In the first wave of synthetic biology, genetic elements, combined into simple circuits, are used to control individual cellular functions. In the second wave of synthetic biology, the simple ...circuits, combined into complex circuits, form systems-level functions. However, efforts to construct complex circuits are often impeded by our limited knowledge of the optimal combination of individual circuits. For example, a fundamental question in most metabolic engineering projects is the optimal level of enzymes for maximizing the output. To address this point, combinatorial optimization approaches have been established, allowing automatic optimization without prior knowledge of the best combination of expression levels of individual genes. This review focuses on current combinatorial optimization methods and emerging technologies facilitating their applications.
Flavonoids are a large family of plant and fungal natural products, among which many have been found to possess outstanding biological activities. Utilization of engineered microbes as surrogate ...hosts for heterologous biosynthesis of flavonoids has been investigated extensively. However, current microbial biosynthesis strategies mostly rely on using one microbial strain to accommodate the long and complicated flavonoid pathways, which presents a major challenge for production optimization. Here, we adapt the emerging modular co-culture engineering approach to rationally design, establish and optimize an
Escherichia coli
co-culture for de novo biosynthesis of flavonoid sakuranetin from simple carbon substrate glucose. Specifically, two
E. coli
strains were employed to accommodate the sakuranetin biosynthesis pathway. The upstream strain was engineered for pathway intermediate
p-
coumaric acid production, whereas the downstream strain converted
p-
coumaric acid to sakuranetin. Through step
-
wise optimization of the co-culture system, we were able to produce 29.7 mg/L sakuranetin from 5 g/L glucose within 48 h, which is significantly higher than the production by the conventional monoculture-based approach. The co-culture biosynthesis was successfully scaled up in a fed-batch bioreactor, resulting in the production of 79.0 mg/L sakuranetin. To our knowledge, this is the highest bioproduction concentration reported so far for de novo sakuranetin biosynthesis using the heterologous host
E. coli
. The findings of this work expand the applicability of modular co-culture engineering for addressing the challenges associated with heterologous biosynthesis of complex natural products.
Key points
• De novo biosynthesis of sakuranetin was achieved using E. coli-E. coli co-cultures.
• Sakuranetin production by co-cultures was significantly higher than the mono-culture controls.
• The co-culture system was optimized by multiple metabolic engineering strategies.
• The co-culture biosynthesis was scaled up in fed-batch bioreactor.
In this review, we address recent advances made in pathway engineering, directed evolution, and systems/synthetic biology approaches employed in the production and modification of flavonoids from ...microbial cells. The review is divided into two major parts. In the first, various metabolic engineering and system/synthetic biology approaches used for production of flavonoids and derivatives are discussed broadly. All the manipulations/engineering accomplished on the microorganisms since 2000 are described in detail along with the biosynthetic pathway enzymes, their sources, structures of the compounds, and yield of each product. In the second part of the review, post-modifications of flavonoids by four major reactions, namely glycosylations, methylations, hydroxylations and prenylations using recombinant strains are described.
Engineering cell metabolism for bioproduction not only consumes building blocks and energy molecules (e.g., ATP) but also triggers energetic inefficiency inside the cell. The metabolic burdens on ...microbial workhorses lead to undesirable physiological changes, placing hidden constraints on host productivity. We discuss cell physiological responses to metabolic burdens, as well as strategies to identify and resolve the carbon and energy burden problems, including metabolic balancing, enhancing respiration, dynamic regulatory systems, chromosomal engineering, decoupling cell growth with production phases, and co-utilization of nutrient resources. To design robust strains with high chances of success in industrial settings, novel genome-scale models (GSMs),13 C-metabolic flux analysis (MFA), and machine-learning approaches are needed for weighting, standardizing, and predicting metabolic costs.
