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1.
A machine learning Automated Recommendation Tool for synthetic biology.
Radivojević, T, Costello, Z, Workman, K, Garcia Martin, H
Nature communications. 2020;(1):4879
Abstract
Synthetic biology allows us to bioengineer cells to synthesize novel valuable molecules such as renewable biofuels or anticancer drugs. However, traditional synthetic biology approaches involve ad-hoc engineering practices, which lead to long development times. Here, we present the Automated Recommendation Tool (ART), a tool that leverages machine learning and probabilistic modeling techniques to guide synthetic biology in a systematic fashion, without the need for a full mechanistic understanding of the biological system. Using sampling-based optimization, ART provides a set of recommended strains to be built in the next engineering cycle, alongside probabilistic predictions of their production levels. We demonstrate the capabilities of ART on simulated data sets, as well as experimental data from real metabolic engineering projects producing renewable biofuels, hoppy flavored beer without hops, fatty acids, and tryptophan. Finally, we discuss the limitations of this approach, and the practical consequences of the underlying assumptions failing.
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2.
CRISPR-based metabolic editing: Next-generation metabolic engineering in plants.
Sabzehzari, M, Zeinali, M, Naghavi, MR
Gene. 2020;:144993
Abstract
Plants generate many secondary metabolites, so called phyto-metabolites, which can be used as toxins, dyes, drugs, and insecticides in bio-warfare plus bio-terrorism, industry, medicine, and agriculture, respectively. To 2013, the first generation metabolic engineering approaches like miRNA-based manipulation were widely adopted by researchers in biosciences. However, the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR) genome editing system revolutionized metabolic engineering due to its unique features so that scientists could manipulate the biosynthetic pathways of phyto-metabolites through approaches like miRNA-mediated CRISPR-Cas9. According to the increasing importance of the genome editing in plant sciences, we discussed the current findings on CRISPR-based manipulation of phyto-metabolites in plants, especially medicinal ones, and suggested the ideas to phyto-metabolic editing.
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3.
Mannitol: physiological functionalities, determination methods, biotechnological production, and applications.
Chen, M, Zhang, W, Wu, H, Guang, C, Mu, W
Applied microbiology and biotechnology. 2020;(16):6941-6951
Abstract
Mannitol is a naturally occurring six-carbon sugar alcohol that has wide applications in the food and pharmaceutical industry because of its many properties, namely being a natural sweetener with a low metabolism and no glycemic index. The increasing demand for mannitol has spurred many studies of its production. Compared with its chemical synthesis and extraction from plants, both of which are difficult to satisfy for industrial requirements, biotechnological production of mannitol has received considerably more attention and interest from scientists because of its known advantages over those two methods. Accordingly, in this review, we summarize recent advances made in the production of mannitol through various biotechnological methods. The physicochemical properties, sources, and physiological functionalities and applications of mannitol are systematically covered and presented. Then, different determination methods for mannitol are also described and compared. Furthermore, different biotechnological strategies for the production of mannitol via fermentation engineering, protein engineering, and metabolic engineering receive a detailed overview in terms of mannitol-producing strains, enzymes, and their key reaction parameters and conditions. KEY POINTS • Physiological functionalities and applications of mannitol are presented in detail. • Different determination methods for mannitol are also described and compared. • Various biotechnological strategies for the production of mannitol are reviewed.
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4.
Plant Synthetic Metabolic Engineering for Enhancing Crop Nutritional Quality.
Zhu, Q, Wang, B, Tan, J, Liu, T, Li, L, Liu, YG
Plant communications. 2020;(1):100017
Abstract
Nutrient deficiencies in crops are a serious threat to human health, especially for populations in poor areas. To overcome this problem, the development of crops with nutrient-enhanced traits is imperative. Biofortification of crops to improve nutritional quality helps combat nutrient deficiencies by increasing the levels of specific nutrient components. Compared with agronomic practices and conventional plant breeding, plant metabolic engineering and synthetic biology strategies are more effective and accurate in synthesizing specific micronutrients, phytonutrients, and/or bioactive components in crops. In this review, we discuss recent progress in the field of plant synthetic metabolic engineering, specifically in terms of research strategies of multigene stacking tools and engineering complex metabolic pathways, with a focus on improving traits related to micronutrients, phytonutrients, and bioactive components. Advances and innovations in plant synthetic metabolic engineering would facilitate the development of nutrient-enriched crops to meet the nutritional needs of humans.
