremediation: A master of many trades.

Ever since its discovery in 1957, Corynebacterium glutamicum has become a well-established industrial strain and is known for its massive capability of producing various amino acids (like L-lysine and L-glutamate) and other value-added chemicals. With the rising demand for these bio-based products, the revelation of the whole genome sequences of the wild type strains, and the astounding advancements made in the fields of metabolic engineering and systems biology, our perspective of C. glutamicum has been revolutionized and has expanded our understanding of its strain development. With these advancements, a new era for C. glutamicum supremacy in the field of industrial biotechnology began. This led to remarkable progress in the enhancement of tailor-made over-producing strains and further development of the substrate spectrum of the bacterium, to easily accessible, economical, and renewable resources. C. glutamicum has also been metabolically engineered and used in the degradation/assimilation of highly toxic and ubiquitous environmental contaminant, arsenic, present in water or soil. Here, we review the history, current knowledge, progress, achievements, and future trends relating to the versatile metabolic factory, C. glutamicum. This review paper is devoted to C. glutamicum which is one of the leading industrial microbes, and one of the most promising and versatile candidates to be developed. It can be used not only as a platform microorganism to produce different value-added chemicals and recombinant proteins, but also as a tool for bioremediation, allowing to enhance specific properties, for example in situ bioremediation.

Bioprospecting of microbial enzymes: current trends in industry and healthcare.

Microbial enzymes have an indispensable role in producing foods, pharmaceuticals, and other commercial goods. Many novel enzymes have been reported from all domains of life, such as plants, microbes, and animals. Nonetheless, industrially desirable enzymes of microbial origin are limited. This review article discusses the classifications, applications, sources, and challenges of most demanded industrial enzymes such as pectinases, cellulase, lipase, and protease. In addition, the production of novel enzymes through protein engineering technologies such as directed evolution, rational, and de novo design, for the improvement of existing industrial enzymes is also explored. We have also explored the role of metagenomics, nanotechnology, OMICs, and machine learning approaches in the bioprospecting of novel enzymes. Overall, this review covers the basics of biocatalysts in industrial and healthcare applications and provides an overview of existing microbial enzyme optimization tools. KEY POINTS: • Microbial bioactive molecules are vital for therapeutic and industrial applications. • High-throughput OMIC is the most proficient approach for novel enzyme discovery. • Comprehensive databases and efficient machine learning models are the need of the hour to fast forward de novo enzyme design and discovery.

Novel thermophilic polyhydroxyalkanoates producing strain Aneurinibacillus thermoaerophilus CCM 8960.

Aneurinibacillus thermoaerophilus CCM 8960 is a thermophilic bacterium isolated from compost in Brno. The bacterium accumulates polyhydroxyalkanoates (PHAs), a biodegradable and renewable alternative to petrochemical polymers. The bacterium reveals several features that make it a very interesting candidate for the industrial production of PHA. At first, due to its thermophilic character, the bacterium can be utilized in agreement with the concept of next-generation industrial biotechnology (NGIB), which relies on extremophiles. Second, the bacterium is capable of producing PHA copolymers containing a very high portion of 4-hydroxybutyrate (4HB). Such materials possess unique properties and can be advantageously used in multiple applications, including but not limited to medicine and healthcare. Therefore, this work focuses on the in-depth characterization of A. thermoaerophilus CCM 8960. In particular, we sequenced and assembled the genome of the bacterium and identified its most important genetic features, such as the presence of plasmids, prophages, CRISPR arrays, antibiotic-resistant genes, and restriction-modification (R-M) systems, which might be crucial for the development of genome editing tools. Furthermore, we focused on genes directly involved in PHA metabolism. We also experimentally studied the kinetics of glycerol and 1,4-butanediol (1,4BD) utilization as well as biomass growth and PHA production during cultivation. Based on these data, we constructed a metabolic model to reveal metabolic fluxes and nodes of glycerol and 1,4BD concerning their incorporation into the poly(3-hydroxybutyrate-co-4-hydroxybutyrate (P(3HB-co-4HB)) structure. KEY POINTS: • Aneurinibacillus sp. H1 was identified as Aneurinibacillus thermoaerophilus. • PHA metabolism pathway with associated genes was presented. • Unique monomer composition of produced PHAs was reported.