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While algae demonstrate potential as a sustainable fuel source, low productivities limit the economic realization of algal biofuels. High-throughput strain engineering, omics-informed genome-scale modeling, and microbiome engineering are key technologies for enabling algal biofuels. High-throughput strain engineering efforts generate improved traits, including high biomass productivity and lipid content, in diverse algal species. Genome-scale models, constructed with the aid of omics data, provide insight into metabolic limitations and guide rational algal strain engineering efforts. As outdoor cultivation systems introduce exogenous organisms, microbiome engineering seeks to eliminate harmful organisms and introduce beneficial species. Optimizing algal biomass production and lipid content using these technologies may overcome the productivity barrier for the commercialization of algal biofuels.

Operon prediction in prokaryotes is critical not only for understanding the regulation of endogenous gene expression, but also for exogenous targeting of genes using newly developed tools such as CRISPR-based gene modulation. A number of methods have used transcriptomics data to predict operons, based on the premise that contiguous genes in an operon will be expressed at similar levels. While promising results have been observed using these methods, most of them do not address uncertainty caused by technical variability between experiments, which is especially relevant when the amount of data available is small. In addition, many existing methods do not provide the flexibility to determine the stringency with which genes should be evaluated for being in an operon pair. We present OperonSEQer, a set of machine learning algorithms that uses the statistic and p-value from a non-parametric analysis of variance test (Kruskal-Wallis) to determine the likelihood that two adjacent genes are expressed from the same RNA molecule. We implement a voting system to allow users to choose the stringency of operon calls depending on whether your priority is high recall or high specificity. In addition, we provide the code so that users can retrain the algorithm and re-establish hyperparameters based on any data they choose, allowing for this method to be expanded as additional data is generated. We show that our approach detects operon pairs that are missed by current methods by comparing our predictions to publicly available long-read sequencing data. OperonSEQer therefore improves on existing methods in terms of accuracy, flexibility, and adaptability., Author summary Bacteria and archaea, single-cell organisms collectively known as prokaryotes, live in all imaginable environments and comprise the majority of living organisms on this planet. Prokaryotes play a critical role in the homeostasis of multicellular organisms (such as animals and plants) and ecosystems. In addition, bacteria can be pathogenic and cause a variety of diseases in these same hosts and ecosystems. In short, understanding the biology and molecular functions of bacteria and archaea and devising mechanisms to engineer and optimize their properties are critical scientific endeavors with significant implications in healthcare, agriculture, manufacturing, and climate science among others. One major molecular difference between unicellular and multicellular organisms is the way they express genes–multicellular organisms make individual RNA molecules for each gene while, prokaryotes express operons (i.e., a group of genes coding functionally related proteins) in contiguous polycistronic RNA molecules. Understanding which genes exist within operons is critical for elucidating basic biology and for engineering organisms. In this work, we use a combination of statistical and machine learning-based methods to use next-generation sequencing data to predict operon structure across a range of prokaryotes. Our method provides an easily implemented, robust, accurate, and flexible way to determine operon structure in an organism-agnostic manner using readily available data.

The anthropogenic release of toxic metals into the environment poses danger to the health of both humans and the local ecosystem. Biosensors for the detection of metals have been developed to improve our ability to monitor these environmental contaminants, yet most of these sensors use heterotrophic bacterial hosts, which require a fixed carbon source and do not typically grow in natural waterways. In this study, we constructed and characterized metal sensors for development of a photoautotrophic biosensor using Synechococcus sp. PCC 7002. We characterized gold and copper sensors based on modified MerR transcriptional activators: GolSA113T, with improved gold binding, and GolSCL, containing the metal-binding loop from CueR which binds both gold and copper. The metal-sensing constructs were first optimized and characterized in Escherichia coli MG1655. The addition of a strong ribosome binding site to the optical reporter protein increased translation of the fluorescent reporter, and expression of golSA113T from the rbc promoter of Synechococcus sp. PCC 7002 improved the response to gold in MG1655. In rich medium, the GolSA113T-based E. coli sensor detected gold at concentrations as low as 100 nM, while the GolSCL-based E. coli sensor detected gold and copper at sensitivities of 100 nM and 10 μM, respectively. Both E. coli sensors responded to gold and copper yet showed no detectable response to other metals. Abiotic factors, such as medium complexity, were found to influence the response of the E. coli sensors, with minimal medium resulting in higher sensitivities of detection. Expression of the GolSA113T- and GolSCL-based sensor constructs in the cyanobacterium Synechococcus sp. PCC 7002 resulted in photoautotrophic gold sensors, but these biosensors failed to produce a significant response to copper. Moreover, the fluorescence response of the cyanobacterial sensors to gold was significantly reduced compared to that of analogous E. coli sensors. While this effort demonstrates feasibility for the development of photoautotrophic biosensors, additional efforts to optimize sensor performance will be required.

