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Biological homeostasis is a dynamic and elastic equilibrium of countless interlinked biochemical reactions. A key goal of life sciences is to understand these dynamics; bioengineers seek to reconfigure such networks. Both goals require the ability to monitor the concentration of individual intracellular metabolites with sufficient spatiotemporal resolution. To achieve this, a range of protein or protein/DNA signalling circuits with optical readouts have been constructed. Protein biosensors can provide quantitative information at subsecond temporal and suborganelle spatial resolution. However, their construction is fraught with difficulties related to integrating the affinity- and selectivity-endowing components with the signal reporters. We argue that development of efficient approaches for construction of chemically induced dimerisation systems and reporter domains with large dynamic ranges will solve these problems. Biosensors enable quantification of analytes of choice inside living cells. Protein biosensors can transform the way we monitor and engineer metabolism. The key challenges are to develop biosensors with large dynamic range and tuneable specificity. Ultimately, we seek to democratise this technology so that it is readily available to the scientific community. [ABSTRACT FROM AUTHOR]

Strigolactones (SLs) control plant development, enhance symbioses, and act as germination stimulants for some of the most destructive species of parasitic weeds, making SLs a potential tool to improve crop productivity and resilience. Field trials demonstrate the potential use of SLs as agrochemicals or genetic targets in breeding programs, with applications in improving drought tolerance, increasing yields, and controlling parasitic weeds. However, for effective translation of SLs into agriculture, understanding and exploiting SL diversity and the development of economically viable sources of SL analogs will be critical. Here we review how manipulation of SL signaling can be used when developing new tools and crop varieties to address some critical challenges, such as nutrient acquisition, resource allocation, stress tolerance, and plant–parasite interactions. Proof of principle for strigolactone (SL) application in agriculture has now been demonstrated at field-trial scale for parasitic weed control, increasing fruit yield, enhancing nutrient uptake, and improving yield under drought conditions. The agricultural potential of many emerging and established activities of SLs remains under-investigated. Specificity of activity in the landscape of many complex SL responses can be achieved through exploitation of differences in SL structures and receptor specificities. [ABSTRACT FROM AUTHOR]

Isoprenoids (terpenes/terpenoids) have many useful industrial applications, but are often not produced at industrially viable level in their natural sources. Synthetic biology approaches have been used extensively to reconstruct metabolic pathways in tractable microbial hosts such as yeast and re-engineer pathways and networks to increase yields. Here we review recent advances in this field, focusing on central carbon metabolism engineering to increase precursor supply, re-directing carbon flux for production of C10, C15, or C20 isoprenoids, and chemical decoration of high value diterpenoids (C20). We also overview other novel synthetic biology strategies that have potential utility in yeast isoprenoid pathway engineering. Finally, we address the question of what is required in the future to move the field forwards. [ABSTRACT FROM AUTHOR]

The chemical properties and diversity of volatile isoprenoids lends them to a broad variety of biological roles. It also lends them to a host of biotechnological applications, both by taking advantage of their natural functions and by using them as industrial chemicals/chemical feedstocks. Natural functions include roles as insect attractants and repellents, abiotic stress protectants in pathogen defense, etc. Industrial applications include use as pharmaceuticals, flavours, fragrances, fuels, fuel additives, etc. Here we will examine the ways in which researchers have so far found to exploit volatile isoprenoids using biotechnology. Production and/or modification of volatiles using metabolic engineering in both plants and microorganisms are reviewed, including engineering through both mevalonate and methylerythritol diphosphate pathways. Recent advances are illustrated using several case studies (herbivores and bodyguards, isoprene, and monoterpene production in microbes). Systems and synthetic biology tools with particular utility for metabolic engineering are also reviewed. Finally, we discuss the practical realities of various applications in modern biotechnology, explore possible future applications, and examine the challenges of moving these technologies forward so that they can deliver tangible benefits. While this review focuses on volatile isoprenoids, many of the engineering approaches described here are also applicable to non-isoprenoid volatiles and to non-volatile isoprenoids. [ABSTRACT FROM AUTHOR]

Background Manipulations in Saccharomyces cerevisiae classically depend on use of auxotrophy selection markers. There are several disadvantages to this in a microbial cell factory setting: (1) auxotrophies must first be engineered in prototrophic strains, and many industrial strains are polyploid/aneuploid prototrophs (2) available strain auxotrophies must be paired with available repair plasmids (3) remaining auxotrophies must be repaired prior to development of industrial bioprocesses. Use of dominant antibiotic resistance markers can circumvent these problems. However, there are relatively few yeast antibiotic resistance marker vectors available; furthermore, available vectors contain only one expression cassette, and it is often desirable to introduce more than one gene at a time. Results To overcome these problems, eight new shuttle vectors have been developed. The plasmids are maintained in yeast under a 2 μm ori and in E. coli by a pUC ori. They contain two yeast expression cassettes driven by either (1) the constitutive TEF1 and PGK1 promoters, or (2) the constitutive TEF1 promoter and the inducible GAL10 or HXT7 promoters. Expression strength of these promoters over a typical production time frame in glucose/galactose medium was examined, and identified the TEF1 and HXT7 promoters as preferred promoters over long term fermentations. Selection is provided by either aphA1 (conferring resistance to G418 in yeast and kanamycin/neomycin in E. coli) or ble (conferring resistance to phleomycin in both yeast and E. coli). Selection conditions for these plasmids/antibiotics in defined media were examined, and selections considerations are reviewed. In particular, medium pH has a strong effect on both G418 and phleomycin selection. Conclusions These vectors allow manipulations in prototrophic yeast strains with expression of two gene cassettes per plasmid, and will be particularly useful for metabolic engineering applications. The vector set expands the (currently limited) selection of antibiotic marker plasmids available for use in yeast, and in addition makes available dual gene expression cassettes on individual plasmids using antibiotic selection. The resistance gene cassettes are flanked by loxP recognition sites to allow CreA-mediated marker removal and recycling, providing the potential for genomic integration of multiple genes. Guidelines for selection using G418 and phleomycin are provided. [ABSTRACT FROM AUTHOR]

