Biosystem design of Corynebacterium glutamicum for bioproduction.

Corynebacterium glutamicum, a natural glutamate-producing bacterium adopted for industrial production of amino acids, has been extensively explored recently for high-level biosynthesis of amino acid derivatives, bulk chemicals such as organic acids and short-chain alcohols, aromatics, and natural products, including polyphenols and terpenoids. Here, we review the recent advances with a focus on biosystem design principles, metabolic characterization and modeling, omics analysis, utilization of nonmodel feedstock, emerging CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) tools for Corynebacterium strain engineering, biosensors, and novel strains of C. glutamicum. Future research directions for developing C. glutamicum cell factories are also discussed.

Utilization of microbial cocultures for converting mixed substrates to valuable bioproducts.

Utilization of microbial cocultures has been found to be a powerful approach for biochemical production. Cultivation of microbial co-culturescocultures on mixed substrates provides new opportunities and flexibility to control the growth and biosynthesis behavior of coculture members, and thus adds a new dimension for microbial coculture engineering. More generally, recruitment of microbial cocultures allows for efficient utilization of substrates to produce complex end products, which is challenging to achieve by monoculture approaches, which has been the traditional microbial engineering approach. To this end, significant achievements have been made in recent years to advance this new approach in metabolic engineering. In this review, we highlight representative groups of bioproducts that are produced from mixed substrates using various microbial cocultures. The challenges and opportunities of this approach are also discussed.

Harnessing electrical-to-biochemical conversion for microbial synthesis.

Electrical-to-biochemical conversion (E2BC) drives cell metabolism for biosynthesis and has become a promising way to realize green biomanufacturing. This review discusses the following aspects: 1. the natural E2BC processes and their underlying E2BC mechanism; 2. development of artificial E2BC for tunable microbial electrosynthesis; 3. design of electrobiochemical systems using self-powered, light-assisted, and nano-biohybrid approaches; 4. synthetic biology methods for efficient microbial electrosynthesis. This review also compares E2BC with electrocatalysis-biochemical conversion (EC2BC), as both strategies may lead to future carbon negative green biomanufacturing.

Mitigation of host cell mutations and regime shift during microbial fermentation: a perspective from flux memory.

Microbial engineering forces flux redistribution to accommodate higher production rates, straining the cellular supply chain and leading to growth deficiency. Thus, there is a selective pressure to alleviate metabolic burden and revert towards the innate flux distribution ('flux memory') via mutations. Suboptimal fermentation exacerbates this phenomenon as increased number of generations prolong the selection window for the underlying flux memory to generate faster growing non-producers. New strategies to mitigate host genetic instability include laboratory evolution, high-resolution genome resequencing combined with phenotype screening, mismatch repair protein engineering, and advanced synthetic biology approaches (e.g. oscillators and biosensor regulators). Moreover, 13C-metabolic flux analysis can quantify flux suboptimality driven by metabolic burdens and cultivation stresses. Elucidation of correlations between metabolic suboptimality and host mutation rates/spectra may lead to early stage risk assessments of culture-population's regime shift during process scale-up as well as strategies to boost bioproductions.

Making brilliant colors by microorganisms.

Anthocyanins, the colorful molecules found in plants, have positive health effects in humans, and are used as food colorants and nutraceuticals. Currently, the industrial supply of anthocyanins largely depends on extraction from plants, a method that lacks robustness and is potentially unsustainable. A promising alternative is biosynthesis by metabolically engineered microbes, which has achieved considerable success. Here, we review recent progress on anthocyanin biosynthesis in engineered microorganisms and the engineering approaches for enhancing anthocyanin production. The de novo anthocyanin production strategies and microbial production of unusual anthocyanins such as deuterated cyanidin 3-O-glucoside and pyranoanthocyanins are also covered. These engineering strategies will provide a guidance to microbial production of anthocyanins. Existing problems and future directions are also discussed.

Recent advances in modular co-culture engineering for synthesis of natural products.