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5.
Rewiring yeast metabolism to synthesize products beyond ethanol.
Gambacorta, FV, Dietrich, JJ, Yan, Q, Pfleger, BF
Current opinion in chemical biology. 2020;:182-192
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Abstract
Saccharomyces cerevisiae, Baker's yeast, is the industrial workhorse for producing ethanol and the subject of substantial metabolic engineering research in both industry and academia. S. cerevisiae has been used to demonstrate production of a wide range of chemical products from glucose. However, in many cases, the demonstrations report titers and yields that fall below thresholds for industrial feasibility. Ethanol synthesis is a central part of S. cerevisiae metabolism, and redirecting flux to other products remains a barrier to industrialize strains for producing other molecules. Removing ethanol producing pathways leads to poor fitness, such as impaired growth on glucose. Here, we review metabolic engineering efforts aimed at restoring growth in non-ethanol producing strains with emphasis on relieving glucose repression associated with the Crabtree effect and rewiring metabolism to provide access to critical cellular building blocks. Substantial progress has been made in the past decade, but many opportunities for improvement remain.
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6.
Designing an Artificial Pathway for the Biosynthesis of a Novel Phenazine N-Oxide in Pseudomonas chlororaphis HT66.
Guo, S, Liu, R, Wang, W, Hu, H, Li, Z, Zhang, X
ACS synthetic biology. 2020;(4):883-892
Abstract
Aromatic N-oxides are valuable due to their versatile chemical, pharmaceutical, and agricultural applications. Natural phenazine N-oxides possess potent biological activities and can be applied in many ways; however, few N-oxides have been identified. Herein, we developed a microbial system to synthesize phenazine N-oxides via an artificial pathway. First, the N-monooxygenase NaphzNO1 was predicted and screened in Nocardiopsis sp. 13-12-13 through a product comparison and gene sequencing. Subsequently, according to similarities in the chemical structures of substrates, an artificial pathway for the synthesis of a phenazine N-oxide in Pseudomonas chlororaphis HT66 was designed and established using three heterologous enzymes, a monooxygenase (PhzS) from P. aeruginosa PAO1, a monooxygenase (PhzO) from P. chlororaphis GP72, and the N-monooxygenase NaphzNO1. A novel phenazine derivative, 1-hydroxyphenazine N'10-oxide, was obtained in an engineered strain, P. chlororaphis HT66-SN. The phenazine N-monooxygenase NaphzNO1 was identified by metabolically engineering the phenazine-producing platform P. chlororaphis HT66. Moreover, the function of NaphzNO1, which can catalyze the conversion of 1-hydroxyphenazine but not that of 2-hydroxyphenazine, was confirmed in vitro. Additionally, 1-hydroxyphenazine N'10-oxide demonstrated substantial cytotoxic activity against two human cancer cell lines, MCF-7 and HT-29. Furthermore, the highest microbial production of 1-hydroxyphenazine N'10-oxide to date was achieved at 143.4 mg/L in the metabolically engineered strain P3-SN. These findings demonstrate that P. chlororaphis HT66 has the potential to be engineered as a platform for phenazine-modifying gene identification and derivative production. The present study also provides a promising alternative for the sustainable synthesis of aromatic N-oxides with unique chemical structures by N-monooxygenase.
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7.
A combined experimental and modelling approach for the Weimberg pathway optimisation.