Background: Monoterpenes are a class of natural C10 compounds with a range of potential applications including use as fuel additives, fragrances, and chemical feedstocks. Biosynthesis of monoterpenes in heterologous systems is yet to reach commercially-viable levels, and therefore is the subject of strain engineering and fermentation optimization studies. Detection of monoterpenes typically relies on gas chromatography/mass spectrometry; this represents a significant analytical bottleneck which limits the potential to analyse combinatorial sets of conditions. To address this, we developed a high-throughput method for pre-screening monoterpene biosynthesis. Results: An optimised DPPH assay was developed for detecting monoterpenes from two-phase microbial cultures using dodecane as the extraction solvent. The assay was useful for reproducible qualitative ranking of monoterpene concentrations, and detected standard preparations of myrcene and γ-terpinene dissolved in dodecane at concentrations as low as 10 and 15 μM, respectively, and limonene as low as 200 μM. The assay could not be used quantitatively due to technical difficulties in capturing the initial reaction rate in a multi-well plate and the presence of minor DPPH-reactive contaminants. Initially, limonene biosynthesis in Saccharomyces cerevisiae was tested using two different limonene synthase enzymes and three medium compositions. The assay indicated that limonene biosynthesis was enhanced in a supplemented YP medium and that the Citrus limon limonene synthase (CLLS) was more effective than the Mentha spicata limonene synthase (MSLS). GC-MS analysis revealed that the DPPH assay had correctly identified the best limonene synthase (CLLS) and culture medium (supplemented YP medium). Because only traces of limonene were detected in SD medium, we subsequently identified medium components that improved limonene production and developed a defined medium based on these findings. The best limonene titres obtained were 1.48 ± 0.22 mg limonene per L in supplemented YP medium and 0.9 ± 0.15 mg limonene per L in a pH-adjusted supplemented SD medium. Conclusions: The DPPH assay is useful for detecting biosynthesis of limonene. Although the assay cannot be used quantitatively, it proved successful in ranking limonene production conditions qualitatively and thus is suitable as a first-tier screen. The DPPH assay will likely be applicable in detecting biosynthesis of several other monoterpenes and for screening libraries of monoterpene-producing strains. [ABSTRACT FROM AUTHOR]

Isoprene is a highly reactive gas, and is emitted in such large quantities from the biosphere that it substantially affects the oxidizing potential of the atmosphere. Relatively little is known about the control of isoprene emission at the molecular level. Using transgenic tobacco lines harbouring a poplar isoprene synthase gene, we examined control of isoprene emission. Isoprene synthase required chloroplastic localization for catalytic activity, and isoprene was produced via the methyl erythritol (MEP) pathway from recently assimilated carbon. Emission patterns in transgenic tobacco plants were remarkably similar to naturally emitting plants under a wide variety of conditions. Emissions correlated with photosynthetic rates in developing and mature leaves, and with the amount of isoprene synthase protein in mature leaves. Isoprene synthase protein levels did not change under short-term increase in heat/light, despite an increase in emissions under these conditions. A robust circadian pattern could be observed in emissions from long-day plants. The data support the idea that substrate supply and changes in enzyme kinetics (rather than changes in isoprene synthase levels or post-translational regulation of activity) are the primary controls on isoprene emission in mature transgenic tobacco leaves. [ABSTRACT FROM AUTHOR]

The sessile nature of plants has resulted in the evolution of an extraordinarily diverse suite of protective mechanisms against biotic and abiotic stresses. Though volatile isoprenoids are known to be involved in many types of biotic interactions, they also play important but relatively unappreciated roles in abiotic stress responses. We review those roles, discuss the proposed mechanistic explanations and examine the evolutionary significance of volatile isoprenoid emission. We note that abiotic stress responses generically involve production of reactive oxygen species in plant cells, and volatile isoprenoids mitigate the effects of oxidative stress by mediating the oxidative status of the plant. On the basis of these observations, we propose a 'single biochemical mechanism for multiple physiological stressors' model, whereby the protective effect against abiotic stress is exerted through direct or indirect improvement in resistance to damage by reactive oxygen species. [ABSTRACT FROM AUTHOR]

Isoprene emission represents a significant loss of carbon to those plant species that synthesize this highly volatile and reactive compound. As a tool for studying the role of isoprene in plant physiology and biochemistry, we developed transgenic tobacco plants capable of emitting isoprene in a similar manner to and at rates comparable to a naturally emitting species. Thermotolerance of photosynthesis against transient high-temperature episodes could only be observed in lines emitting high levels of isoprene; the effect was very mild and could only be identified over repetitive stress events. However, isoprene-emitting plants were highly resistant to ozone-induced oxidative damage compared with their non-emitting azygous controls. In ozone-treated plants, accumulation of toxic reactive oxygen species (ROS) was inhibited, and antioxidant levels were higher. Isoprene-emitting plants showed remarkably decreased foliar damage and higher rates of photosynthesis compared to non-emitting plants immediately following oxidative stress events. An inhibition of hydrogen peroxide accumulation in isoprene-emitting plants may stall the programmed cell death response which would otherwise lead to foliar necrosis. These results demonstrate that endogenously produced isoprene provides protection from oxidative damage. [ABSTRACT FROM AUTHOR]

In this article the authors discuss the challenges involving chassis cells which are self-replicating and can be modified for the creation of certain chemicals. It says that new technologies for biocatalyst design are crucial in furthering the field. However, it states that these processes cannot have an impact if they are not largely accessible. It adds that the chassis cell challenge will catalyze a faster move from a worldwide economy that is petrochemical-based to one that is bio-based.

Enzyme spatial organization is an evolved mechanism for facilitating multi‐step biocatalysis and can play an important role in the regulation of promiscuous enzymes. The latter function suggests that artificial spatial organization can be an untapped avenue for controlling the specificity of bioengineered metabolic pathways. A promiscuous terpene synthase (nerolidol synthase) is co‐localized and spatially organized with the preceding enzyme (farnesyl diphosphate synthase) in a heterologous production pathway, via translational protein fusion and/or co‐encapsulation in a self‐assembling protein cage. Spatial organization enhances nerolidol production by ≈11‐ to ≈62‐fold relative to unorganized enzymes. More interestingly, striking differences in the ratio of end products (nerolidol and linalool) are observed with each spatial organization approach. This demonstrates that artificial spatial organization approaches can be harnessed to modulate the product profiles of promiscuous enzymes in engineered pathways in vivo. This extends the application of spatial organization beyond situations where multiple enzymes compete for a single substrate to cases where there is competition among multiple substrates for a single enzyme. [ABSTRACT FROM AUTHOR]

The multiple fundamental and applied facets of synthetic biology When synthetic biology emerged as an independent scientific field, scientists devoted quite some effort in developing tools for engineering living systems. We hope that such efforts will be shared by the global synthetic biology community beyond territorial and conceptual borders, especially when synthetic biology is scaled-up to offer global solutions (de Lorenzo and Marlière, 2016). Synthetic biology has targeted this circuitry at all levels, engineering it towards a wide array of applications - but also expanding our understanding fundamental aspects of the cell. As synthetic biology continues to expand horizons, the importance of a special issue that covers a breadth of topics in the field cannot be overstated. [Extracted from the article]