Industrial accidents, such as the Fukushima and Chernobyl disasters, release harmful chemicals into the environment, covering large geographical areas. Natural flora may serve as biological sensors for detecting metal contamination, such as cesium. Spectral detection of plant stresses typically employs a few select wavelengths and often cannot distinguish between different stress phenotypes. In this study, we apply hyperspectral reflectance imaging in the visible and near-infrared along with multivariate curve resolution (MCR) analysis to identify unique spectral signatures of three stresses in Arabidopsis thaliana: salt, copper, and cesium. While all stress conditions result in common stress physiology, hyperspectral reflectance imaging and MCR analysis produced unique spectral signatures that enabled classification of each stress. As the level of potassium was previously shown to affect cesium stress in plants, the response of A. thaliana to cesium stress under variable levels of potassium was also investigated. Increased levels of potassium reduced the spectral response of A. thaliana to cesium and prevented changes to chloroplast cellular organization. While metal stress mechanisms may vary under different environmental conditions, this study demonstrates that hyperspectral reflectance imaging with MCR analysis can distinguish metal stress phenotypes, providing the potential to detect metal contamination across large geographical areas.

While recent studies have demonstrated direct photosynthetic production of biofuels via engineered cyanobacteria, biofuel yields from cyanobacteria remain at low levels. As with heterotrophic biofuel production, product toxicity is likely a limiting factor. Some mechanisms of toxicity may be similar to those studied in common heterotrophic hosts; however, the photosynthesis-dependent pathways for carbon fixation and energy production in cyanobacteria present unique targets for biofuel toxicity. This study investigates biofuel toxicity for three model cyanobacterial strains: Synechococcus elongatus PCC 7942, Synechocystis sp. PCC 6803, and Synechococcus sp. PCC 7002. While cyanobacterial biofuel tolerances were generally lower than that of heterotrophic hosts, the marine strain Synechococcus sp. PCC 7002 showed increased tolerance to short chain alcohols, and long-chain biofuel products, such as fatty alcohols, saturated free fatty acids, alkanes, and alkenes had minimal toxicity for all three cyanobacteria. Targeted mutants were generated to explore natural mechanisms of biofuel tolerance in cyanobacteria, such as cell membrane composition, reactive oxygen species degradation, and efflux pumps. These mutants confirmed the influence of cell membrane composition on cyanobacterial tolerance to short-chain alcohols. This study provides data to guide both biofuel product and cyanobacterial host selection and further identifies potential targets for improving biofuel tolerance in cyanobacteria.

Photosynthetically derived fuels, such as those produced by microalgae, are touted as a future renewable energy source and a means for achieving energy independence. Realization of these claims, however, will require fuel production rates beyond the native capabilities of these microorganisms. The development of a metabolic engineering toolkit for microalgae will be key for reaching the production rates necessary for fuel production. This work advances the toolkit for cyanobacterial fuels by exploring the use of eukaryotic algal gene sources for free fatty acid biosynthesis rather than the traditional bacterial and plant sources. Many species of eukaryotic algae naturally accumulate high levels of triacylglycerol, a compound requiring three fatty acid side chains. Triacylglycerol accumulation implies that eukaryotic algae have naturally efficient enzymes for free fatty acid production, representing an unexplored resource for metabolic engineering targets. In this work, the model cyanobacterium, Synechococcus elongatus PCC7942, was engineered for free fatty acid production by targeting three main rate-limiting steps: (1) fatty acid release, catalyzed by a thioesterase, (2) fixation of carbon by ribulose-1,5-bisphosphate carboxylase/oxygenase, and (3) the first committed step in fatty acid biosynthesis, acetyl-CoA carboxylase. The recombinant acyl-ACP thioesterase and acetyl-CoA carboxylase were derived from the model green alga, Chlamydomonas reinhardtii CC-503. By targeting these proposed rate-determining steps, free fatty acid production was improved on a cell weight basis; however, the overall concentration of excreted free fatty acid did not increase. Recombinant gene expression was optimized by using native promoters, and while expression improved, the free fatty acid yield did not likewise increase. From physiological measurements, it was determined that free fatty acid production in S. elongatus PCC7942 is ultimately limited by the negative physiological effects associated with free fatty acid synthesis rather than bottlenecks within the metabolic pathway. This work demonstrates the successful expression of algal genes in a cyanobacterial host, but further improvement in free fatty acid yields will only be possible when the negative effects of free fatty acid production are mitigated.

