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Abstract Industrial biotechnology employs the controlled use of microorganisms for the production of synthetic chemicals or simple biomass that can further be used in a diverse array of applications that span the pharmaceutical, chemical and nutraceutical industries. Recent advances in metagenomics and in the incorporation of entire biosynthetic pathways into Saccharomyces cerevisiae have greatly expanded both the fitness and the repertoire of biochemicals that can be synthesized from this popular microorganism. Further, the availability of the S. cerevisiae entire genome sequence allows the application of systems biology approaches for improving its enormous biosynthetic potential. In this review, we will describe some of the efforts on using S. cerevisiae as a cell factory for the biosynthesis of high-value natural products that belong to the families of isoprenoids, flavonoids and long chain polyunsaturated fatty acids. As natural products are increasingly becoming the center of attention of the pharmaceutical and nutraceutical industries, the use of S. cerevisiae for their production is only expected to expand in the future, further allowing the biosynthesis of novel molecular structures with unique properties.

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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, 13 C-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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (© 2019 The Authors.)

During microbial applications, metabolic burdens can lead to a significant drop in cell performance. Novel synthetic biology tools or multi-step bioprocessing (e.g., fermentation followed by chemical conversions) are therefore needed to avoid compromised biochemical productivity from over-burdened cells. A possible solution to address metabolic burden is Division of Labor (DoL) via natural and synthetic microbial consortia. In particular, consolidated bioprocesses and metabolic cooperation for detoxification or cross feeding (e.g., vitamin C fermentation) have shown numerous successes in industrial level applications. However, distributing a metabolic pathway among proper hosts remains an engineering conundrum due to several challenges: complex subpopulation dynamics/interactions with a short time-window for stable production, suboptimal cultivation of microbial communities, proliferation of cheaters or low-producers, intermediate metabolite dilution, transport barriers between species, and breaks in metabolite channeling through biosynthesis pathways. To develop stable consortia, optimization of strain inoculations, nutritional divergence and crossing feeding, evolution of mutualistic growth, cell immobilization, and biosensors may potentially be used to control cell populations. Another opportunity is direct integration of non-bioprocesses (e.g., microbial electrosynthesis) to power cell metabolism and improve carbon efficiency. Additionally, metabolic modeling and 13 C-metabolic flux analysis of mixed culture metabolism and cross-feeding offers a computational approach to complement experimental research for improved consortia performance.

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Heparin, an anticoagulant drug, is biosynthesized in selected animal cells. The heparin biosynthetic enzymes mainly consist of sulfotransferases and all are integral transmembrane glycoproteins. These enzymes are generally produced in engineered Escherichia coli as without their transmembrane domains as non-glycosylated fusion proteins. In this study, we used the yeast, Komagataella pastoris, to prepare four sulfotransferases involved in heparin biosynthesis as glycoproteins. While the yields of these yeast-expressed enzymes were considerably lower than E. coli-expressed enzymes, these enzymes were secreted into the fermentation media simplifying their purification and were endotoxin free. The activities of these sulfotransferases, expressed as glycoproteins in yeast, were compared to the bacterially expressed proteins. The yeast-expressed sulfotransferase glycoproteins showed improved kinetic properties than the bacterially expressed proteins.

Background: Anthocyanins are a class of brightly colored, glycosylated flavonoid pigments that imbue their flower and fruit host tissues with hues of predominantly red, orange, purple, and blue. Although all anthocyanins exhibit pH-responsive photochemical changes, distinct structural decorations on the core anthocyanin skeleton also cause dramatic color shifts, in addition to improved stabilities and unique pharmacological properties. In this work, we report for the first time the extension of the reconstituted plant anthocyanin pathway from (+)-catechin to O-methylated anthocyanins in a microbial production system, an effort which requires simultaneous co-option of the endogenous metabolites UDP-glucose and S-adenosyl-L-methionine (SAM or AdoMet)., Results: Anthocyanin O-methyltransferase (AOMT) orthologs from various plant sources were co-expressed in Escherichia coli with Petunia hybrida anthocyanidin synthase (PhANS) and Arabidopsis thaliana anthocyanidin 3-O-glucosyltransferase (At3GT). Vitis vinifera AOMT (VvAOMT1) and fragrant cyclamen 'Kaori-no-mai' AOMT (CkmOMT2) were found to be the most effective AOMTs for production of the 3'-O-methylated product peonidin 3-O-glucoside (P3G), attaining the highest titers at 2.4 and 2.7 mg/L, respectively. Following modulation of plasmid copy number and optimization of VvAOMT1 and CkmOMT2 expression conditions, production was further improved to 23 mg/L using VvAOMT1. Finally, CRISPRi was utilized to silence the transcriptional repressor MetJ in order to deregulate the methionine biosynthetic pathway and improve SAM availability for O-methylation of cyanidin 3-O-glucoside (C3G), the biosynthetic precursor to P3G. MetJ repression led to a final titer of 51 mg/L (56 mg/L upon scale-up to shake flask), representing a twofold improvement over the non-targeting CRISPRi control strain and 21-fold improvement overall., Conclusions: An E. coli strain was engineered for production of the specialty anthocyanin P3G using the abundant and comparatively inexpensive flavonol precursor, (+)-catechin. Furthermore, dCas9-mediated transcriptional repression of metJ alleviated a limiting SAM pool size, enhancing titers of the methylated anthocyanin product. While microbial production of P3G and other O-methylated anthocyanin pigments will likely be valuable to the food industry as natural food and beverage colorants, we expect that the strain constructed here will also prove useful to the ornamental plant industry as a platform for evaluating putative anthocyanin O-methyltransferases in pursuit of bespoke flower pigment compositions.

