Efficient De Novo Biosynthesis of Heme by Membrane Engineering in Escherichia coli.

Heme is of great significance in food nutrition and food coloring, and the successful launch of artificial meat has greatly improved the application of heme in meat products. The precursor of heme, 5-aminolevulinic acid (ALA), has a wide range of applications in the agricultural and medical fields, including in the treatment of corona virus disease 2019 (COVID-19). In this study, E. coli recombinants capable of heme production were developed by metabolic engineering and membrane engineering. Firstly, by optimizing the key genes of the heme synthesis pathway and the screening of hosts and plasmids, the recombinant strain EJM-pCD-AL produced 4.34 ± 0.02 mg/L heme. Then, the transport genes of heme precursors CysG, hemX and CyoE were knocked out, and the extracellular transport pathways of heme Dpp and Ccm were strengthened, obtaining the strain EJM-ΔCyoE-pCD-AL that produced 9.43 ± 0.03 mg/L heme. Finally, fed-batch fermentation was performed in a 3-L fermenter and reached 28.20 ± 0.77 mg/L heme and 303 ± 1.21 mg/L ALA. This study indicates that E. coli recombinant strains show a promising future in the field of heme and ALA production.

Versatile tools of synthetic biology applied to drug discovery and production.

Although synthetic biology is an emerging research field, which has come to prominence within the last decade, it already has many practical applications. Its applications cover the areas of pharmaceutical biotechnology and drug discovery, bringing essential novel methods and strategies such as metabolic engineering, reprogramming the cell fate, drug production in genetically modified organisms, molecular glues, functional nucleic acids and genome editing. This review discusses the main avenues for synthetic biology application in pharmaceutical biotechnology. The authors believe that synthetic biology will reshape drug development and drug production to a similar extent as the advances in organic chemical synthesis in the 20th century. Therefore, synthetic biology already plays an essential role in pharmaceutical, biotechnology, which is the main focus of this review.

Efficient cell factories for the production of N-methylated amino acids and for methanol-based amino acid production.

The growing world needs commodity amino acids such as L-glutamate and L-lysine for use as food and feed, and specialty amino acids for dedicated applications. To meet the supply a paradigm shift regarding their production is required. On the one hand, the use of sustainable and cheap raw materials is necessary to sustain low production cost and decrease detrimental effects of sugar-based feedstock on soil health and food security caused by competing uses of crops in the feed and food industries. On the other hand, the biotechnological methods to produce functionalized amino acids need to be developed further, and titres enhanced to become competitive with chemical synthesis methods. In the current review, we present successful strain mutagenesis and rational metabolic engineering examples leading to the construction of recombinant bacterial strains for the production of amino acids such as L-glutamate, L-lysine, L-threonine and their derivatives from methanol as sole carbon source. In addition, the fermentative routes for bioproduction of N-methylated amino acids are highlighted, with focus on three strategies: partial transfer of methylamine catabolism, S-adenosyl-L-methionine dependent alkylation and reductive methylamination of 2-oxoacids.

An Engineered Cytidine Deaminase for Biocatalytic Production of a Key Intermediate of the Covid-19 Antiviral Molnupiravir.

The Covid-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue recently approved as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of an efficient biocatalyst for the production of N-hydroxy-cytidine through evolutionary adaption of the hydrolytic enzyme cytidine deaminase. This engineered biocatalyst performs >85 000 turnovers in less than 3 h, operates at 180 g/L substrate loading, and benefits from in situ crystallization of the N-hydroxy-cytidine product (85% yield), which can be converted to Molnupiravir by a selective 5'-acylation using Novozym 435.

Mini-synplastomes for plastid genetic engineering.

In the age of synthetic biology, plastid engineering requires a nimble platform to introduce novel synthetic circuits in plants. While effective for integrating relatively small constructs into the plastome, plastid engineering via homologous recombination of transgenes is over 30 years old. Here we show the design-build-test of a novel synthetic genome structure that does not disturb the native plastome: the 'mini-synplastome'. The mini-synplastome was inspired by dinoflagellate plastome organization, which is comprised of numerous minicircles residing in the plastid instead of a single organellar genome molecule. The first mini-synplastome in plants was developed in vitro to meet the following criteria: (i) episomal replication in plastids; (ii) facile cloning; (iii) predictable transgene expression in plastids; (iv) non-integration of vector sequences into the endogenous plastome; and (v) autonomous persistence in the plant over generations in the absence of exogenous selection pressure. Mini-synplastomes are anticipated to revolutionize chloroplast biotechnology, enable facile marker-free plastid engineering, and provide an unparalleled platform for one-step metabolic engineering in plants.