The Editorial on the Research Topic Cyanobacteria: The GreenE. coli As the world struggles to reduce its dependence on fossil fuels and curb greenhouse gas emissions, industrial biotechnology is also “going green.” Escherichia coli has long been used as a model Gram-negative bacterium, not only for fundamental research but also for industrial applications. Recently, however, cyanobacteria have emerged as candidate chassis for the production of commodity fuels and chemicals, using CO2 and sunlight as their main sources of carbon and energy (Ducat et al., 2011; Machado and Atsumi, 2012). In addition to their potential for reducing greenhouse gas emissions and lowering production costs, cyanobacteria have naturally efficient pathways for the production of carbohydrates and proteins as well as metabolites such as vitamins and carotenoids, which are of importance in the nutraceutical industry (Wang et al., 2014). The unique metabolic and regulatory pathways present in cyanobacteria present new challenges for metabolic engineers and synthetic biologists. Moreover, their requirement for light and the dynamic regulatory mechanisms of the diurnal cycle further complicate the development and application of cyanobacteria for industrial applications. Consequently, significant advancements in cyanobacterial engineering and strain development are necessary for the development of a truly “green E. coli.” This Research Topic highlights cyanobacteria as organisms of emerging industrial relevance and discusses unique challenges posed by these photosynthetic hosts. Original Research articles in this issue demonstrate the production of energy-dense biofuels in engineered cyanobacterial hosts, including alkanes (Klahn et al.), free fatty acids (Ruffing; Work et al.), and terpenes (Davies et al.). The use of cyanobacteria for the production of plant secondary metabolites is reviewed by Xue and He. In addition to the production of metabolites, Jensen and Leister present their Opinion article on exploiting cyanobacteria for modifying photosynthesis, allowing for more rapid modification and characterization compared to plant hosts. Collectively, these articles provide a broad overview of the potential applications enabled by cyanobacterial hosts, yet this is certainly not an exhaustive list. Cyanobacteria have been successfully engineered in other applications including biosensing, bioremediation, protein production, and hydrogen production (Ruffing, 2011), and diverse, new applications for cyanobacteria are most certainly under development. A common challenge in engineering cyanobacteria is often the paucity of characterized genetic tools available for cyanobacterial hosts. Oftentimes, genetic tools developed for E. coli may be applied in a cyanobacterial host, yet the function of these E. coli elements can vary greatly when expressed in a cyanobacterium (Markley et al., 2015). The synthetic constructs may also be more effective when integrated with the cyanobacterial metabolism, requiring the development of new tools that interface with the circadian rhythm of cyanobacteria and the diurnal nature of photosynthetic metabolism. This Research Topic includes a review by Camsund and Lindblad of transcriptional tools and design principles for engineering transcriptional systems in cyanobacteria. Further, Branco dos Santos et al. present the compelling perspective that mitigation of carbon emissions is more likely to succeed if cyanobacteria are viewed as cell factories for bioproducts, similar to E. coli, rather than solely as a means for CO2 capture. In addition to genetic tool availability for cyanobacteria, other unique challenges result from the use of these photosynthetic hosts in biotechnology applications. Metabolite production, particularly biofuel synthesis, may have toxic or detrimental effects on the physiology of the cyanobacterial host, as described in an Original Research article by Pei et al., analyzing the effects of exogenous biofuels on the protein network in Synechocystis sp. PCC 6803. The photosynthetic constraints of light sensing and light harvesting are discussed in a Perspective by Montgomery. In his Hypothesis and Theory article, Burnap analyzes systems-level constraints of autotrophic productivity in cyanobacteria and argues that intracellular crowding imposes limitations on the allocation of proteomic resources. An interesting case-in-point may be the recent rediscovery of a fast-growing cyanobacterium, with a generation time of ~2 h that apparently does so in part by not devoting resources to glycogen synthesis during early stages of growth (Yu et al., 2015). Lastly, Jones provides an important Perspective on a largely ignored problem in cyanobacterial engineering, namely that of genetic instability. As illustrated by the articles in this Research Topic, the successes and challenges of developing cyanobacteria as cell factories make this an exciting time for the green E. coli.