Cutinase thermostability is important so that the enzymes can function above the glass transition of what are often rigid polymer substrates. A detailed thermal inactivation analysis was performed for two well-characterized cutinases, Aspergillus oryzae Cutinase (AoC) and Thiellavia terrestris Cutinase (TtC). Both AoC and TtC are prone to thermal aggregation upon unfolding at high temperature, which was found to be a major reason for irreversible loss of enzyme activity. Our study demonstrates that glycosylation stabilizes TtC expressed in Pichia pastoris by inhibiting its thermal aggregation. Based on the comparative thermal inactivation analyses of non-glycosylated AoC, glycosylated (TtC-G), and non-glycosylated TtC (TtC-NG), a unified model for thermal inactivation is proposed that accounts for thermal aggregation and may be applicable to other cutinase homologues. Inspired by glycosylated TtC, we successfully employed glycosylation site engineering to inhibit AoC thermal aggregation. Indeed, the inhibition of thermal aggregation by AoC glycosylation was greater than that achieved by conventional use of trehalose under a typical condition. Collectively, this study demonstrates the excellent potential of implementing glycosylation site engineering for thermal aggregation inhibition, which is one of the most common reasons for the irreversible thermal inactivation of cutinases and many proteins. Biotechnol. Bioeng. 2017;114: 63-73. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Methanol is an attractive substrate for biological production of chemicals and fuels. Engineering methylotrophic Escherichia coli as a platform organism for converting methanol to metabolites is desirable. Prior efforts to engineer methylotrophic E. coli were limited by methanol dehydrogenases (Mdhs) with unfavorable enzyme kinetics. We engineered E. coli to utilize methanol using a superior NAD-dependent Mdh from Bacillus stearothermophilus and ribulose monophosphate (RuMP) pathway enzymes from B. methanolicus. Using 13 C-labeling, we demonstrate this E. coli strain converts methanol into biomass components. For example, the key TCA cycle intermediates, succinate and malate, exhibit labeling up to 39%, while the lower glycolytic intermediate, 3-phosphoglycerate, up to 53%. Multiple carbons are labeled for each compound, demonstrating a cycling RuMP pathway for methanol assimilation to support growth. By incorporating the pathway to synthesize the flavanone naringenin, we demonstrate the first example of in vivo conversion of methanol into a specialty chemical in E. coli., (Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

We report here the 4.092-Mb high-quality draft genome assembly of a newly isolated poly-γ-glutamic acid-producing strain, Bacillus subtilis Ia1a. The genome sequence is considered a critical tool to facilitate the engineering of improved production strains. Exopolysaccharides and many industrially important enzymes can be produced by this new strain utilizing different carbon sources., (Copyright © 2016 Ibrahim et al.)