The end-to-end fusion of enzymes that catalyse successive steps in a reaction pathway is a metabolic engineering strategy that has been successfully applied in a variety of pathways and is particularly common in terpene bioproduction. Despite its popularity, limited work has been done to interrogate the mechanism of metabolic enhancement from enzyme fusion. We observed a remarkable >110-fold improvement in nerolidol production upon translational fusion of nerolidol synthase (a sesquiterpene synthase) to farnesyl diphosphate synthase. This delivered a titre increase from 29.6 mg/L up to 4.2 g/L nerolidol in a single engineering step. Whole-cell proteomic analysis revealed that nerolidol synthase levels in the fusion strains were greatly elevated compared to the non-fusion control. Similarly, the fusion of nerolidol synthase to non-catalytic domains also produced comparable increases in titre, which coincided with improved enzyme expression. When farnesyl diphosphate synthase was fused to other terpene synthases, we observed more modest improvements in terpene titre (1.9- and 3.8-fold), corresponding with increases of a similar magnitude in terpene synthase levels. Our data demonstrate that increased in vivo enzyme levels – resulting from improved expression and/or improved protein stability – is a major driver of catalytic enhancement from enzyme fusion. • Fusion of farnesyl diphosphate synthase (FPPS) to nerolidol synthase increased nerolidol production by >110-fold. • Fusion of nerolidol synthase to metabolically inactive proteins also increased titres. • Fusion of FPPS to other terpene synthases produced smaller increases in titre. • Increases in titre coincided with elevated levels of terpene synthase. [ABSTRACT FROM AUTHOR]

Cyanobacteria are photosynthetic prokaryotes with strong potential to be used for industrial terpenoid production. However, the key enzymes forming the principal terpenoid building blocks, called short‐chain prenyltransferases (SPTs), are insufficiently characterized. Here, we examined SPTs in the model cyanobacteria Synechococcus elongatus sp. PCC 7942 and Synechocystis sp. PCC 6803. Each species has a single putative SPT (SeCrtE and SyCrtE, respectively). Sequence analysis identified these as type‐II geranylgeranyl pyrophosphate synthases (GGPPSs) with high homology to GGPPSs found in the plastids of green plants and other photosynthetic organisms. In vitro analysis demonstrated that SyCrtE is multifunctional, producing geranylgeranyl pyrophosphate (GGPP; C20) primarily but also significant amounts of farnesyl pyrophosphate (FPP, C15) and geranyl pyrophosphate (GPP, C10); whereas SeCrtE appears to produce only GGPP. The crystal structures were solved to 2.02 and 1.37 Å, respectively, and the superposition of the structures against the GGPPS of Synechococcus elongatus sp. PCC 7002 yield a root mean square deviation of 0.8 Å (SeCrtE) and 1.1 Å (SyCrtE). We also discovered that SeCrtE is co‐encoded in an operon with a functional GGPP phosphatase, suggesting metabolic pairing of these two activities and a putative function in tocopherol biosynthesis. This work sheds light on the activity of SPTs and terpenoid synthesis in cyanobacteria. Understanding native prenyl phosphate metabolism is an important step in developing approaches to engineering the production of different chain‐length terpenoids in cyanobacteria. [ABSTRACT FROM AUTHOR]

Summary: The strigolactone (SL) class of phytohormones shows broad chemical diversity, the functional importance of which remains to be fully elucidated, along with the enzymes responsible for the diversification of the SL structure. Here we explore the functional evolution of the highly conserved CYP711A P450 family, members of which catalyze several key monooxygenation reactions in the strigolactone pathway.Ancestral sequence reconstruction was utilized to infer ancestral CYP711A sequences based on a comprehensive set of extant CYP711 sequences. Eleven ancestral enzymes, corresponding to key points in the CYP711A phylogenetic tree, were resurrected and their activity was characterized towards the native substrate carlactone and the pure enantiomers of the synthetic strigolactone analogue, GR24.The ancestral and extant CYP711As tested accepted GR24 as a substrate and catalyzed several diversifying oxidation reactions on the structure. Evidence was obtained for functional divergence in the CYP711A family. The monocot group 3 ancestor, arising from gene duplication events within monocot grasses, showed both increased catalytic activity towards GR24 and high stereoselectivity towards the GR24 isomer resembling strigol‐type SLs.These results are consistent with a role for CYP711As in strigolactone diversification in early land plants, which may have extended to the diversification of strigol‐type SLs. [ABSTRACT FROM AUTHOR]

Temporal control of heterologous pathway expression is critical to achieve optimal efficiency in microbial metabolic engineering. The broadly-used GAL promoter system for engineered yeast (Saccharomyces cerevisiae) suffers from several drawbacks; specifically, unintended induction during laboratory development, and unintended repression in industrial production applications, which decreases overall production capacity. Eukaryotic synthetic circuits have not been well examined to address these problems. Here, we explore a modularised engineering method to deploy new genetic circuits applicable for expanding the control of GAL promoter-driven heterologous pathways in S. cerevisiae. Trans- and cis- modules, including eukaryotic trans-activating-and-repressing mechanisms, were characterised to provide new and better tools for circuit design. A eukaryote-like tetracycline-mediated circuit that delivers stringent repression was engineered to minimise metabolic burden during strain development and maintenance. This was combined with a novel 37 °C induction circuit to relief glucose-mediated repression on the GAL promoter during the bioprocess. This delivered a 44% increase in production of the terpenoid nerolidol, to 2.54 g L−1 in flask cultivation. These negative/positive transcriptional regulatory circuits expand global strategies of metabolic control to facilitate laboratory maintenance and for industry applications. Eukaryotic trans-regulatory mechanisms are exploited to synthesize a stringent tetracycline-repressible circuit and a heat-inducible circuit for negative/positive metabolic controls using the GAL system in Saccharomyces cerevisiae. [ABSTRACT FROM AUTHOR]