The high productivity of fluidically mixed open ponds for algal biofuel production is accompanied by high environmental and temporal variability. Therefore, a recognized need exists for rapid monitoring of open ponds to quantify algal growth rates, assess algal stress, detect the presence of invading species, and determine the optimum time for harvesting. Multispectral/hyperspectral approaches are now being used to optimize conventional agriculture practices and similar remote sensing techniques can potentially be used for monitoring algal ponds. In this work, we assess the application of remote techniques for algal biofuel production by using a dual-channel spectroradiometer to monitor the laboratory-scale growth of Nannochloropsis salina , a popular microalgal candidate for biofuels. One channel of the spectroradiometer measures the downwelling irradiance while the second channel simultaneously monitors the upwelling radiance, and the reflectance is calculated by ratioing these two signals. A detailed reflectance model is developed to interpret the acquired spectra, enabling a remote assessment the culture's optical depth as well as the relative optical activity of different algal pigments in N. salina .

The direct conversion of carbon dioxide into biofuels by photosynthetic microorganisms is a promising alternative energy solution. In this study, a model cyanobacterium, Synechococcus elongatus PCC 7942, is engineered to produce free fatty acids (FFA), potential biodiesel precursors, via gene knockout of the FFA-recycling acyl-ACP synthetase and expression of a thioesterase for release of the FFA. Similar to previous efforts, the engineered strains produce and excrete FFA, but the yields are too low for large-scale production. While other efforts have applied additional metabolic engineering strategies in an attempt to boost FFA production, we focus on characterizing the engineered strains to identify the physiological effects that limit cell growth and FFA synthesis. The strains engineered for FFA-production show reduced photosynthetic yields, chlorophyll-a degradation, and changes in the cellular localization of the light-harvesting pigments, phycocyanin and allophycocyanin. Possible causes of these physiological effects are also identified. The addition of exogenous linolenic acid, a polyunsaturated FFA, to cultures of S. elongatus 7942 yielded a physiological response similar to that observed in the FFA-producing strains with only one notable difference. In addition, the lipid constituents of the cell and thylakoid membranes in the FFA-producing strains show changes in both the relative amounts of lipid components and the degree of saturation of the fatty acid side chains. These changes in lipid composition may affect membrane integrity and structure, the binding and diffusion of phycobilisomes, and the activity of membrane-bound enzymes including those involved in photosynthesis. Thus, the toxicity of unsaturated FFA and changes in membrane composition may be responsible for the physiological effects observed in FFA-producing S. elongatus 7942. These issues must be addressed to enable the high yields of FFA synthesis necessary for large-scale biofuel production.

Agrobacterium sp. ATCC 31749 was previously shown to be an advantageous host for oligosaccharide production. Unexpectedly, the addition of citrate to the oligosaccharide synthesis reaction resulted in up to a sixfold improvement in the production N-aceytl-lactosamine, a disaccharide. The possible mechanisms for this citrate-induced stimulation of oligosaccharide production were investigated, including the consumption of citrate as a carbon and energy source, enhanced metal ion solubility from citrate chelation, and the ability of citrate to act as a buffer. The main mechanisms for the effect of citrate on oligosaccharide production were determined to be carbon and energy provision from citrate consumption and pH maintenance. ATCC 31749 was shown to co-metabolize citrate along with sucrose, a preferred carbon source, indicating the lack of a catabolite repression system in this Agrobacterium. Metabolic flux analysis suggested an increase in flux through TCA cycle for the citrate-containing reaction, which may provide additional energy supply to support enhanced oligosaccharide production. The citrate stimulation of oligosaccharide synthesis was shown to be unique to the Agrobacterium strain, as a similarly engineered Escherichia coli strain did not show significant improvement in oligosaccharide production with citrate addition. This work provides insight into the metabolism of Agrobacterium sp. ATCC 31749 and highlights important factors in whole-cell oligosaccharide synthesis.