Robust gene circuit construction requires use of promoters exhibiting low crosstalk. Orthogonal promoters have been engineered utilizing an assortment of natural and synthetic transcription factors, but design of large orthogonal promoter-repressor sets is complicated, labor-intensive, and often results in unanticipated crosstalk. The specificity and ease of targeting the RNA-guided DNA-binding protein dCas9 to any 20 bp user-defined DNA sequence makes it a promising candidate for orthogonal promoter regulation. Here, we rapidly construct orthogonal variants of the classic T7-lac promoter using site-directed mutagenesis, generating a panel of inducible hybrid promoters regulated by both LacI and dCas9. Remarkably, orthogonality is mediated by only two to three nucleotide mismatches in a narrow window of the RNA:DNA hybrid, neighboring the protospacer adjacent motif. We demonstrate that, contrary to many reports, one PAM-proximal mismatch is insufficient to abolish dCas9-mediated repression, and we show for the first time that mismatch tolerance is a function of target copy number. Finally, these promoters were incorporated into the branched violacein biosynthetic pathway as dCas9-dependent switches capable of throttling and selectively redirecting carbon flux in Escherichia coli We anticipate this strategy is relevant for any promoter and will be adopted for many applications at the interface of synthetic biology and metabolic engineering., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Metabolic engineering strives to develop microbial strains that are capable of high-titer production of a variety of industrially significant pharmaceuticals, nutraceuticals, commodity, and high-value compounds. Despite extensive success with many proof-of-concept systems there is still the need for optimization to achieve industrially relevant titers, yields, and productivities. The field of metabolic pathway optimization and balancing has formed to address this need using a scientific and systematic approach. In this chapter, we aim to outline various pathway optimization and system balancing strategies while giving insights and tips into the systems and procedures that have demonstrated recent success in the peer-reviewed literature., (© 2016 Elsevier Inc. All rights reserved.)

Flavonoids are a growing class of bioactive natural products with distinct and interesting bioactivity both in vitro and in vivo. The extraction of flavonoids from plant sources is limited by their low natural abundance and commonly results in a mixture of products that are difficult to separate. However, due to recent advances, the microbial production of plant natural products has developed as a promising alternative for flavonoid production. Through optimization of media, induction temperature, induction point, and substrate delay time, we demonstrate the highest conversion of naringenin to eriodictyol (62.7 ± 2.7 mg/L) to date, using the native E. coli hydroxylase complex, HpaBC. We also show the first evidence of in vivo HpaBC activity towards the monohydroxylated flavan-3-ol afzelechin with catechin product titers of 34.7 ± 1.5 mg/L. This work confirms the wide applicability of HpaBC towards realizing efficient de novo production of various orthohydroxylated flavonoids and flavonoid derived products in E. coli., (© 2015 American Institute of Chemical Engineers.)

Plant polyphenols are known to have varying antimicrobial potencies, including direct antibacterial activity, synergism with antibiotics and suppression of bacterial virulence. We performed the in vitro oligomerization of resveratrol catalyzed by soybean peroxidase, and the two isomers (resveratrol-trans-dihydrodimer and pallidol) produced were tested for antimicrobial activity. The resveratrol-trans-dihydrodimer displayed antimicrobial activity against the Gram-positive bacteria Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus (minimum inhibitory concentration (MIC) = 15.0, 125, and 62.0 μM, respectively) and against Gram-negative Escherichia coli (MIC = 123 μM, upon addition of the efflux pump inhibitor Phe-Arg-β-naphthylamide). In contrast, pallidol had no observable antimicrobial activity against all tested strains. Transcriptomic analysis implied downregulation of ABC transporters, genes involved in cell division and DNA binding proteins. Flow cytometric analysis of treated cells revealed a rapid collapse in membrane potential and a substantial decrease in total DNA content. The active dimer showed >90% inhibition of DNA gyrase activity, in vitro, by blocking the ATP binding site of the enzyme. We thus propose that the resveratrol-trans-dihydrodimer acts to: (1) disrupt membrane potential; and (2) inhibit DNA synthesis. In summary, we introduce the mechanisms of action and the initial evaluation of an active bactericide, and a platform for the development of polyphenolic antimicrobials., (© 2015 Wiley Periodicals, Inc.)

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Programmable control over an addressable global regulator would enable simultaneous repression of multiple genes and would have tremendous impact on the field of synthetic biology. It has recently been established that CRISPR/Cas systems can be engineered to repress gene transcription at nearly any desired location in a sequence-specific manner, but there remain only a handful of applications described to date. In this work, we report development of a vector possessing a CRISPathBrick feature, enabling rapid modular assembly of natural type II-A CRISPR arrays capable of simultaneously repressing multiple target genes in Escherichia coli. Iterative incorporation of spacers into this CRISPathBrick feature facilitates the combinatorial construction of arrays, from a small number of DNA parts, which can be utilized to generate a suite of complex phenotypes corresponding to an encoded genetic program. We show that CRISPathBrick can be used to tune expression of plasmid-based genes and repress chromosomal targets in probiotic, virulent, and commonly engineered E. coli strains. Furthermore, we describe development of pCRISPReporter, a fluorescent reporter plasmid utilized to quantify dCas9-mediated repression from endogenous promoters. Finally, we demonstrate that dCas9-mediated repression can be harnessed to assess the effect of downregulating both novel and computationally predicted metabolic engineering targets, improving the yield of a heterologous phytochemical through repression of endogenous genes. These tools provide a platform for rapid evaluation of multiplex metabolic engineering interventions.