Plasticity in substrate acceptance is a well-characterised phenomenon for disaccharide transporters. Sucrose, a non-reducing disaccharide, is usually metabolised via either the permease-mediated chromosomally-encoded sucrose catabolism (csc) regulon or the sucrose phosphotransferase system (PTS). E. coli W is a fast-growing strain which efficiently utilises sucrose at concentrations above 1% via the csc regulon. To examine if sucrose could be metabolised via other routes, a library of transposon mutants was generated and screened on 0.2% sucrose. One mutant identified from this library had an insertion in the repressor for the regulon controlling catabolism of the disaccharide trehalose (treR). A series of mutants was constructed to elucidate the mechanism of sucrose utilization in the treR insertion strain. Analysis of these mutants provided evidence that deletion of TreR enables uptake of sucrose via TreB, an enzyme II protein required for PTS-mediated uptake of trehalose. Once inside the cell, this sucrose is not processed by the TreC hydrolase, nor is it sufficient for growth of the strain. QRT-PCR analysis showed that levels of cscA (invertase) transcript increased in the WΔtreR mutant relative to the wild-type strain when grown under low sucrose conditions. This result suggests that the intracellular sucrose provided by TreB can facilitate de-repression of the csc regulon, leading to increased gene expression, sucrose uptake and sucrose utilization in the treR mutant. [ABSTRACT FROM AUTHOR]

Summary: The yeast Saccharomyces cerevisiae uses the pyruvate dehydrogenase‐bypass for acetyl‐CoA biosynthesis. This relatively inefficient pathway limits production potential for acetyl‐CoA‐derived biochemical due to carbon loss and the cost of two high‐energy phosphate bonds per molecule of acetyl‐CoA. Here, we attempted to improve acetyl‐CoA production efficiency by introducing heterologous acetylating aldehyde dehydrogenase and phosphoketolase pathways for acetyl‐CoA synthesis to enhance production of the sesquiterpene trans‐nerolidol. In addition, we introduced auxin‐mediated degradation of the glucose‐dependent repressor Mig1p to allow induced expression of GAL promoters on glucose so that production potential on glucose could be examined. The novel genes that we used to reconstruct the heterologous acetyl‐CoA pathways did not sufficiently complement the loss of endogenous acetyl‐CoA pathways, indicating that superior heterologous enzymes are necessary to establish fully functional synthetic acetyl‐CoA pathways and properly explore their potential for nerolidol synthesis. Notwithstanding this, nerolidol production was improved twofold to a titre of ˜ 900 mg l−1 in flask cultivation using a combination of heterologous acetyl‐CoA pathways and Mig1p degradation. Conditional Mig1p depletion is presented as a valuable strategy to improve the productivities in the strains engineered with GAL promoters‐controlled pathways when growing on glucose. [ABSTRACT FROM AUTHOR]

Sucrose is an industrially important carbon source for microbial fermentation. Sucrose utilization in Escherichia coli, however, is poorly understood, and most industrial strains cannot utilize sucrose. The roles of the chromosomally encoded sucrose catabolism (csc) genes in E. coli W were examined by knockout and overexpression experiments. At low sucrose concentrations, the csc genes are repressed and cells cannot grow. Removal of either the repressor protein (cscR) or the fructokinase (cscK) gene facilitated derepression. Furthermore, combinatorial knockout of cscR and cscKconferred an improved growth rate on low sucrose. The invertase (cscA) and sucrose transporter (cscB) genes are essential for sucrose catabolism in E. coli W, demonstrating that no other genes can provide sucrose transport or inversion activities. However, cscKis not essential for sucrose utilization. Fructose is excreted into the medium by the cscK-knockout strain in the presence of high sucrose, whereas at low sucrose (when carbon availability is limiting), fructose is utilized by the cell. Overexpression of cscA, cscAK, or cscAB could complement the WΔcscRKAB knockout mutant or confer growth on a K-12 strain which could not naturally utilize sucrose. However, phenotypic stability and relatively good growth rates were observed in the K-12 strain only when overexpressing cscAB, and full growth rate complementation in WΔcscRKAB also required cscAB. Our understanding of sucrose utilization can be used to improve E. coli Wand engineer sucrose utilization in strains which do not naturally utilize sucrose, allowing substitution of sucrose for other, less desirable carbon sources in industrial fermentations. [ABSTRACT FROM AUTHOR]

Isoprene (C5H8, 2-methyl 1,3-butadiene) is synthesized and emitted by many, but not all, plants. Unlike other related volatile organic compounds (monoterpenes and sesquiterpenes), isoprene has not been shown to mediate plant–herbivore interactions. Here, for the first time, we show, in feeding choice tests using isoprene-emitting transgenic tobacco plants ( Nicotiana tabacum cv. Samsun) and non-emitting azygous control plants, that isoprene deters Manduca sexta caterpillars from feeding. This avoidance behaviour was confirmed using an artificial (isoprene-emitting and non-emitting control) diet. Both in vivo and in vitro experiments showed that isoprene can activate feeding avoidance behaviour in this system with a dose–response effect on caterpillar behaviour and an isoprene emission threshold level of <6 nmol m−2 s−1. [ABSTRACT FROM AUTHOR]

Previous studies have shown that the promoter from the barley (Hordeum vulgare) phosphate transporter gene, HυPht1;1, activates high levels of expression in rice (Oryza sativa) roots and that the expression level was induced by up to 4-fold in response to phosphorus (P) deprivation. To identify promoter regions controlling gene regulation specificities, successive promoter truncations were made and attached to reporter genes. Promoters of between 856 and 1,400 nucleotides activated gene expression in a number of cell types but with maximal expression in trichoblast (root hair) cells. For shorter promoters the trichoblast specificity was lost, but in other tissues the distribution pattern was unchanged. The low P induction response was unaffected by promoter length. Domain exchange experiments subsequently identified that the region between -856 and -547 nucleotides (relative to the translational start) is required for epidermal cell expression. A second region located between 0 and -195 nucleotides controls root-tip expression. The HυPht1;1 promoter contains one PHO-like motif and three motifs similar to the dicot P1BS element. Analysis of promoters from which the PHO-like element was eliminated (by truncation) showed no change in the gene induction response to P deficiency. In contrast, mutation of the P1BS elements eliminated any induction of gene expression in response to low P. An internal HυPht1;1 promoter fragment, incorporating a single P1BS element, had an increased response to P deprivation in comparison with the unmodified promoter (containing three elements). Together these findings further our understanding of the regulation of the HυPht1;1 gene and provide direct evidence for a functional role of the P1BS element in the expression of P-regulated genes. [ABSTRACT FROM AUTHOR]

A biofoundry provides automation and analytics infrastructure to support the engineering of biological systems. It allows scientists to perform synthetic biology and aligned experimentation on a high-throughput scale, massively increasing the solution space that can be examined for any given problem or question. However, establishing a biofoundry is a challenging undertaking, with numerous technical and operational considerations that must be addressed. Using collated learnings, here we outline several considerations that should be addressed prior to and during establishment. These include drivers for establishment, institutional models, funding and revenue models, personnel, hardware and software, data management, interoperability, client engagement and biosecurity issues. The high cost of establishment and operation means that developing a long-term business model for biofoundry sustainability in the context of funding frameworks, actual and potential client base, and costing structure is critical. Moreover, since biofoundries are leading a conceptual shift in experimental design for bioengineering, sustained outreach and engagement with the research community are needed to grow the client base. Recognition of the significant, long-term financial investment required and an understanding of the complexities of operationalization is critical for a sustainable biofoundry venture. To ensure state-of-the-art technology is integrated into planning, extensive engagement with existing facilities and community groups, such as the Global Biofoundries Alliance, is recommended. [ABSTRACT FROM AUTHOR]