To determine the effect of Liquidoff on bacteria, three bacterial strains were tested: Escherichia coli DH5α, Synechococcus sp. PCC 7002, and Synechococcus elongatus PCC 7942. E. coli DH5α is a Gramnegative, aerobic bacterium that is often found in normal gut flora and is commonly used the laboratory due to its fast growth rate. Synechococcus sp. PCC 7002 and S. elongatus PCC 7942 are Gram-negative, aquatic, autophototrophic cyanobacteria. Synechococcus sp. PCC 7002 is a marine cyanobacterium isolated from ‘fish pens’ on Magueyes Island, Puerto Rico in 1962, while S. elongatus PCC 7942 is a freshwater cyanobacterium. It should be noted that no Gram-positive bacterium was tested in this study.

Microbial free fatty acids (FFAs) have been proposed as a potential feedstock for renewable energy. The ability to directly convert carbon dioxide into FFAs makes cyanobacteria ideal hosts for renewable FFA production. Previous metabolic engineering efforts using the cyanobacterial hosts Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 have demonstrated this direct conversion of carbon dioxide into FFAs; however, FFA yields in these hosts are limited by the negative impact of FFA production on the host cell physiology. This work investigates the use of Synechococcus sp. PCC 7002 as a cyanobacterial host for FFA production. In comparison to S. elongatus PCC 7942, Synechococcus sp. PCC 7002 strains produced and excreted FFAs at similar concentrations but without the detrimental effects on host physiology. The enhanced tolerance to FFA production with Synechococcus sp. PCC 7002 was found to be temperature-dependent, with physiological effects such as reduced photosynthetic yield and decreased photosynthetic pigments observed at higher temperatures. Additional genetic manipulations were targeted for increased FFA production, including thioesterases and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Overexpression of non-native RuBisCO subunits (rbcLS) from a psbAI promoter resulted in more than a threefold increase in FFA production, with excreted FFA concentrations reaching >130 mg/L. This work illustrates the importance of host strain selection for cyanobacterial biofuel production and demonstrates that the FFA tolerance of Synechococcus sp. PCC 7002 can allow for high yields of excreted FFA.

The world’s supply of petroleum hydrocarbons, which serve as feedstock for the fuel and chemical industries, is rapidly diminishing to satisfy the global demand for energy and consumer goods. In response to this increasing demand and limited supply, the cost of crude oil has risen to over $100 per barrel in 2012, a 10-fold increase compared to prices in the late 1990s [1]. As fossil fuels are nonrenewable resources, the price of oil is only expected to increase in the future. This unavoidable reality necessitates the development of renewable energy sources in order to maintain the current standard of living. Among the alternative energy options under development, biofuels are anticipated to supplement and eventually replace the petroleum-based fuels that supply the transportation and chemical industries. Currently, first generation biofuels like corn-based ethanol are blended into conventional petroleum fuels, with biofuels supplying 2.7% of the world’s transportation fuel in 2010 [2]. It appears that biofuels are on their way to becoming a viable renewable energy source, yet technological and biological advancements are necessary for sustainable and economical biofuel production at the scales necessary to support the world’s energy needs.

Algal biofuels are a renewable energy source with the potential to replace conventional petroleum-based fuels, while simultaneously reducing greenhouse gas emissions. The economic feasibility of commercial algal fuel production, however, is limited by low productivity of the natural algal strains. The project described in this SAND report addresses this low algal productivity by genetically engineering cyanobacteria (i.e. blue-green algae) to produce free fatty acids as fuel precursors. The engineered strains were characterized using Sandia's unique imaging capabilities along with cutting-edge RNA-seq technology. These tools are applied to identify additional genetic targets for improving fuel production in cyanobacteria. This proof-of-concept study demonstrates successful fuel production from engineered cyanobacteria, identifies potential limitations, and investigates several strategies to overcome these limitations. This project was funded from FY10-FY13 through the President Harry S. Truman Fellowship in National Security Science and Engineering, a program sponsored by the LDRD office at Sandia National Laboratories.