Anthocyanins are water-soluble colored pigments found in terrestrial plants and are responsible for the red, blue, and purple coloration of many flowers and fruits. In addition to the plethora of health benefits associated with anthocyanins (cardioprotective, anti-inflammatory, antioxidant, and antiaging properties), these compounds have attracted widespread attention due to their promising potential as natural food colorants. Previously, we reported the biotransformation of anthocyanin, specifically cyanidin 3-O-glucoside (C3G), from the substrate (+)-catechin in Escherichia coli. In the present work, we set out to systematically improve C3G titers by enhancing substrate and precursor availability, balancing gene expression level, and optimizing cultivation and induction parameters. We first identified E. coli transporter proteins that are responsible for the uptake of catechin and secretion of C3G. We then improved the expression of the heterologous pathway enzymes anthocyanidin synthase (ANS) and 3-O-glycosyltransferase (3GT) using a bicistronic expression cassette. Next, we augmented the intracellular availability of the critical precursor UDP-glucose, which has been known as the rate-limiting precursor to produce glucoside compounds. Further optimization of culture and induction conditions led to a final titer of 350 mg/liter of C3G. We also developed a convenient colorimetric assay for easy screening of C3G overproducers. The work reported here constitutes a promising foundation to develop a cost-effective process for large-scale production of plant-derived anthocyanin from recombinant microorganisms., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The ability to fine tune gene expression has created the field of metabolic pathway optimization and balancing where a variety of factors affecting flux balance are carefully modulated to improve product titers, yields, and productivity. Using a library of isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible mutant T7 promoters of varied strength a combinatorial method was developed for transcriptional balancing of the violacein pathway. Violacein biosynthesis involves a complex five-gene pathway that is an excellent model for exploratory metabolic engineering efforts into pathway regulation and control due to many colorful intermediates and side products allowing for easy analysis and strain comparison. Upon screening approximately 4% of the total initial library, several high-titer mutants were discovered that resulted in up to a 63-fold improvement over the control strain. With further fermentation optimization, titers were improved to 1829 ± 46 mg/L; a 2.6-fold improvement in titer and a 30-fold improvement in productivity from previous literature reports.

In the last decade, metabolic engineering benefited greatly from systems and synthetic biology due to substantial advancements in those fields. As a result, technologies and methods evolved to be more complex and controllable than ever. In this review, we highlight up-to-date case studies using these techniques, examine their potential, and stress their importance for production of compounds such as fatty acids, alcohols, and high value chemicals. Beginning with basic rational control techniques and continuing with advanced level modern approaches, we review the vast number of possibilities for controlling metabolic fluxes. Our aim is to give a brief and informative insight about commonly used tools and universalized methodologies for metabolic pathway balancing and optimization., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A novel C17 resorcylic acid was synthesized by a structure-guided Vitis vinifera stilbene synthase (STS) mutant, in which threonine 197 was replaced with glycine (T197G). Altering the architecture of the coumaroyl binding and cyclization pocket of the enzyme led to the attachment of an extra acetyl unit, derived from malonyl-CoA, to p-coumaroyl-CoA. The resulting novel pentaketide can be produced strictly by STS-like enzymes and not by Chalcone synthase-like type III polyketide synthases; due to the unique thioesterase like activity of STS-like enzymes. We utilized a liquid chromatography mass spectrometry-based data analysis approach to directly compare the reaction products of the mutant and wild type STS. The findings suggest an easy to employ platform for precursor-directed biosynthesis and identification of unnatural polyketides by structure-guided mutation of STS-like enzymes., (© 2014 The Protein Society.)