Metabolic engineering consistently demands to produce the maximum carbon and energy flux to target chemicals. To balance metabolic flux, gene expression levels of artificially synthesized pathways ...usually fine‐tuned using multimodular optimization strategy. However, forward construction is an engineering conundrum because a vast number of possible pathway combinations need to be constructed and analyzed. Here, an iterative high‐throughput balancing (IHTB) strategy was established to thoroughly fine‐tune the (2S)‐naringenin biosynthetic pathway. A series of gradient constitutive promoters from Escherichia coli were randomly cloned upstream of pathway genes, and the resulting library was screened using an ultraviolet spectrophotometry–fluorescence spectrophotometry high‐throughput method, which was established based on the interactions between AlCl3 and (2S)‐naringenin. The metabolic flux of the screened high‐titer strains was analyzed and iterative rounds of screening were performed based on the analysis results. After several rounds, the metabolic flux of the (2S)‐naringenin synthetic pathway was balanced, reaching a final titer of 191.9 mg/L with 29.2 mg/L p‐coumaric acid accumulation. Chalcone synthase was speculated to be the rate‐limiting enzyme because its expression level was closely related to the production of both (2S)‐naringenin and p‐coumaric acid. The established IHTB strategy can be used to efficiently balance multigene pathways, which will accelerate the development of efficient recombinant strains.
An iterative high‐throughput balancing (IHTB) strategy was established to rapidly and efficiently fine‐tuning the multi‐gene pathways by combined with dozens of gradient strength promoters. Based on the IHTB strategy, Zhou and coworkers have optimized the (2S)‐naringenin synthetic pathway, and they find that chalcone synthase is the rate‐limiting step. The described IHTB strategy provided potential applications in balancing heterologous pathways to enhance some specific metabolic fluxes for increasing the titer of target compounds.
Abstract
The increasing prevalence of antibiotic-resistant bacteria portends an impending postantibiotic age, characterized by diminishing efficacy of common antibiotics and routine application of ...multifaceted, complementary therapeutic approaches to treat bacterial infections, particularly multidrug-resistant organisms. The first line of defense for most bacterial pathogens consists of a physical and immunologic barrier known as the capsule, commonly composed of a viscous layer of carbohydrates that are covalently bound to the cell wall in Gram-positive bacteria or often to lipids of the outer membrane in many Gram-negative bacteria. Bacterial capsular polysaccharides are a diverse class of high molecular weight polysaccharides contributing to virulence of many human pathogens in the gut, respiratory tree, urinary tract, and other host tissues, by hiding cell surface components that might otherwise elicit host immune response. This review highlights capsular polysaccharides that are structurally identical or similar to polysaccharides found in mammalian tissues, including polysialic acid and glycosaminoglycan capsules hyaluronan, heparosan, and chondroitin. Such nonimmunogenic coatings render pathogens insensitive to certain immune responses, effectively increasing residence time in host tissues and enabling pathologically relevant population densities to be reached. Biosynthetic pathways and capsular involvement in immune system evasion are described, providing a basis for potential therapies aimed at supplementing or replacing antibiotic treatment.
Bacterial pathogens bearing capsular polysaccharides identical to mammalian glycans benefit from an additional level of protection from host immune response.
l-Serine is a non-essential amino acid that has wide and expanding applications in industry with a fast-growing market demand. Currently, extraction and enzymatic catalysis are the main processes for ...l-serine production. However, such approaches limit the industrial-scale applications of this important amino acid. Therefore, shifting to the direct fermentative production of l-serine from renewable feedstocks has attracted increasing attention. This review details the current status of microbial production of l-serine from renewable feedstocks. We also summarize the current trends in metabolic engineering strategies and techniques for the typical industrial organisms Corynebacterium glutamicum and Escherichia coli that have been developed to address and overcome major challenges in the l-serine production process.
Identifying exporters for l-serine can reduce l-serine feedback inhibition and therefore enhance l-serine production.
High-throughput screening using biosensors can be developed for l-serine and used for strain engineering.