Cyanobacteria and algae are photosynthetic microorganisms that are emerging as attractive hosts for synthetic biology applications, ranging from metabolic engineering for the production of industrial biochemicals to microbial energy storage. Photosynthetic microbes have a unique combination of characteristics that drives their consideration for synthetic biology, including relatively simple physiology, fast photosynthetic growth, an availability of model systems, and useful subcellular structures. However, their development as synthetic biology hosts/chassis has been relatively slow compared to that of model heterotrophs. Here we review current synthetic biology tools and applications in photosynthetic microbes and identify opportunities and challenges moving into the future. We focus on cyanobacteria as model photosynthetic prokaryotes, specifically the freshwater unicellular species Synechocystis sp PCC 6803 and Synechococcus elongatus PCC 7942, the marine unicellular species Synechococcus sp PCC 7002, and the nitrogen fixing filamentous species Nostoc sp PCC 7120. Moreover, we present algae as model photosynthetic eukaryotes, specifically the unicellular green alga Chlamydomonas reinhardtii and the diatom Phaeodactylum tricornutum. Finally, we include an examination of some emerging systems. For each organism, the toolbox of genetic parts, high-throughput assembly systems, and other methods that are key components to facilitate synthetic biology are described. Challenges and future directions for synthetic biology and its applications in photosynthetic microorganisms are also discussed. [ABSTRACT FROM AUTHOR]

Terpenoids are a group of natural products that have a variety of essential and non-essential roles in metabolism, in biotic and abiotic interactions, as well as commercial applications such as pharmaceuticals, food additives, and chemical feedstocks. Economic viability for commercial applications is commonly not achievable by using natural source organisms or chemical synthesis. Engineered bio-production in suitable heterologous hosts is often required to achieve commercial viability. However, our poor understanding of regulatory mechanisms and other biochemical processes makes obtaining efficient conversion yields from feedstocks challenging. Moreover, production from carbon dioxide via photosynthesis would significantly increase the environmental and potentially the economic credentials of these processes by disintermediating biomass feedstocks. In this paper, we briefly review terpenoid metabolism, outline some recent advances in terpenoid metabolic engineering, and discuss why photosynthetic unicellular organisms—such as algae and cyanobacteria—might be preferred production platforms for the expression of some of the more challenging terpenoid pathways. [ABSTRACT FROM AUTHOR]

Isoprene and other plastidial isoprenoids are produced primarily from recently assimilated photosynthates via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. However, when environmental conditions limit photosynthesis, a fraction of carbon for MEP pathway can come from extrachloroplastic sources. The flow of extrachloroplastic carbon depends on the species and on leaf developmental and environmental conditions. The exchange of common phosphorylated intermediates between the MEP pathway and other metabolic pathways can occur via plastidic phosphate translocators. C 1 and C 2 carbon intermediates can contribute to chloroplastic metabolism, including photosynthesis and isoprenoid synthesis. Integration of these metabolic processes provide an example of metabolic flexibility, and results in the synthesis of primary metabolites for plant growth and secondary metabolites for plant defense, allowing effective use of environmental resources under multiple stresses. Highlights The high emission rates of isoprene under conditions of restricted photosynthesis can be associated with an increase in the availability of alternative carbon sources for the MEP pathway. The proposed shunt of carbon around the Calvin–Benson cycle involving glucose-6 phosphate could, at least partly, explain the incomplete 13C labeling of isoprene, which so far has been interpreted as uncoupling between isoprene and photosynthetic metabolism. The differences in the isoprene emission rate at different leaf developmental stages are related to overall changes in carbon partitioning between isoprenoid synthesis and primary metabolic pathways, and within the isoprenoid synthesis pathway, to dimethylallyl diphosphate (DMADP) by isoprene synthase and downstream prenyltransferase reactions. Limited availability of energetic cofactors and reducing equivalents can inhibit MEP pathway flux, resulting in an accumulation of MEP pathway intermediates and decreased carbon availability for isoprene production. [ABSTRACT FROM AUTHOR]

Monoterpene production in Saccharomyces cerevisae requires the introduction of heterologous monoterpene synthases (MTSs). The endogenous farnesyl pyrosphosphate synthase (FPPS; Erg20p) competes with MTSs for the precursor geranyl pyrophosphate (GPP), which limits the production of monoterpenes. ERG20 is an essential gene that cannot be deleted and transcriptional down-regulation of ERG20 has failed to improve monoterpene production. Here, we investigated an N-degron-dependent protein degradation strategy to down-regulate Erg20p activity. Degron tagging decreased GFP protein half-life drastically to 1 h (degron K3K15) or 15 min (degrons KN113 and KN119). Degron tagging of ERG20 was therefore paired with a sterol responsive promoter to ensure sufficient metabolic flux to essential downstream sterols despite the severe destabilisation effect of degron tagging. A dual monoterpene/sesquiterpene (linalool/nerolidol) synthase, AcNES1 , was used as a reporter of intracellular GPP and FPP production. Transcription of the synthetic pathway was controlled by either constitutive or diauxie-inducible promoters. A combination of degron K3K15 and the ERG1 promoter increased linalool titre by 27-fold to 11 mg L −1 in the strain with constitutive promoter constructs, and by 17-fold to 18 mg L −1 in the strain with diauxie-inducible promoter constructs. The sesquiterpene nerolidol remained the major product in both strains. The same strategies were applied to construct a limonene-producing strain, which produced 76 mg L −1 in batch cultivation. The FPPS regulation method developed here successfully redirected metabolic flux toward monoterpene production. Examination of growth defects in various strains suggested that the intracellular FPP concentration had a significant effect on growth rate. Further strategies are required to balance intracellular production of FPP and GPP so as to maximise monoterpene production without impacting on cellular growth. [ABSTRACT FROM AUTHOR]