As a result of the discovery that flavonoids are directly or indirectly connected to health, flavonoid metabolism and its fascinating molecules that are natural products in plants, have attracted the attention of both the industry and researchers involved in plant science, nutrition, bio/chemistry, chemical bioengineering, pharmacy, medicine, etc. Subsequently, in the past few years, flavonoids became a top story in the pharmaceutical industry, which is continually seeking novel ways to produce safe and efficient drugs. Microbial cell cultures can act as workhorse bio-factories by offering their metabolic machinery for the purpose of optimizing the conditions and increasing the productivity of a selective flavonoid. Furthermore, metabolic engineering methodology is used to reinforce what nature does best by correcting the inadequacies and dead-ends of a metabolic pathway. Combinatorial biosynthesis techniques led to the discovery of novel ways of producing natural and even unnatural plant flavonoids, while, in addition, metabolic engineering provided the industry with the opportunity to invest in synthetic biology in order to overcome the currently existing restricted diversification and productivity issues in synthetic chemistry protocols. In this review, is presented an update on the rationalized approaches to the production of natural or unnatural flavonoids through biotechnology, analyzing the significance of combinatorial biosynthesis of agricultural/pharmaceutical compounds produced in heterologous organisms. Also mentioned are strategies and achievements that have so far thrived in the area of synthetic biology, with an emphasis on metabolic engineering targeting the cellular optimization of microorganisms and plants that produce flavonoids, while stressing the advances in flux dynamic control and optimization. Finally, the involvement of the rapidly increasing numbers of assembled genomes that contribute to the gene- or pathway-mining in order to identify the gene(s) responsible for producing species-specific secondary metabolites is also considered herein.

Steam explosion is the most promising technology to replace conventional acid hydrolysis of lignocellulose for biomass pretreatment. In this paper, a new screw-steam-explosive extruder was designed and explored for xylose production and lignocellulose biorefinery at the pilot scale. We investigated the effect of different chemicals on xylose yield in the screw-steam-explosive extrusion process, and the xylose production process was optimized as followings: After pre-impregnation with sulfuric acid at 80 °C for 3 h, corncob was treated at 1.55 MPa with 9 mg sulfuric acid/g dry corncob (DC) for 5.5 min, followed by countercurrent extraction (3 recycles), decoloration (activated carbon dosage 0.07 g/g sugar, 75 °C for 40 min), and ion exchange (2 batches). Using this process, 3.575 kg of crystal xylose was produced from 22 kg corncob, almost 90 % of hemicellulose was released as monomeric sugar, and only a small amount of by-products was released (formic acid, acetic acid, fural, 5-hydroxymethylfurfural, and phenolic compounds were 0.17, 1.14, 0.53, 0.19, and 1.75 g/100 g DC, respectively). All results indicated that the screw-steam-explosive extrusion provides a more effective way to convert hemicellulose into xylose and could be an alternative method to traditional sulfuric acid hydrolysis process for lignocellulose biorefinery.

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., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Malonyl-CoA is the rate-limiting precursor involved in the chain elongation reaction of a range of value-added pharmaceuticals and biofuels. Development of malonyl-CoA responsive sensors holds great promise in overcoming critical pathway limitations and optimizing production titers and yields. By incorporating the Bacillus subtilis trans-regulatory protein FapR and the cis-regulatory element fapO, we constructed a hybrid promoter-regulatory system that responds to a broad range of intracellular malonyl-CoA concentrations (from 0.1 to 1.1 nmol/mgDW) in Escherichia coli. Elimination of regulatory protein and nonspecific DNA cross-communication leads to a sensor construct that exhibits malonyl-CoA-dependent linear phase kinetics with increased dynamic response range. The sensors reported in this study could potentially control and optimize carbon flux leading to robust biosynthetic pathways for the production of malonyl-CoA-derived compounds.