The microbial production of natural products has been traditionally accomplished in a single organism engineered to accommodate target biosynthetic pathways. Often times, such approaches result in large metabolic burdens as key cofactors, precursor metabolites and energy are channeled to pathways of structurally complex chemicals. Recently, modular co-culture engineering has emerged as a new approach to efficiently conduct heterologous biosynthesis and greatly enhance the production of natural products. This review highlights recent advances that leverage Escherichia coli-based modular co-culture engineering for making natural products. Potential future perspectives for studies in this promising field are addressed as well.

Advances in the development and application of microbial consortia for metabolic engineering.

Recent advances in metabolic engineering enable the production of high-value chemicals via expressing complex biosynthetic pathways in a single microbial host. However, many engineered strains suffer from poor product yields due to redox imbalance and excess metabolic burden, and require compartmentalization of the pathway for optimal function. To address this problem, significant developments have been made towards co-cultivation of more than one engineered microbial strains to distribute metabolic burden between the co-cultivation partners and improve the product yield. In this emerging approach, metabolic pathway modules can be optimized separately in suitable hosts that will then be combined to enable optimal functionality of the complete pathway. This modular approach broadens the possibilities to fine tune sophisticated production platforms and thus achieve the biosynthesis of very complex compounds. Here, we review the different applications and the overall potential of natural and artificial co-cultivation systems in metabolic engineering in order to improve bioproduction/bioconversion. In addition to the several advantages over monocultures, major challenges and opportunities associated with co-cultivation are also discussed in this review.

The road to animal-free glycosaminoglycan production: current efforts and bottlenecks.

Animal-extraction, despite its limitations, continues to monopolize the fast-growing glycosaminoglycan (GAG) industry. The past few years have seen an increased interest in the development of alternative GAG production methods. Chemical and chemo-enzymatic synthesis and biosynthesis from GAG producing cells, including engineered recombinant strains, are currently under investigation. Despite achieving considerable successes, these alternate approaches cannot yet meet worldwide demands for these important polysaccharides. Bottlenecks associated with achieving high-titers need to be addressed using newly developed tools. Several parameters including chassis choice, analytics, intracellular precursor synthesis, enzyme engineering and use of synthetic biology tools need to be optimized. We envision that new engineering approaches together with advances in the basic biology and chemistry of GAGs will move GAG production beyond its currently limited supply chain.

Microbial production of value-added nutraceuticals.

Nutraceuticals are important natural bioactive compounds that confer health-promoting and medical benefits to humans. Globally growing demands for value-added nutraceuticals for prevention and treatment of human diseases have rendered nutraceuticals a multi-billion dollar market. However, supply limitations and extraction difficulties from natural sources such as plants, animals or fungi, restrict the large-scale use of nutraceuticals. Metabolic engineering via microbial production platforms has been advanced as an eco-friendly alternative approach for production of value-added nutraceuticals from simple carbon sources. Microbial platforms like the most widely used Escherichia coli and Saccharomyces cerevisiae have been engineered as versatile cell factories for production of diverse and complex value-added chemicals such as phytochemicals, prebiotics, polysaccaharides and poly amino acids. This review highlights the recent progresses in biological production of value-added nutraceuticals via metabolic engineering approaches.

Sensitive cells: enabling tools for static and dynamic control of microbial metabolic pathways.

Natural metabolic pathways are dynamically regulated at the transcriptional, translational, and protein levels. Despite this, traditional pathway engineering has relied on static control strategies to engender changes in metabolism, most likely due to ease of implementation and perceived predictability of design outcome. Increasingly in recent years, however, metabolic engineers have drawn inspiration from natural systems and have begun to harness dynamically controlled regulatory machinery to improve design of engineered microorganisms for production of specialty and commodity chemicals. Here, we review recent enabling technologies for engineering static control over pathway expression levels, and we discuss state-of-the-art dynamic control strategies that have yielded improved outcomes in the field of microbial metabolic engineering. Furthermore, we emphasize design of a novel class of genetically encoded controllers that will facilitate automatic, transient tuning of synthetic and endogenous pathways.