The rapid expansion in the number of sequenced genomes and metagenomes provides an exceptional resource for mining of the enzymes with biotechnologically relevant properties. However, the majority of protein production and analysis methods are not sufficiently cost-efficient and scalable to experimentally verify the results of computational genomic mining. Here, we present a pipeline based on Leishmania tarentolae cell-free system that was used to characterize 30 putative thermostable endo -1,4-β-glucanases and xylanases identified in public genomic databases. In order to analyse the recombinant proteins without purification, novel high-throughput assays for glucanase and xylanase activities were developed. The assays rely on solubilisation of labelled particulate substrates performed in multiwell plates. Using this approach both acidophilic and thermophilic enzymes were identified. The developed approach enables rapid discovery of new biotechnologically useful enzymes. [ABSTRACT FROM AUTHOR]

Background: Assembly of heterologous metabolic pathways is commonly required to generate microbial cell factories for industrial production of both commodity chemicals (including biofuels) and high-value chemicals. Promotermediated transcriptional regulation coordinates the expression of the individual components of these heterologous pathways. Expression patterns vary during culture as conditions change, and this can influence yeast physiology and productivity in both positive and negative ways. Well-characterized strategies are required for matching transcriptional regulation with desired output across changing culture conditions. Results: Here, constitutive and inducible regulatory mechanisms were examined to optimize synthetic isoprenoid metabolic pathway modules for production of trans-nerolidol, an acyclic sesquiterpene alcohol, in yeast. The choice of regulatory system significantly affected physiological features (growth and productivity) over batch cultivation. Use of constitutive promoters resulted in poor growth during the exponential phase. Delaying expression of the assembled metabolic modules using the copper-inducible CUP1 promoter resulted in a 1.6-fold increase in the exponentialphase growth rate and a twofold increase in productivity in the post-exponential phase. However, repeated use of the CUP1 promoter in multiple expression cassettes resulted in genetic instability. A diauxie-inducible expression system, based on an engineered GAL regulatory circuit and a set of four different GAL promoters, was characterized and employed to assemble nerolidol synthetic metabolic modules. Nerolidol production was further improved by 60% to 392 mg L-1 using this approach. Various carbon source systems were investigated in batch/fed-batch cultivation to regulate induction through the GAL system; final nerolidol titres of 4-5.5 g L-1 were achieved, depending on the conditions. Conclusion: Direct comparison of different transcriptional regulatory mechanisms clearly demonstrated that coupling the output strength to the fermentation stage is important to optimize the growth fitness and overall productivities of engineered cells in industrially relevant processes. Applying different well-characterized promoters with the same induction behaviour mitigates against the risks of homologous sequence-mediated genetic instability. Using these approaches, we significantly improved sesquiterpene production in yeast. [ABSTRACT FROM AUTHOR]

Sesquiterpenes are C15 isoprenoids with utility as fragrances, flavours, pharmaceuticals, and potential biofuels. Microbial fermentation is being examined as a competitive approach for bulk production of these compounds. Competition for carbon allocation between synthesis of endogenous sterols and production of the introduced sesquiterpene limits yields. Achieving balance between endogenous sterols and heterologous sesquiterpenes is therefore required to achieve economical yields. In the current study, the yeast Saccharomyces cerevisiae was used to produce the acyclic sesquiterpene alcohol, trans-nerolidol. Nerolidol production was first improved by enhancing the upstream mevalonate pathway for the synthesis of the precursor farnesyl pyrophosphate (FPP). However, excess FPP was partially directed towards squalene by squalene synthase (Erg9p), resulting in squalene accumulation to 1% biomass; moreover, the specific growth rate declined. In order to re-direct carbon away from sterol production and towards the desired heterologous sesquiterpene, a novel protein destabilisation approach was developed for Erg9p. It was shown that Erg9p is located on endoplasmic reticulum and lipid droplets through a C-terminal ER-targeted transmembrane peptide. A PEST (rich in Pro, Glu/Asp, Ser, and Thr) sequence-dependent endoplasmic reticulum-associated protein degradation (ERAD) mechanism was established to decrease cellular levels of Erg9p without relying on inducers, repressors or specific repressing conditions. This improved nerolidol titre by 86% to ~100 mg L −1 . In this strain, squalene levels were similar to the wild-type control strain, and downstream ergosterol levels were slightly decreased relative to the control, indicating redirection of carbon away from sterols and towards sesquiterpene production. There was no negative effect on cell growth under these conditions. Protein degradation is an efficient mechanism to control carbon allocation at flux-competing nodes in metabolic engineering applications. This study demonstrates that an engineered ERAD mechanism can be used to balance flux competition between the endogenous sterol pathway and an introduced bio-product pathways at the FPP node. The approach of protein degradation in general might be more widely applied to improve metabolic engineering outcomes. [ABSTRACT FROM AUTHOR]

Background: High-throughput screening methods assume that the output measured is representative of changes in metabolic flux toward the desired product and is not affected by secondary phenotypes. However, metabolic engineering can result in unintended phenotypes that may go unnoticed in initial screening. The red pigment lycopene, a carotenoid with antioxidant properties, has been used as a reporter of isoprenoid pathway flux in metabolic engineering for over a decade. Lycopene production is known to vary between wild-type Escherichia coli hosts, but the reasons behind this variation have never been fully elucidated. Results: In an examination of six E. coli strains we observed that strains also differ in their capacity for increased lycopene production in response to metabolic engineering. A combination of genetic complementation, quantitative SWATH proteomics, and biochemical analysis in closely-related strains was used to examine the mechanistic reasons for variation in lycopene accumulation. This study revealed that rpoS, a gene previously identified in lycopene production association studies, exerts its effect on lycopene accumulation not through modulation of pathway flux, but through alteration of cellular oxidative status. Specifically, absence of rpoS results in increased accumulation of reactive oxygen species during late log and stationary phases. This change in cellular redox has no effect on isoprenoid pathway flux, despite the presence of oxygen-sensitive iron-sulphur cluster enzymes and the heavy redox requirements of the methylerythritol phosphate pathway. Instead, decreased cellular lycopene in the ?rpoS strain is caused by degradation of lycopene in the presence of excess reactive oxygen species. Conclusions: Our results demonstrate that lycopene is not a reliable indicator of isoprenoid pathway flux in the presence of oxidative stress, and suggest that caution should be exercised when using lycopene as a screening tool in genome-wide metabolic engineering studies. More extensive use of systems biology for strain analysis will help elucidate such unpredictable side-effects in metabolic engineering projects. [ABSTRACT FROM AUTHOR]