Natural products have been associated with significant health benefits in preventing and treating various chronic human diseases such as cancer, cardiovascular diseases, diabetes, Alzheimer's disease, and pathogenic infections. However, the isolation, characterization and evaluation of natural products remain a challenge, mainly due to their limited bioavailability. Metabolic engineering and fermentation technology have emerged as alternative approaches for generating natural products under controlled conditions that can be optimized to maximize yields. Optimization of these processes includes the evaluation of factors such as host selection, product biosynthesis interaction with the cell's central metabolism, product degradation, and byproduct formation. This review summarizes the most recent biochemical strategies and advances in expanding and diversifying natural compounds as well as maximizing their production in microbial and plants cells., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Nature has provided us the basis of designing the most integrated and efficacious production platforms. Cell factories via their millions of years of evolution have nearly perfected each of their production systems. We have been trying to imitate, utilize and tweak this system to our advantage by using slightly overlapping and greatly interdependent approaches such as metabolic engineering and systems biology to make nature work for us in an efficient and robust way, without producing toxic waste and/or unnecessary side products. Systems biology, metabolic engineering and 'omics' technologies have paved the way for protein and pathway engineering. To this end we will talk about the recent advances in production of novel pharmaceutical and commodity small molecules by designing novel proteins and pathways., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Over the years, natural products from plants and their non-natural derivatives have shown to be active against different types of chronic diseases. However, isolation of such natural products can be limited due to their low bioavailability, and environmental restrictions. To address these issues, in vivo and in vitro reconstruction of plant metabolic pathways and the metabolic engineering of microbes and plants have been used to generate libraries of compounds. Significant advances have been made through metabolic engineering of microbes and plant cells to generate a variety of compounds (e.g. isoprenoids, flavonoids, or stilbenes) using a diverse array of methods to optimize these processes (e.g. host selection, operational variables, precursor selection, gene modifications). These approaches have been used also to generate non-natural analogues with different bioactivities. In vitro biosynthesis allows the synthesis of intermediates as well as final products avoiding post-translational limitations. Moreover, this strategy allows the use of substrates and the production of metabolites that could be toxic for cells, or expand the biosynthesis into non-conventional media (e.g. organic solvents, supercritical fluids). A perspective is also provided on the challenges for generating novel chemical structures and the potential of combining metabolic engineering and in vitro biocatalysis to produce metabolites with more potent biological activities., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Plant metabolism represents an enormous repository of compounds that are of pharmaceutical and biotechnological importance. Engineering plant metabolism into microbes will provide sustainable solutions to produce pharmaceutical and fuel molecules that could one day replace substantial portions of the current fossil-fuel based economy. Metabolic engineering entails targeted manipulation of biosynthetic pathways to maximize yields of desired products. Recent advances in Systems Biology and the emergence of Synthetic Biology have accelerated our ability to design, construct and optimize cell factories for metabolic engineering applications. Progress in predicting and modeling genome-scale metabolic networks, versatile gene assembly platforms and delicate synthetic pathway optimization strategies has provided us exciting opportunities to exploit the full potential of cell metabolism. In this review, we will discuss how systems and synthetic biology tools can be integrated to create tailor-made cell factories for efficient production of natural products and fuel molecules in microorganisms., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We announce the availability of the 5.023-Mbp high-quality draft assembly of the Escherichia coli strain Nissle 1917 (serovar O6:K5:H1) genome. Short genomic segments from this important probiotic strain have been available in public databases, but the full genome sequence has remained inaccessible. Thus, high-coverage, whole genome sequencing of E. coli Nissle 1917 is presented herein. Reannotation and metabolic reconstruction will enable comparative genomics analysis and model-guided predictions of genetic manipulations leading to increased production of the K5 capsular polysaccharide known as N-acetyl heparosan, a precursor to the anticoagulant pharmaceutical heparin.

We report the 5.101-Mbp high-quality draft assembly of the Escherichia coli strain ATCC 23506 (serovar O10:K5:H4, also known as NCDC Bi 8337-41) genome. This uropathogenic strain, commonly referred to as E. coli K5, produces N-acetyl heparosan, a glycosaminoglycan-like capsular polysaccharide and precursor to the anticoagulant pharmaceutical heparin. Metabolic reconstruction of this genome will enable the prediction of gene deletions and overexpressions that lead to increased heparosan production.

We report the 4.049-Mbp high-quality draft assembly of the Pseudoalteromonas luteoviolacea strain B (ATCC 29581) genome. This marine species is known to biosynthesize several antimicrobial compounds, including the purple pigment violacein. Whole-genome sequencing and genome mining will complement experimental studies aimed at elucidating novel biosynthetic pathways capable of producing pharmaceutically relevant molecules. Based upon 16S rRNA phylogenetic analysis, we propose that strain ATCC 29581 be classified as a distinct phylogenetic species of the genus Pseudoalteromonas.

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.

As an emerging discipline, synthetic biology is becoming increasingly important to design, construct, and optimize metabolic pathways leading to desired phenotypes such as overproduction of biofuels and pharmaceuticals in genetically tractable organisms. We have recently developed a versatile gene assembly platform ePathBricks supporting the modular assembly of multi-gene pathway components and combinatorial generation of pathway diversities. In this protocol, we will detail the process to assemble a seven gene flavonoid pathway (~9 kb) on one single ePathBrick vector. We will also demonstrate that a three-gene flavonoid pathway can be easily diversified to 54 pathway equivalents differing in pathway configuration and gene order; coupled with high-throughput screening techniques, we envision that this combinatorial strategy would greatly improve our ability to exploit the full potential of microbial cell factories for recombinant metabolite production.

Harnessing cell factories for producing biofuel and pharmaceutical molecules has stimulated efforts to develop novel synthetic biology tools customized for modular pathway engineering and optimization. Here we report the development of a set of vectors compatible with BioBrick standards and its application in metabolic engineering. The engineered ePathBrick vectors comprise four compatible restriction enzyme sites allocated on strategic positions so that different regulatory control signals can be reused and manipulation of expression cassette can be streamlined. Specifically, these vectors allow for fine-tuning gene expression by integrating multiple transcriptional activation or repression signals into the operator region. At the same time, ePathBrick vectors support the modular assembly of pathway components and combinatorial generation of pathway diversities with three distinct configurations. We also demonstrated the functionality of a seven-gene pathway (~9 Kb) assembled on one single ePathBrick vector. The ePathBrick vectors presented here provide a versatile platform for rapid design and optimization of metabolic pathways in E. coli.