Shen, L, Kohlhaas, M, Enoki, J, Meier, R, Schönenberger, B, Wohlgemuth, R, Kourist, R, Niemeyer, F, van Niekerk, D, Bräsen, C, et al
Nature communications. 2020;(1):1098
Abstract
The oxidative Weimberg pathway for the five-step pentose degradation to α-ketoglutarate is a key route for sustainable bioconversion of lignocellulosic biomass to added-value products and biofuels. The oxidative pathway from Caulobacter crescentus has been employed in in-vivo metabolic engineering with intact cells and in in-vitro enzyme cascades. The performance of such engineering approaches is often hampered by systems complexity, caused by non-linear kinetics and allosteric regulatory mechanisms. Here we report an iterative approach to construct and validate a quantitative model for the Weimberg pathway. Two sensitive points in pathway performance have been identified as follows: (1) product inhibition of the dehydrogenases (particularly in the absence of an efficient NAD+ recycling mechanism) and (2) balancing the activities of the dehydratases. The resulting model is utilized to design enzyme cascades for optimized conversion and to analyse pathway performance in C. cresensus cell-free extracts.
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8.
Plant Aromatic Prenyltransferases: Tools for Microbial Cell Factories.
de Bruijn, WJC, Levisson, M, Beekwilder, J, van Berkel, WJH, Vincken, JP
Trends in biotechnology. 2020;(8):917-934
Abstract
In plants, prenylation of aromatic compounds, such as (iso)flavonoids and stilbenoids, by membrane-bound prenyltransferases (PTs), is an essential step in the biosynthesis of many bioactive compounds. Prenylated aromatic compounds have various health-beneficial properties that are interesting for industrial applications, but their exploitation is limited due to their low abundance in nature. Harnessing plant aromatic PTs for prenylation in microbial cell factories may be a sustainable and economically viable alternative. Limitations in prenylated aromatic compound production have been identified, including availability of prenyl donor substrate. In this review, we summarize the current knowledge about plant aromatic PTs and discuss promising strategies towards the optimized production of prenylated aromatic compounds by microbial cell factories.
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9.
Metabolic engineering for the production of butanol, a potential advanced biofuel, from renewable resources.
Zhao, C, Zhang, Y, Li, Y
Biochemical Society transactions. 2020;(5):2283-2293
Abstract
Butanol is an important chemical and potential fuel. For more than 100 years, acetone-butanol-ethanol (ABE) fermentation of Clostridium strains has been the most successful process for biological butanol production. In recent years, other microbes have been engineered to produce butanol as well, among which Escherichia coli was the best one. Considering the crude oil price fluctuation, minimizing the cost of butanol production is of highest priority for its industrial application. Therefore, using cheaper feedstocks instead of pure sugars is an important project. In this review, we summarized butanol production from different renewable resources, such as industrial and food waste, lignocellulosic biomass, syngas and other renewable resources. This review will present the current progress in this field and provide insights for further engineering efforts on renewable butanol production.
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10.
Green (cell) factories for advanced production of plant secondary metabolites.
Marchev, AS, Yordanova, ZP, Georgiev, MI
Critical reviews in biotechnology. 2020;(4):443-458
Abstract
For centuries plants have been intensively utilized as reliable sources of food, flavoring, agrochemical and pharmaceutical ingredients. However, plant natural habitats are being rapidly lost due to climate change and agriculture. Plant biotechnology offers a sustainable method for the bioproduction of plant secondary metabolites using plant in vitro systems. The unique structural features of plant-derived secondary metabolites, such as their safety profile, multi-target spectrum and "metabolite likeness," have led to the establishment of many plant-derived drugs, comprising approximately a quarter of all drugs approved by the Food and Drug Administration and/or European Medicinal Agency. However, there are still many challenges to overcome to enhance the production of these metabolites from plant in vitro systems and establish a sustainable large-scale biotechnological process. These challenges are due to the peculiarities of plant cell metabolism, the complexity of plant secondary metabolite pathways, and the correct selection of bioreactor systems and bioprocess optimization. In this review, we present an integrated overview of the possible avenues for enhancing the biosynthesis of high-value marketable molecules produced by plant in vitro systems. These include metabolic engineering and CRISPR/Cas9 technology for the regulation of plant metabolism through overexpression/repression of single or multiple structural genes or transcriptional factors. The use of NMR-based metabolomics for monitoring metabolite concentrations and additionally as a tool to study the dynamics of plant cell metabolism and nutritional management is discussed here. Different types of bioreactor systems, their modification and optimal process parameters for the lab- or industrial-scale production of plant secondary metabolites are specified.