Production of industrial chemicals using renewable biomass feedstock is becoming increasingly important to address limited fossil resources, climate change and other environmental problems. To develop high-performance microbial cell factories, equivalent to chemical plants, microorganisms undergo systematic metabolic engineering to efficiently convert biomass-derived carbon sources into target chemicals. Over the past two decades, many engineered microorganisms capable of producing natural and non-natural chemicals have been developed. This Review details the current status of representative industrial chemicals that are produced through biological and/or chemical reactions. We present a comprehensive bio-based chemicals map that highlights the strategies and pathways of single or multiple biological reactions, chemical reactions and combinations thereof towards production of particular chemicals of interest. Future challenges are also discussed to enable production of even more diverse chemicals and more efficient production of chemicals from renewable feedstocks.

A comprehensive metabolic map for production of bio-based chemicals

The microbiota consists of a dynamic multispecies community of bacteria, fungi, archaea, and protozoans, bringing to the host organism a dowry of cells and genes more numerous than its own. Among the different non-sterile cavities, the human gut harbors the most complex microbiota, with a strong impact on host homeostasis and immunostasis, being thus essential for maintaining the health condition. In this review, we outline the roles of gut microbiota in immunity, starting with the background information supporting the further presentation of the implications of gut microbiota dysbiosis in host susceptibility to infections, hypersensitivity reactions, autoimmunity, chronic inflammation, and cancer. The role of diet and antibiotics in the occurrence of dysbiosis and its pathological consequences, as well as the potential of probiotics to restore eubiosis is also discussed.

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Aspects of Gut Microbiota and Immune System Interactions in Infectious Diseases, Immunopathology, and Cancer.

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

Cellular processes are carried out by many genes, and their study and optimization requires multiple levers by which they can be independently controlled. The most common method is via a genetically encoded sensor that responds to a small molecule. However, these sensors are often suboptimal, exhibiting high background expression and low dynamic range. Further, using multiple sensors in one cell is limited by cross-talk and the taxing of cellular resources. Here, we have developed a directed evolution strategy to simultaneously select for lower background, high dynamic range, increased sensitivity, and low cross-talk. This is applied to generate a set of 12 high-performance sensors that exhibit >100-fold induction with low background and cross-reactivity. These are combined to build a single “sensor array” in the genomes of E. coli MG1655 (wild-type), DH10B (cloning), and BL21 (protein expression). These “Marionette” strains allow for the independent control of gene expression using 12 small-molecule inducers. A directed evolution approach was applied to optimize a set of 12 small-molecule-responsive biosensors, which led to the engineering of “Marionette” strains of Escherichia coli incorporating these sensors for biotechnological applications.

Escherichia coli "Marionette" strains with 12 highly optimized small-molecule sensors.

Most petroleum-derived plastics, as exemplified by poly(ethylene terephthalate) (PET), are chemically inactive and highly resistant to microbial attack. The accumulation of plastic waste results in environmental pollution and threatens ecosystems, referred to as the “microplastic issue”. Recently, PET hydrolytic enzymes (PHEs) have been identified and we reported PET degradation by a microbial consortium and its bacterial resident, Ideonella sakaiensis. Bioremediation may thus provide an alternative solution to recycling plastic waste. The mechanism of PET degradation into benign monomers by PET hydrolase and mono(2-hydroxyethyl) terephthalic acid (MHET) hydrolase from I. sakaiensis has been elucidated; nevertheless, biodegradation may require additional development for commercialization owing to the low catalytic activity of these enzymes. Here, we introduce PET degrading microorganisms and the enzymes involved, along with the evolution of PHEs to address the issues that hamper microbial and enzymatic PE...

Biodegradation of PET: Current Status and Application Aspects

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

CRISPR technologies for bacterial systems: Current achievements and future directions.

Haem has widespread applications in healthcare and food supplement industries. Escherichia coli has previously been engineered to produce a small amount of haem intracellularly through the C4 pathway, requiring extraction for applications. Here we report secretory production of free haem by engineered E. coli strains, using the C5 pathway and the optimized downstream pathway for haem biosynthesis. Furthermore, knocking out ldhA, pta and also yfeX—encoding a putative haem-degrading enzyme—results in 7.88 mg l−1 of total haem with 1.26 mg l−1 of extracellular haem in flask cultivation. Fed-batch fermentations of the engineered strain overexpressing a haem exporter CcmABC from glucose only and glucose supplemented with l-glutamate secrete 73.4 and 151.4 mg l−1 of haem, respectively, which are 63.5% of 115.5 mg l−1 and 63.3% of 239.2 mg l−1 of total haem produced. The engineered E. coli strain reported here will be useful for microbial production of free haem. Microbial production of haem for applications in healthcare and food supplement industry requires high-performing strains. Here, Lee and co-workers report secretory production of free haem by metabolically engineered Escherichia coli strains to produce up to 239 mg l−1 total haem.

Metabolic engineering of Escherichia coli for secretory production of free haem

Systems metabolic engineering, which recently emerged as metabolic engineering integrated with systems biology, synthetic biology, and evolutionary engineering, allows engineering of microorganisms on a systemic level for the production of valuable chemicals far beyond its native capabilities. Here, we review the strategies for systems metabolic engineering and particularly its applications in Escherichia coli. First, we cover the various tools developed for genetic manipulation in E. coli to increase the production titers of desired chemicals. Next, we detail the strategies for systems metabolic engineering in E. coli, covering the engineering of the native metabolism, the expansion of metabolism with synthetic pathways, and the process engineering aspects undertaken to achieve higher production titers of desired chemicals. Finally, we examine a couple of notable products as case studies produced in E. coli strains developed by systems metabolic engineering. The large portfolio of chemical products successfully produced by engineered E. coli listed here demonstrates the sheer capacity of what can be envisioned and achieved with respect to microbial production of chemicals. Systems metabolic engineering is no longer in its infancy; it is now widely employed and is also positioned to further embrace next-generation interdisciplinary principles and innovation for its upgrade. Systems metabolic engineering will play increasingly important roles in developing industrial strains including E. coli that are capable of efficiently producing natural and nonnatural chemicals and materials from renewable nonfood biomass.

Kyeong Rok Choi

Papers

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

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

CRISPR technologies for bacterial systems: Current achievements and future directions.

Metabolic engineering of Escherichia coli for secretory production of free haem

Systems Metabolic Engineering of Escherichia coli.