The search is on for new renewable energy and algal-derived biofuel is a critical piece in the multi-faceted renewable energy puzzle. It has 30x more oil than any terrestrial oilseed crop, ideal composition for biodiesel, no competition with food crops, can be grown in waste water, and is cleaner than petroleum based fuels. This project discusses these three goals: (1) Conduct fundamental research into the effects that dynamic biotic and abiotic stressors have on algal growth and lipid production - Genomics/Transcriptomics, Bioanalytical spectroscopy/Chemical imaging; (2) Discover spectral signatures for algal health at the benchtop and greenhouse scale - Remote sensing, Bioanalytical spectroscopy; and (3) Develop computational model for algal growth and productivity at the raceway scale - Computational modeling.

Cyanobacteria have played an important role in the development of the Earth and have long been studied as model organisms for photosynthesis and the circadian rhythm. Recent developments have led to increased interest in the use of engineered cyanobacteria for the production of protein and chemical products. This review highlights the genetic tools and strategies for manipulation of cyanobacteria as well as previous accomplishments in the development of engineered cyanobacteria for applied use. Particular attention is given to the engineering of cyanobacteria for biofuel production, including both hydrocarbon and hydrogen fuels. Genetic engineering efforts to enhance cyanobacterial fitness are reviewed with an emphasis on physiological improvements for large-scale production. Lastly, a future outlook on engineered cyanobacteria is presented, highlighting the future areas of focus and technical challenges in this field. With the uncertainty of future energy security, it is an exciting time in applied cyanobacterial research, but we must take the time to learn from these past accomplishments before we can capitalize on the potential of these photosynthetic microorganisms.

Background Oligosaccharides containing a terminal Gal-α1,3-Gal moiety are collectively known as α-Gal epitopes. α-Gal epitopes are integral components of several medical treatments under development, including flu and HIV vaccines as well as cancer treatments. The difficulty associated with synthesizing the α-Gal epitope hinders the development and application of these treatments due to the limited availability and high cost of the α-Gal epitope. This work illustrates the development of a whole-cell biocatalyst for synthesizing the α-Gal epitope, Gal-α1,3-Lac. Results Agrobacterium sp. ATCC 31749 was engineered to produce Gal-α1,3-Lac by the introduction of a UDP-galactose 4'-epimerase:α1,3-galactosyltransferase fusion enzyme. The engineered Agrobacterium synthesized 0.4 g/L of the α-Gal epitope. Additional metabolic engineering efforts addressed the factors limiting α-Gal epitope production, namely the availability of the two substrates, lactose and UDP-glucose. Through expression of a lactose permease, the intracellular lactose concentration increased by 60 to 110%, subsequently leading to an improvement in Gal-α1,3-Lac production. Knockout of the curdlan synthase gene increased UDP-glucose availability by eliminating the consumption of UDP-glucose for synthesis of the curdlan polysaccharide. With these additional engineering efforts, the final engineered strain synthesized approximately 1 g/L of Gal-α1,3-Lac. Conclusions The Agrobacterium biocatalyst developed in this work synthesizes gram-scale quantities of α-Gal epitope and does not require expensive cofactors or permeabilization, making it a useful biocatalyst for industrial production of the α-Gal epitope. Furthermore, the engineered Agrobacterium, with increased lactose uptake and improved UDP-glucose availability, is a promising host for the production of other medically-relevant oligosaccharides.