The split between l-serine consumption for cell growth and extracellular l-serine accumulation can be balanced by tunable control of metabolic flows combined with 13C metabolic flux analysis.
Systems biology methods, including transcriptomics, proteomics, and metabolomics analysis, can be used for identifying targets for optimizing l-serine production.
Genome-scale metabolic models can prove to be useful for systematically identifying genetic targets for improving l-serine production.
To reduce the cost for commercializing l-serine production, it is essential to explore alternative and cheaper carbon sources.
Microbial fatty acid-derived fuels have emerged as promising alternatives to petroleum-based transportation fuels. Here we report a modular engineering approach that systematically removed metabolic ...pathway bottlenecks and led to significant titre improvements in a multi-gene fatty acid metabolic pathway. On the basis of central pathway architecture, E. coli fatty acid biosynthesis was re-cast into three modules: the upstream acetyl coenzyme A formation module; the intermediary acetyl-CoA activation module; and the downstream fatty acid synthase module. Combinatorial optimization of transcriptional levels of these three modules led to the identification of conditions that balance the supply of acetyl-CoA and consumption of malonyl-CoA/ACP. Refining protein translation efficiency by customizing ribosome binding sites for both the upstream acetyl coenzyme A formation and fatty acid synthase modules enabled further production improvement. Fed-batch cultivation of the engineered strain resulted in a final fatty acid production of 8.6 g l(-1). The modular engineering strategies demonstrate a generalized approach to engineering cell factories for valuable metabolites production.
The identification of optimal genotypes that result in improved production of recombinant metabolites remains an engineering conundrum. In the present work, various strategies to reengineer central ...metabolism in Escherichia coli were explored for robust synthesis of flavanones, the common precursors of plant flavonoid secondary metabolites. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) pool through the coordinated overexpression of four acetyl-CoA carboxylase (ACC) subunits from Photorhabdus luminescens (PlACC) under a constitutive promoter resulted in an increase in flavanone production up to 576%. Exploration of macromolecule complexes to optimize metabolic efficiency demonstrated that auxiliary expression of PlACC with biotin ligase from the same species (BirAPl) further elevated flavanone synthesis up to 1,166%. However, the coexpression of PlACC with Escherichia coli BirA (BirAEc) caused a marked decrease in flavanone production. Activity improvement was reconstituted with the coexpression of PlACC with a chimeric BirA consisting of the N terminus of BirAEc and the C terminus of BirAPl. In another approach, high levels of flavanone synthesis were achieved through the amplification of acetate assimilation pathways combined with the overexpression of ACC. Overall, the metabolic engineering of central metabolic pathways described in the present work increased the production of pinocembrin, naringenin, and eriodictyol in 36 h up to 1,379%, 183%, and 373%, respectively, over production with the strains expressing only the flavonoid pathway, which corresponded to 429 mg/liter, 119 mg/liter, and 52 mg/liter, respectively.
Chondroitin sulfates are linear sulfated polysaccharides called glycosaminoglycans. They are important nutraceutical and pharmaceutical products that are biosynthesized through the action of ...chondroitin sulfotransferases on either an unsulfated chondroitin or a dermatan polysaccharide precursor. While the enzymes involved in the biosynthesis of chondroitin sulfates are well known, the cloning end expression of these membrane-bound Golgi enzymes continue to pose challenges. The major chondroitin-4-sulfotransferase,
Homo sapiens
C4ST-1, had been previously cloned and expressed from mammalian CHO, COS-7, and HEK 293 cells, and its activity was shown to require glycosylation. In the current study, a C4ST-1 construct was designed and expressed in both
Escherichia coli
and
Pichia pastoris
in its non-glycosylated and glycosylated forms. Both constructs showed similar activity albeit different kinetic parameters when acting on a microbially prepared unsulfated chondroitin substrate. Moreover, the glycosylated form of C4ST-1 showed lower stability than the non-glycosylated form.
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