Marika Ziesack

Water splitting–biosynthetic system with CO2 reduction efficiencies exceeding photosynthesis

Artificial photosynthesis steps up Photosynthesis fixes CO2 from the air by using sunlight. Industrial mimics of photosynthesis seek to convert CO2 directly into biomass, fuels, or other useful products. Improving on a previous artificial photosynthesis design, Liu et al. combined the hydrogen-oxidizing bacterium Raistonia eutropha with a cobalt-phosphorus water-splitting catalyst. This biocompatible self-healing electrode circumvented the toxicity challenges of previous designs and allowed it to operate aerobically. When combined with solar photovoltaic cells, solar-to-chemical conversion rates should become nearly an order of magnitude more efficient than natural photosynthesis. Science, this issue p. 1210 A biocompatible catalyst in an artificial photosynthesis system leads to efficient solar-to-fuel conversion. Artificial photosynthetic systems can store solar energy and chemically reduce CO2. We developed a hybrid water splitting–biosynthetic system based on a biocompatible Earth-abundant inorganic catalyst system to split water into molecular hydrogen and oxygen (H2 and O2) at low driving voltages. When grown in contact with these catalysts, Ralstonia eutropha consumed the produced H2 to synthesize biomass and fuels or chemical products from low CO2 concentration in the presence of O2. This scalable system has a CO2 reduction energy efficiency of ~50% when producing bacterial biomass and liquid fusel alcohols, scrubbing 180 grams of CO2 per kilowatt-hour of electricity. Coupling this hybrid device to existing photovoltaic systems would yield a CO2 reduction energy efficiency of ~10%, exceeding that of natural photosynthetic systems.

Better together: engineering and application of microbial symbioses.

Symbioses provide a way to surpass the limitations of individual microbes. Natural communities exemplify this in symbioses like lichens and biofilms that are robust to perturbations, an essential feature in fluctuating environments. Metabolic capabilities also expand in consortia enabling the division of labor across organisms as seen in photosynthetic and methanogenic communities. In engineered consortia, the external environment provides levers of control for microbes repurposed from nature or engineered to interact through synthetic biology. Consortia have successfully been applied to real-world problems including remediation and energy, however there are still fundamental questions to be answered. It is clear that continued study is necessary for the understanding and engineering of microbial systems that are more than the sum of their parts.

Expression of the sub-pathways of the Chloroflexus aurantiacus 3-hydroxypropionate carbon fixation bicycle in E. coli: Toward horizontal transfer of autotrophic growth

The 3-hydroxypropionate (3-HPA) bicycle is unique among CO2-fixing systems in that none of its enzymes appear to be affected by oxygen. Moreover, the bicycle includes a number of enzymes that produce novel intermediates of biotechnological interest, and the CO2-fixing steps in this pathway are relatively rapid. We expressed portions of the 3-HPA bicycle in a heterologous organism, E. coli K12. We subdivided the 3-HPA bicycle into four sub-pathways: (1) synthesis of propionyl-CoA from acetyl-CoA, (2) synthesis of succinate from propionyl-CoA, (3) glyoxylate production and regeneration of acetyl-CoA, and (4) assimilation of glyoxylate and propionyl-CoA to form pyruvate and regenerate acetyl-CoA. We expressed the novel enzymes of the 3-HPA bicycle in operon form and used phenotypic tests for activity. Sub-pathway 1 activated a propionate-specific biosensor. Sub-pathway 2, found in non-CO2-fixing bacteria, was reassembled in E. coli using genes from diverse sources. Sub-pathway 3, operating in reverse, generated succinyl-CoA sufficient to rescue a sucAD(-) double mutant of its diaminopimelic acid (DAP) auxotrophy. Sub-pathway 4 was able to reduce the toxicity of propionate and allow propionate to contribute to cell biomass in a prpC(-)(2 methylcitrate synthase) mutant strain. These results indicate that all of the sub-pathways of the 3-HPA bicycle can function to some extent in vivo in a heterologous organism, as indicated by growth tests. Overexpression of certain enzymes was deleterious to cell growth, and, in particular, expression of MMC-CoA lyase caused a mucoid phenotype. These results have implications for metabolic engineering and for bacterial evolution through horizontal gene transfer.