Curdlan-producing Agrobacterium sp. is unique in possessing a highly efficient UDP-glucose regeneration system. A broad-host-range expression strategy was successfully developed to exploit the unique metabolic capability for UDP-galactose regeneration during oligosaccharide synthesis. The engineered Agrobacterium cells functioned as a UDP-galactose regeneration system, allowing galactose-containing disaccharides to be synthesized from glucose or other simple sugars. Unexpectedly, a lag period of 24 h preceded the active synthesis, which could be eliminated with rifampicin. An intracellular nucleotide profiling revealed that the UMP level was elevated by 3.8 fold in the presence of rifampicin, suggesting that rifampicin simulated a nitrogen-limitation condition that triggered the metabolic change. Product selectivity was improved nearly 40-fold by using high acceptor concentration and restricting glucose supply. N -acetyllactosamine concentration near 20 mM (7.5 g/l) was obtained, demonstrating the effectiveness of the engineered strain in UDP-galactose regeneration. This organism could be engineered to regenerate other UDP-sugar nucleotides using the same strategy as illustrated here.

Metabolic engineering has recently been embraced as an effective tool for developing whole-cell biocatalysts for oligosaccharide and polysaccharide synthesis. Microbial catalysts now provide a practical means to derive many valuable oligosaccharides, previously inaccessible through other methods, in sufficient quantities to support research and clinical applications. The synthesis process based upon these microbes is scalable as it avoids expensive starting materials. Most impressive is the high product concentrations (up to 188 g/L) achieved through microbe-catalyzed synthesis. The overall cost for selected molecules has been brought to a reasonable range (estimated $ 30–50/g). Microbial synthesis of oligosaccharides and polysaccharides is a carbon-intensive and energy-intensive process, presenting some unique challenges in metabolic engineering. Unlike nicotinamide cofactors, the required sugar nucleotides are products of multiple interacting pathways, adding significant complexity to the metabolic engineering effort. Besides the challenge of providing the necessary mammalian-originated glycosyltransferases in active form, an adequate uptake of sugar acceptors can be an issue when another sugar is necessary as a carbon and energy source. These challenges are analyzed, and various strategies used to overcome these difficulties are reviewed in this article. Despite the impressive success of the microbial coupling strategy, there is a need to develop a single strain that can achieve at least the same efficiency. Host selection and the manner with which the synthesis interacts with the central metabolism are two important factors in the design of microbial catalysts. Additionally, unlike in vitro enzymatic synthesis, product degradation and byproduct formation are challenges of whole-cell systems that require additional engineering. A systematic approach that accounts for various and often conflicting requirements of the synthesis holds the key to deriving an efficient catalyst. Metabolic engineering strategies applied to selected polysaccharides (hyaluronan, alginate, and exopolysaccharides for food use) are reviewed in this article to highlight the recent progress in this area and similarity to challenges in oligosaccharide synthesis. Many naturally occurring microbes possess highly efficient mechanisms for polysaccharide synthesis. These mechanisms could potentially be engineered into a microbe for oligosaccharide and polysaccharide synthesis with enhanced efficiency.

Agrobacterium sp. ATCC 31749 is an industrial strain for the commercial production of curdlan, an important exopolysaccharide with food and medical applications. Here we report the genome sequence of the curdlan-producing strain ATCC 31749. Genome sequencing is the first step toward the understanding of regulation of curdlan biosynthesis.

Background High-energy-density biofuels are typically derived from the fatty acid pathway, thus establishing free fatty acids (FFAs) as important fuel precursors. FFA production using photosynthetic microorganisms like cyanobacteria allows for direct conversion of carbon dioxide into fuel precursors. Recent studies investigating cyanobacterial FFA production have demonstrated the potential of this process, yet FFA production was also shown to have negative physiological effects on the cyanobacterial host, ultimately limiting high yields of FFAs. Results Cyanobacterial FFA production was shown to generate reactive oxygen species (ROS) and lead to increased cell membrane permeability. To identify genetic targets that may mitigate these toxic effects, RNA-seq analysis was used to investigate the host response of Synechococcus elongatus PCC 7942. Stress response, nitrogen metabolism, photosynthesis, and protein folding genes were up-regulated during FFA production while genes involved in carbon and hydrogen metabolisms were down-regulated. Select genes were targeted for mutagenesis to confirm their role in mitigating FFA toxicity. Gene knockout of two porins and the overexpression of ROS-degrading proteins and hypothetical proteins reduced the toxic effects of FFA production, allowing for improved growth, physiology, and FFA yields. Comparative transcriptomics, analyzing gene expression changes associated with FFA production and other stress conditions, identified additional key genes involved in cyanobacterial stress response. Conclusions A total of 15 gene targets were identified to reduce the toxic effects of FFA production. While single-gene targeted mutagenesis led to minor increases in FFA production, the combination of these targeted mutations may yield additional improvement, advancing the development of high-energy-density fuels derived from cyanobacteria.