Background: Predictable control of gene expression is necessary for the rational design and optimization of cell factories. In the yeast Saccharomyces cerevisiae, the promoter is one of the most important tools available for controlling gene expression. However, the complex expression patterns of yeast promoters have not been fully characterised and compared on different carbon sources (glucose, sucrose, galactose and ethanol) and across the diauxic shift in glucose batch cultivation. These conditions are of importance to yeast cell factory design because they are commonly used and encountered in industrial processes. Here, the activities of a series of "constitutive" and inducible promoters were characterised in single cells throughout the fermentation using green fluorescent protein (GFP) as a reporter. Results: The "constitutive" promoters, including glycolytic promoters, transcription elongation factor promoters and ribosomal promoters, differed in their response patterns to different carbon sources; however, in glucose batch cultivation, expression driven by these promoters decreased sharply as glucose was depleted and cells moved towards the diauxic shift. Promoters induced at low-glucose levels (PHXT7, PSSA1 and PADH2) varied in induction strength on non-glucose carbon sources (sucrose, galactose and ethanol); in contrast to the "constitutive" promoters, GFP expression increased as glucose decreased and cells moved towards the diauxic shift. While lower than several "constitutive" promoters during the exponential phase, expression from the SSA1 promoter was higher in the post-diauxic phase than the commonly-used TEF1 promoter. The galactose-inducible GAL1 promoter provided the highest GFP expression on galactose, and the copper-inducible CUP1 promoter provided the highest induced GFP expression following the diauxic shift. Conclusions: The data provides a foundation for predictable and optimised control of gene expression levels on different carbon sources and throughout batch fermentation, including during and after the diauxic shift. This information can be applied for designing expression approaches to improve yields, rates and titres in yeast cell factories. [ABSTRACT FROM AUTHOR]

Background: Engineering dynamic, environmentally- and temporally-responsive control of gene expression is one of the principle objectives in the field of synthetic biology. Dynamic regulation is desirable because many engineered functions conflict with endogenous processes which have evolved to facilitate growth and survival, and minimising conflict between growth and production phases can improve product titres in microbial cell factories. There are a limited number of mechanisms that enable dynamic regulation in yeast, and fewer still that are appropriate for application in an industrial setting. Results: To address this problem we have identified promoters that are repressed during growth on glucose, and activated during growth on sucrose. Catabolite repression and preferential glucose utilisation allows active growth on glucose before switching to production on sucrose. Using sucrose as an activator of gene expression circumvents the need for expensive inducer compounds and enables gene expression to be triggered during growth on a fermentable, high energy-yield carbon source. The ability to fine-tune the timing and population density at which gene expression is activated from the SUC2 promoter was demonstrated by varying the ratio of glucose to sucrose in the growth medium. Finally, we demonstrated that the system could also be used to repress gene expression (a process also required for many engineering projects). We used the glucose/sucrose system to control a heterologous RNA interference module and dynamically repress the expression of a constitutively regulated GFP gene. Conclusions: The low noise levels and high dynamic range of the SUC2 promoter make it a promising option for implementing dynamic regulation in yeast. The capacity to repress gene expression using RNA interference makes the system highly versatile, with great potential for metabolic engineering applications. [ABSTRACT FROM AUTHOR]

The volatile gas isoprene is emitted in teragrams per annum quantities from the terrestrial biosphere and exerts a large effect on atmospheric chemistry. Isoprene is made primarily from recently fixed photosynthate; however, alternate carbon sources play an important role, particularly when photosynthate is limiting. We examined the relative contribution of these alternate carbon sources under changes in light and temperature, the two environmental conditions that have the strongest influence over isoprene emission. Using a novel real-time analytical approach that allowed us to examine dynamic changes in carbon sources, we observed that relative contributions do not change as a function of light intensity. We found that the classical uncoupling of isoprene emission from net photosynthesis at elevated leaf temperatures is associated with an increased contribution of alternate carbon. We also observed a rapid compensatory response where alternate carbon sources compensated for transient decreases in recently fixed carbon during thermal ramping, thereby maintaining overall increases in isoprene production rates at high temperatures. Photorespiration is known to contribute to the decline in net photosynthesis at high leaf temperatures. A reduction in the temperature at which the contribution of alternate carbon sources increased was observed under photorespiratory conditions, while photosynthetic conditions increased this temperature. Feeding [2-13C]glycine (a photorespiratory intermediate) stimulated emissions of [13C1-5]isoprene and 13CO2, supporting the possibility that photorespiration can provide an alternate source of carbon for isoprene synthesis. Our observations have important implications for establishing improved mechanistic predictions of isoprene emissions and primary carbon metabolism, particularly under the predicted increases in future global temperatures. [ABSTRACT FROM AUTHOR]

Isoprene protects the photosynthetic apparatus of isoprene-emitting plants from oxidative stress. The role of isoprene in the response of plants to drought is less clear., Water was withheld from transgenic isoprene-emitting and non-emitting tobacco ( Nicotiana tabacum) plants, to examine: the response of isoprene emission to plant water deficit; a possible relationship between concentrations of the drought-induced phytohormone abscisic acid ( ABA) and isoprene; and whether isoprene affected foliar reactive oxygen species ( ROS) and lipid peroxidation levels., Isoprene emission did not affect whole-plant water use, foliar ABA concentration or leaf water potential under water deficit. Compared with well-watered controls, droughted non-emitting plants significantly increased ROS content (31-46%) and lipid peroxidation (30-47%), concomitant with decreased operating and maximum efficiencies of photosystem II photochemistry and lower leaf and whole-plant water use efficiency ( WUE). Droughted isoprene-emitting plants showed no increase in ROS content or lipid peroxidation relative to well-watered controls, despite isoprene emission decreasing before leaf wilting., Although isoprene emission protected the photosynthetic apparatus and enhanced leaf and whole-plant WUE, non-emitting plants had 8-24% more biomass under drought, implying that isoprene emission incurred a yield penalty. [ABSTRACT FROM AUTHOR]