7-O-Methyl aromadendrin (7-OMA) is an aglycone moiety of one of the important flavonoid-glycosides found in several plants, such as Populus alba and Eucalyptus maculata, with various medicinal applications. To produce such valuable natural flavonoids in large quantity, an Escherichia coli cell factory has been developed to employ various plant biosynthetic pathways. Here, we report the generation of 7-OMA from its precursor, p-coumaric acid, in E. coli for the first time. Primarily, naringenin (NRN) (flavanone) synthesis was achieved by feeding p-coumaric acid and reconstructing the plant biosynthetic pathway by introducing the following structural genes: 4-coumarate-coenzyme A (CoA) ligase from Petroselinum crispum, chalcone synthase from Petunia hybrida, and chalcone isomerase from Medicago sativa. In order to increase the availability of malonyl-CoA, a critical precursor of 7-OMA, genes for the acyl-CoA carboxylase α and β subunits (nfa9890 and nfa9940), biotin ligase (nfa9950), and acetyl-CoA synthetase (nfa3550) from Nocardia farcinica were also introduced. Thus, produced NRN was hydroxylated at position 3 by flavanone-3-hydroxylase from Arabidopsis thaliana, which was further methylated at position 7 to produce 7-OMA in the presence of 7-O-methyltransferase from Streptomyces avermitilis. Dihydrokaempferol (DHK) (aromadendrin) and sakuranetin (SKN) were produced as intermediate products. Overexpression of the genes for flavanone biosynthesis and modification pathways, along with malonyl-CoA overproduction in E. coli, produced 2.7 mg/liter (8.9 μM) 7-OMA upon supplementation with 500 μM p-coumaric acid in 24 h, whereas the strain expressing only the flavanone modification enzymes yielded 30 mg/liter (99.2 μM) 7-OMA from 500 μM NRN in 24 h.

Malonyl-coenzyme A is an important precursor metabolite for the biosynthesis of polyketides, flavonoids and biofuels. However, malonyl-CoA naturally synthesized in microorganisms is consumed for the production of fatty acids and phospholipids leaving only a small amount available for the production of other metabolic targets in recombinant biosynthesis. Here we present an integrated computational and experimental approach aimed at improving the intracellular availability of malonyl-CoA in Escherichia coli. We used a customized version of the recently developed OptForce methodology to predict a minimal set of genetic interventions that guarantee a prespecified yield of malonyl-CoA in E. coli strain BL21 Star™. In order to validate the model predictions, we have successfully constructed an E. coli recombinant strain that exhibits a 4-fold increase in the levels of intracellular malonyl-CoA compared to the wild type strain. Furthermore, we demonstrate the potential of this E. coli strain for the production of plant-specific secondary metabolites naringenin (474mg/L) with the highest yield ever achieved in a lab-scale fermentation process. Combined effect of the genetic interventions was found to be synergistic based on a developed analysis method that correlates genetic modification to cell phenotype, specifically the identified knockout targets (ΔfumC and ΔsucC) and overexpression targets (ACC, PGK, GAPD and PDH) can cooperatively force carbon flux towards malonyl-CoA. The presented strategy can also be readily expanded for the production of other malonyl-CoA-derived compounds like polyketides and biofuels., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Plant polyphenols have been the subject of several recent scientific investigations since many of the molecules in this class have been found to be highly active in the human body, with a plethora of health-promoting activities against a variety of diseases, including heart disease, diabetes, and cancer, and with even the potential to slow aging. Further development of these potent natural therapeutics hinges on the formation of robust industrial production platforms designed using specifically selected as well as engineered protein sources along with the construction of optimal expression platforms. In this work, we first report the investigation of various stilbene synthases from an array of plant species considering structure-activity relationships, their expression efficiency in microorganisms, and their ability to synthesize resveratrol. Second, we looked into the construct environment of recombinantly expressed stilbene synthases, including different promoters, construct designs, and host strains, to create an Escherichia coli strain capable of producing superior resveratrol titers sufficient for commercial usage. Further improvement of metabolic capabilities of the recombinant strain aimed at improving the intracellular malonyl-coenzyme A pool, a resveratrol precursor, resulted in a final improved titer of 2.3 g/liter resveratrol.

