Metabolic engineering of microbes for oligosaccharide and polysaccharide synthesis

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

Metabolic engineering of Agrobacterium sp. strain ATCC 31749 for production of an α-Gal epitope

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p><it>Agrobacterium </it>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 <it>Agrobacterium </it>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.</p> <p>Conclusions</p> <p>The <it>Agrobacterium </it>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 <it>Agrobacterium</it>, with increased lactose uptake and improved UDP-glucose availability, is a promising host for the production of other medically-relevant oligosaccharides.</p

Transcriptome profiling of a curdlan-producing Agrobacterium reveals conserved regulatory mechanisms of exopolysaccharide biosynthesis

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>We report here the transcriptome profiling of <it>Agrobacterium </it>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.</p> <p>Conclusions</p> <p>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 <it>Agrobacterium </it>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.</p

Genetic engineering of cyanobacteria as biodiesel feedstock.

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

Metabolic engineering and omics analysis of Agrobacterium sp. ATCC 31749 for oligosaccharide synthesis

Oligosaccharides are important biomolecules that are targets and also components of many medical treatments, including treatments for cancer, HIV, and inflammation. While the demand for medically-relevant oligosaccharides is increasing, these compounds have proven difficult to synthesize. Whole-cell oligosaccharide synthesis is a promising method that requires relatively inexpensive substrates and can complete the synthesis in just one step. However, whole-cell oligosaccharide synthesis employing common microorganisms like E. coli have been plagued by low yields. This dissertation investigates an alternative microorganism for oligosaccharide production: Agrobacterium sp. ATCC 31749. This Agrobacterium strain produces high levels of curdlan polysaccharide, demonstrating its natural ability to produce the sugar nucleotide precursor for oligosaccharide production. The two main objectives of this dissertation are 1) to develop biocatalysts for oligosaccharide synthesis by engineering ATCC 31749 and 2) to determine what factors affect poly- and oligosaccharide production in this Agrobacterium strain. ATCC 31749 was engineered to produce two oligosaccharides of medical importance: N-acetyllactosamine and galactose-α 1,3-lactose. Oligosaccharide production in the biocatalyst was further improved with additional metabolic engineering. Substrate uptake was increased through expression of a lactose permease, and availability of the sugar nucleotide substrate improved with gene knockout of the curdlan synthase gene. Both of these engineering efforts led to increased oligosaccharide synthesis in the Agrobacterium biocatalyst. Overall, the engineered Agrobacterium strains synthesized gram-scale quantities of the oligosaccharide products in just one step and requiring only a few inexpensive substrates and cofactors. Additional improvement of the oligosaccharide-producing biocatalysts required further investigation of the factors influencing poly- and oligosaccharide production in ATCC 31749. In this dissertation, several environmental and intracellular factors are identified that affect both oligosaccharide and curdlan production. Sucrose was the preferred carbon source for oligosaccharide synthesis, and the addition of citrate to the synthesis reaction led to significant improvement in oligosaccharide production. To identify the genetic factors and possible mechanisms regulating curdlan production, the genome of ATCC 31749 was sequenced. The genome sequence was utilized for transcriptome analysis of ATCC 31749. In the transcriptome analysis, genes significantly up- and down-regulated during curdlan production were identified. Subsequent gene knockout experiments showed several factors to be important for curdlan synthesis, namely the nitrogen signaling cascade, polyphosphate, and the GTP-derived second messengers (p)ppGpp and c-di-GMP. In addition to the development of biocatalysts for oligosaccharide production, this investigation provides insight into the complex mechanisms regulating exopolysaccharide synthesis.Ph.D.Committee Chair: Chen, Rachel; Committee Member: Doyle, Donald; Committee Member: Grover, Martha; Committee Member: Prausntiz, Mark; Committee Member: Spain, Ji

Cyanobacteria: The Green E. 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, utilizing CO2 and sunlight as the main nutrient requirements. In addition to their potential for reducing greenhouse gas emissions and lowering production costs, cyanobacteria have naturally efficient pathways for the production metabolites such as carotenoids, which are of importance in the nutraceutical industry. 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 ‘green E. coli’. This Research Topic will focus on cyanobacteria as organisms of emerging industrial relevance, including research focused on the development of genetic tools for cyanobacteria, the investigation of new cyanobacterial strains, the construction of novel cyanobacterial strains via genetic engineering, the application of ‘omics’ tools to advance the understanding of engineered cyanobacteria, and the development of computational models for cyanobacterial strain development