Background: Metabolic engineering projects often require integration of multiple genes in order to control the desired phenotype. However, this often requires iterative rounds of engineering because many current insertion approaches are limited by the size of the DNA that can be transferred onto the chromosome. Consequently, construction of highly engineered strains is very time-consuming. A lack of well-characterised insertion loci is also problematic. Results: A series of knock-in/knock-out (KIKO) vectors was constructed for integration of large DNA sequences onto the E. coli chromosome at well-defined loci. The KIKO plasmids target three nonessential genes/operons as insertion sites: arsB (an arsenite transporter); lacZ (β-galactosidase); and rbsA-rbsR (a ribose metabolism operon). Two homologous 'arms' target each insertion locus; insertion is mediated by λ Red recombinase through these arms. Between the arms is a multiple cloning site for the introduction of exogenous sequences and an antibiotic resistance marker (either chloramphenicol or kanamycin) for selection of positive recombinants. The resistance marker can subsequently be removed by flippase-mediated recombination. The insertion cassette is flanked by hairpin loops to isolate it from the effects of external transcription at the integration locus. To characterize each target locus, a xylanase reporter gene (xynA) was integrated onto the chromosomes of E. coli strains W and K-12 using the KIKO vectors. Expression levels varied between loci, with the arsB locus consistently showing the highest level of expression. To demonstrate the simultaneous use of all three loci in one strain, xynA, green fluorescent protein (gfp) and a sucrose catabolic operon (cscAKB) were introduced into lacZ, arsB and rbsAR respectively, and shown to be functional. Conclusions: The KIKO plasmids are a useful tool for efficient integration of large DNA fragments (including multiple genes and pathways) into E. coli. Chromosomal insertion provides stable expression without the need for continuous antibiotic selection. Three non-essential loci have been characterised as insertion loci; combinatorial insertion at all three loci can be performed in one strain. The largest insertion at a single site described here was 5.4 kb; we have used this method in other studies to insert a total of 7.3 kb at one locus and 11.3 kb across two loci. These vectors are particularly useful for integration of multigene cassettes for metabolic engineering applications. [ABSTRACT FROM AUTHOR]

Abstract: Sucrose has economic and environmental advantages over glucose as a feedstock for bioprocesses. E. coli is widely used in industry, but the majority of current industrial E. coli strains cannot utilize sucrose. Previous attempts to transfer sucrose catabolic capabilities into non-sucrose-utilizing strains have met with limited success due to low growth rates on sucrose and phenotypic instability of the engineered strains. To address these problems, we developed a transferrable sucrose utilization cassette which confers efficient sucrose catabolism when integrated onto the E. coli chromosome. The cassette was based on the csc genes from E. coli W, a strain which grows very quickly on sucrose. Both plasmid-borne expression and chromosomal integration of a repressor-less sucrose utilizing cassette were investigated in E. coli strains K-12, B and C. In contrast to previous studies, strains harboring chromosomal cassettes could grow at the same rate as they do on glucose. Interestingly, we also discovered that spontaneous chromosomal integration of the csc genes was required to allow efficient growth from plasmid-transformed strains. The ability to engineer industrial strains for efficient sucrose utilization will allow substitution of sucrose for glucose in industrial fermentations. This will encourage the use of sucrose as a carbon source and assist in transition of our petrochemical-based economy to a bio-based economy. [Copyright &y& Elsevier]

The article offers information on the study conducted by researchers including J.R. Wicks, N.B. Oldridge, and L.K. Nielsen on HR Index, a procedure to measure oxygen uptake by humanbeings during physical activities. Under HR Index method, the consumption of oxygen is predicted on the basis of HR measurements. This process has simplified the work of analyzing energy uptake, because it does not require other things such as gas analysis, moving-recording devices, and exercise equipments. Scientists also studied the link between HR Index and metabolic equivalent (MET), and revealed that energy cost of physical activities can be ascertained using both together.

Background: Escherichia coli is a model prokaryote, an important pathogen, and a key organism for industrial biotechnology. E. coli W (ATCC 9637), one of four strains designated as safe for laboratory purposes, has not been sequenced. E. coli W is a fast-growing strain and is the only safe strain that can utilize sucrose as a carbon source. Lifecycle analysis has demonstrated that sucrose from sugarcane is a preferred carbon source for industrial bioprocesses. Results: We have sequenced and annotated the genome of E. coli W. The chromosome is 4,900,968 bp and encodes 4,764 ORFs. Two plasmids, pRK1 (102,536 bp) and pRK2 (5,360 bp), are also present. W has unique features relative to other sequenced laboratory strains (K-12, B and Crooks): it has a larger genome and belongs to phylogroup B1 rather than A. W also grows on a much broader range of carbon sources than does K-12. A genome-scale reconstruction was developed and validated in order to interrogate metabolic properties. Conclusions: The genome of W is more similar to commensal and pathogenic B1 strains than phylogroup A strains, and therefore has greater utility for comparative analyses with these strains. W should therefore be the strain of choice, or 'type strain' for group B1 comparative analyses. The genome annotation and tools created here are expected to allow further utilization and development of E. coli W as an industrial organism for sucrose-based bioprocesses. Refinements in our E. coli metabolic reconstruction allow it to more accurately define E. coli metabolism relative to previous models. [ABSTRACT FROM AUTHOR]

Abstract: Sucrose has several advantages over glucose as a feedstock for bioprocesses, both environmentally and economically. However, most industrial Escherichia coli strains are unable to utilize sucrose. E. coli W can grow on sucrose but stops growing when sucrose concentrations become low. This is undesirable in fed-batch conditions where sugar levels are low between feeding pulses. Sucrose uptake rates were improved by removal of the cscR gene, which encodes a protein that represses expression of the sucrose utilization genes at low sucrose concentrations. Poly-3-hydroxybutyrate (PHB) was used as a model compound in order to assess the effect of improved sugar utilization on bio-production. In the cscR knockout strain, production from sucrose was improved by 50%; this strain also produced 30% more PHB than the wild-type using glucose. This result demonstrates the feasibility of utilizing sucrose as an industrial feedstock for E. coli-based bioprocesses in high cell density culture. [Copyright &y& Elsevier]

Sucrose is one of the most promising carbon sources for industrial fermentation. To achieve sucrose catabolism, the sucrose utilization operons have been introduced into microorganisms that are not able to utilize sucrose. However, the rates of growth and sucrose uptake of these engineered strains were relatively low to be successfully employed for industrial applications. Here, we report a practical example of developing sucrose-utilizing microorganisms using Escherichia coli K-12 as a model system. The sucrose utilizing ability was acquired by introducing only β-fructofuranosidase from three different sucrose-utilizing organisms ( Mannheimia succiniciproducens, E. coli W, and Bacillus subtilis). Among them, the M. succiniciproducens β-fructofuranosidase was found to be the most effective for sucrose utilization. Analyses of the underlying mechanism revealed that sucrose was hydrolyzed into glucose and fructose in the extracellular space and both liberated hexoses could be transported by their respective uptake systems in E. coli K-12. To prove that this system can also be applied for the production of useful metabolites, the M. succiniciproducens β-fructofuranosidase was introduced into the engineered l-threonine production strain of E. coli K-12. This recombinant strain was able to produce 51.1 g/L l-threonine by fed-batch culture, resulting in an overall yield of 0.284 g l-threonine per g sucrose. This simple approach to make E. coli K-12 to acquire sucrose-utilizing ability and its successful biotechnological application can be employed to develop sustainable bioprocesses using renewable biomass. [ABSTRACT FROM AUTHOR]