Computational and Synthetic Biology Approaches for the Biosynthesis of Antiviral and Anticancer Terpenoids from Bacillus subtilis.

Recent advancements in medicinal research have identified several antiviral and anticancer terpenoids that are usually deployed as a source of flavor, fragrances and pharmaceuticals. Under the current COVID-19 pandemic conditions, natural therapeutics with the least side effects are the need of the hour to save the patients, especially, which are pre-affected with other medical complications. Although plants are the major sources of terpenoids; however, for the environmental concerns, the global interest has shifted to the biocatalytic production of molecules from microbial sources. The gram-positive bacterium Bacillus subtilis is a suitable host in this regard due to its GRAS (generally regarded as safe) status, ease in genetic manipulations and wide industrial acceptability. The B. subtilis synthesizes its terpenoid molecules from 1-deoxy-d-xylulose-5-phosphate (DXP) pathway, a common route in almost all microbial strains. Here, we summarize the computational and synthetic biology approaches to improve the production of terpenoid-based therapeutics from B. subtilis by utilizing DXP pathway. We focus on the in-silico approaches for screening the functionally improved enzyme-variants of the two crucial enzymes namely, the DXP synthase (DXS) and Farnesyl Pyrophosphate Synthase (FPPS). The approaches for engineering the active sites are subsequently explained. It will be helpful to construct the functionally improved enzymes for the high-yield production of terpenoid-based anticancer and antiviral metabolites, which would help to reduce the cost and improve the availability of such therapeutics for the humankind.

Endophytes: the novel sources for plant terpenoid biosynthesis.

Terpenoids are natural compounds predominantly present in plants. They have many pharmaceutical and/or nutritional functions, and have been widely applied in medical, food, and cosmetics industries. Recently, terpenoids have been used in the clinical treatment of COVID-19 due to the good antiviral activities. The increasing demand for terpenoids in international markets poses a serious threat to many plant species. For environmentally sustainable development, microbial cell factories have been utilized as the promising platform to produce terpenoids. Nevertheless, the bioproduction of most terpenoids cannot meet commercial requirements due to the low cost-benefit ratio until now. The biosynthetic potential of endophytes has gained attention in recent decades owing to the continual discovery of endophytes capable of synthesizing plant bioactive compounds. Accordingly, endophytes could be alternative sources of terpenoid-producing strains or terpenoid synthetic genes. In this review, we summarized the research progress describing the main and supporting roles of endophytes in terpenoid biosynthesis and biotransformation, and discussed the current problems and challenges which may prevent the further exploitation. This review will improve our understanding of endophyte resources for terpenoid production in industry in the future. The four main research interests on endophytes for terpenoid production. A: Isolation of terpenoid-producing endophytes; B: The heterologous expression of endophyte-derived terpenoid synthetic genes; C: Endophytes promoting their hosts' terpenoid production. The blue dashed arrows indicate signal transduction; D: Biotransformation of terpenoids by endophytes or their enzymes. Key points• The mechanisms employed by endophytes in terpenoid synthesis in vivo and in vitro.• Endophytes have the commercial potentials in terpenoid bioproduction and biotransformation.• Synthetic biology and multiomics will improve terpenoid bioproduction in engineered cell factories.

Shattering barriers toward clinically meaningful MSC therapies.

More than 1050 clinical trials are registered at FDA.gov that explore multipotent mesenchymal stromal cells (MSCs) for nearly every clinical application imaginable, including neurodegenerative and cardiac disorders, perianal fistulas, graft-versus-host disease, COVID-19, and cancer. Several companies have or are in the process of commercializing MSC-based therapies. However, most of the clinical-stage MSC therapies have been unable to meet primary efficacy end points. The innate therapeutic functions of MSCs administered to humans are not as robust as demonstrated in preclinical studies, and in general, the translation of cell-based therapy is impaired by a myriad of steps that introduce heterogeneity. In this review, we discuss the major clinical challenges with MSC therapies, the details of these challenges, and the potential bioengineering approaches that leverage the unique biology of MSCs to overcome the challenges and achieve more potent and versatile therapies.