Laccase activity in plants results in the formation of a number of brown pigments, often referred to as tannins. Laccase-dependent pigment production is also catalogued in numerous fungal and bacterial species. The laccase of the haploid yeast Cryptococcus neoformans forms melanin-like pigmentation outside the cell wall in the presence of exogenous substrates. While this process is a contributing factor to its virulence in humans, the evolutionary intent for the laccase function remains a mystery. We show here that C. neoformans and Bacillus subtilis have the ability to create melanin-like pigments from a variety of flavonoid molecules across a range of conformations, preferring those with 3',4'-dihydroxylations. Since flavonoids are ubiquitous plant molecules and often-considered antimicrobial agents, we postulate that they are the intended natural targets of laccase activity and result in the formation of a defensive melanin-like coat. These results suggests a new mechanism by which flavonoid-melanin formation may occur, using not only A- and C-ring linkages, but also monomer links through the B-ring of the flavonoid structure. We also show that resveratrol and other non- and mono-hydroxylated polyphenol substrates have the ability to restrict pigment formation and may be potent inhibitors of laccase activity., (Copyright © 2010 John Wiley & Sons, Ltd.)

With growing concerns over multidrug resistance microorganisms, particularly strains of bacteria and fungi, evolving to become resistant to the antimicrobial agents used against them, the identification of new molecular targets becomes paramount for novel treatment options. Recently, the use of new treatments containing multiple active ingredients has been shown to increase the effectiveness of existing molecules for some infections, often with these added compounds enabling the transport of a toxic molecule into the infecting species. Flavonoids are among the most abundant plant secondary metabolites and have been shown to have natural abilities as microbial deterrents and anti-infection agents in plants. Combining these ideas we first sought to investigate the potency of natural flavonoids in the presence of efflux pump inhibitors to limit Escherichia coli growth. Then we used the natural flavonoid scaffold to synthesize non-natural flavanone molecules and further evaluate their antimicrobial efficacy on Escherichia coli, Bacillus subtilis and the fungal pathogens Cryptococcus neoformans and Aspergillus fumigatus. Of those screened, we identified the synthetic molecule 4-chloro-flavanone as the most potent antimicrobial compound with a MIC value of 70 µg/mL in E. coli when combined with the inhibitor Phe-Arg-ß-naphthylamide, and MICs of 30 µg/mL in S. cerevesiae and 30 µg/mL in C. neoformans when used alone. Through this study we have demonstrated that combinatorial synthesis of non-natural flavonones can identify novel antimicrobial agents with activity against bacteria and fungi but with minimal toxicity to human cells.

Isoflavonoids possess enormous potential for human health with potential impact on heart disease and cancer, and some display striking affinities for steroid receptors. Synthesized primarily by legumes, isoflavonoids are present in low and variable abundance within complex mixtures, complicating efforts to assess their clinical potential. To satisfy the need for controlled, efficient, and flexible biosynthesis of isoflavonoids, a three-enzyme system has been constructed in yeast that can convert natural and synthetic flavanones into their corresponding isoflavones in practical quantities. Based on the determination of the substrate requirements of isoflavone synthase, a series of natural and nonnatural isoflavones were prepared and their binding affinities for the human estrogen receptors (ER alpha and ER beta) were determined. Structure activity relationships are suggested based on changes to binding affinities related to small variations on the isoflavone structure., ((c) 2010 Elsevier Ltd. All rights reserved.)

With microbial production becoming the primary choice for natural product synthesis, increasing precursor and cofactor availability has become a chief hurdle for the generation of efficient production platforms. As such, we employed a stoichiometric-based model to identify combinations of gene knockouts for improving NADPH availability in Escherichia coli. Specifically, two different model objectives were used to identify possible genotypes that exhibited either improved overall NADPH production or an improved flux through an artificial reaction coupling NADPH yield to biomass. The top single, double and triple gene deletion candidates were constructed and as a case study evaluated for their ability to produce two polyphenols, leucocyanidin and (+)-catechin. Each is derived from their common precursor dihydroquercetin using two recombinant NADPH-dependent enzymes: dihydroflavonol 4-reductase and leucoanthocyanidin reductase. The best engineered strain carrying Delta pgi, Delta ppc and Delta pldA deletions accumulated up to 817 mg/L of leucocyanidin and 39 mg/L (+)-catechin in batch culture with 10 g/L glucose in modified M9 medium, a 4-fold and 2-fold increase, respectively, compared to the wild-type control., ((c) 2009 Elsevier Inc. All rights reserved.)