Background The ability to synthesize exopolysaccharides (EPS) is widespread among microorganisms, and microbial EPS play important roles in biofilm formation, pathogen persistence, and applications in the food and medical industries. Although it is well established that EPS synthesis is invariably in response to environmental cues, it remains largely unknown how various environmental signals trigger activation of the biochemical synthesis machinery. Results We report here the transcriptome profiling of Agrobacterium sp. ATCC 31749, a microorganism that produces large amounts of a glucose polymer known as curdlan under nitrogen starvation. Transcriptome analysis revealed a nearly 100-fold upregulation of the curdlan synthesis operon upon transition to nitrogen starvation, thus establishing the prominent role that transcriptional regulation plays in the EPS synthesis. In addition to known mechanisms of EPS regulation such as activation by c-di-GMP, we identify novel mechanisms of regulation in ATCC 31749, including RpoN-independent NtrC regulation and intracellular pH regulation by acidocalcisomes. Furthermore, we show evidence that curdlan synthesis is also regulated by conserved cell stress responses, including polyphosphate accumulation and the stringent response. In fact, the stringent response signal, pppGpp, appears to be indispensible for transcriptional activation of curdlan biosynthesis. Conclusions This study identifies several mechanisms regulating the synthesis of curdlan, an EPS with numerous applications. These mechanisms are potential metabolic engineering targets for improving the industrial production of curdlan from Agrobacterium sp. ATCC 31749. Furthermore, many of the genes identified in this study are highly conserved across microbial genomes, and we propose that the molecular elements identified in this study may serve as universal regulators of microbial EPS synthesis.

Background Successful implementation of modified cyanobacteria as hosts for industrial applications requires the development of a cyanobacterial chassis. The cyanobacterium Synechococcus sp. PCC 7002 embodies key attributes for an industrial host, including a fast growth rate and high salt, light, and temperature tolerances. This study addresses key limitations in the advancement of Synechococcus sp. PCC 7002 as an industrial chassis. Results Tools for genome integration were developed and characterized, including several putative neutral sites for genome integration. The minimum homology arm length for genome integration in Synechococcus sp. PCC 7002 was determined to be approximately 250 bp. Three fluorescent protein reporters (hGFP, Ypet, and mOrange) were characterized for gene expression, microscopy, and flow cytometry applications in Synechococcus sp. PCC 7002. Of these three proteins, the yellow fluorescent protein (Ypet) had the best optical properties for minimal interference with the native photosynthetic pigments and for detection using standard microscopy and flow cytometry optics. Twenty-five native promoters were characterized as tools for recombinant gene expression in Synechococcus sp. PCC 7002 based on previous RNA-seq results. This characterization included comparisons of protein and mRNA levels as well as expression under both continuous and diurnal light conditions. Promoters A2520 and A2579 were found to have strong expression in Synechococcus sp. PCC 7002 while promoters A1930, A1961, A2531, and A2813 had moderate expression. Promoters A2520 and A2813 showed more than twofold increases in gene expression under light conditions compared to dark, suggesting these promoters may be useful tools for engineering diurnal regulation. Conclusions The genome integration, fluorescent protein, and promoter tools developed in this study will help to advance Synechococcus sp. PCC 7002 as a cyanobacterial chassis. The long minimum homology arm length for Synechococcus sp. PCC 7002 genome integration indicates native exonuclease activity or a low efficiency of homologous recombination. Low correlation between transcript and protein levels in Synechococcus sp. PCC 7002 suggests that transcriptomic data are poor selection criteria for promoter tool development. Lastly, the conventional strategy of using promoters from photosynthetic operons as strong promoter tools is debunked, as promoters from hypothetical proteins (A2520 and A2579) were found to have much higher expression levels. Electronic supplementary material The online version of this article (doi:10.1186/s12934-016-0584-6) contains supplementary material, which is available to authorized users.