Production of fatty acids in Ralstonia eutropha H16 by engineering β-oxidation and carbon storage

Ralstonia eutropha H16 is a facultatively autotrophic hydrogen-oxidizing bacterium capable of producing polyhydroxybutyrate (PHB)-based bioplastics. As PHB’s physical properties may be improved by incorporation of medium-chain-length fatty acids (MCFAs), and MCFAs are valuable on their own as fuel and chemical intermediates, we engineered R. eutropha for MCFA production. Expression of UcFatB2, a medium-chain-length-specific acyl-ACP thioesterase, resulted in production of 14 mg/L laurate in wild-type R. eutropha. Total fatty acid production (22 mg/L) could be increased up to 2.5-fold by knocking out PHB synthesis, a major sink for acetyl-CoA, or by knocking out the acyl-CoA ligase fadD3, an entry point for fatty acids into β-oxidation. As ΔfadD3 mutants still consumed laurate, and because the R. eutropha genome is predicted to encode over 50 acyl-CoA ligases, we employed RNA-Seq to identify acyl-CoA ligases upregulated during growth on laurate. Knockouts of the three most highly upregulated acyl-CoA ligases increased fatty acid yield significantly, with one strain (ΔA2794) producing up to 62 mg/L free fatty acid. This study demonstrates that homologous β-oxidation systems can be rationally engineered to enhance fatty acid production, a strategy that may be employed to increase yield for a range of fuels, chemicals, and PHB derivatives in R. eutropha.

Phenotypic memory in Bacillus subtilis links dormancy entry and exit by a spore quantity-quality tradeoff

Some bacteria, such as Bacillus subtilis, withstand starvation by forming dormant spores that revive when nutrients become available. Although sporulation and spore revival jointly determine survival in fluctuating environments, the relationship between them has been unclear. Here we show that these two processes are linked by a phenotypic “memory” that arises from a carry-over of molecules from the vegetative cell into the spore. By imaging life histories of individual B. subtilis cells using fluorescent reporters, we demonstrate that sporulation timing controls nutrient-induced spore revival. Alanine dehydrogenase contributes to spore memory and controls alanine-induced outgrowth, thereby coupling a spore’s revival capacity to the gene expression and growth history of its progenitors. A theoretical analysis, and experiments with signaling mutants exhibiting altered sporulation timing, support the hypothesis that such an intrinsically generated memory leads to a tradeoff between spore quantity and spore quality, which could drive the emergence of complex microbial traits.Bacillus subtilis withstands starvation by forming dormant spores that revive when nutrients become available. Here, Mutlu et al. show that sporulation timing controls spore revival through a phenotypic ‘memory’ that arises from the carry-over of a metabolic enzyme from the vegetative cell into the spore.

A synthetic bacterial information transfer system functions in the mammalian gut

The gut microbiome is intricately involved with establishing and maintaining the health of the host. Engineering of gut microbes aims to add new functions and expand the scope of control over the gut microbiome. To create systems that can perform increasingly complex tasks in the gut with multiple engineered strains it is necessary to program communication among these bacteria in the gut. Towards this goal, we engineered an information transfer system for inter-cellular communication, using native gut Escherichia coli and attenuated Salmonella enterica serovar Typhimurium. Specifically, we have taken two genetic circuits-one for signaling from the quorum sensing system and the other for memory from the bacteriophage genetic switch–and integrated them into a robust system that can report on successful communication in the mammalian gut. Our system provides a basis for the construction of a programmable gut consortia as well as a basis for further understanding of bacterial interactions in an otherwise hard-to-study environment.

Automatic Single-Cell Segmentation and Tracking of Bacterial Cells in Fluorescence Microscopy Images

Automatic single-cell image analysis allows gaining deeper insights into biological processes. We present an approach for single-cell segmentation and tracking of bacterial cells in time-lapse microscopy image data. For cell segmentation we use linear feature detection and a probability map combined with schemes for cell splitting. For cell tracking we propose an approach based on the maximal overlapping area between cells, which is robust regarding cell rotation and accurately detects cell divisions. Our approach was successfully applied to segment and track cells in time-lapse images of the life cycle of Bacillus subtilis. We also quantitatively evaluated the performance of the segmentation and tracking approaches.

Inducible Cooperation in a Synthetic Gut Bacterial Consortium Introduces Population Balance and Stability

Abstract In nature, microbes interact antagonistically, neutrally or beneficially. To shed light on the effects of positive interactions in microbial consortia we introduced metabolic dependencies and metabolite overproduction into four bacterial species. While antagonistic interactions govern the wildtype consortium behavior, the genetic modifications alleviated antagonistic interactions and resulted in beneficial interactions. Engineered cross-feeding increased population evenness, a component of ecological diversity, in different environments including in a more complex gnotobiotic mouse gut environment. Our findings suggest that metabolite cross-feeding could be used as a tool for intentionally shaping microbial consortia in complex environments. Importance Microbial communities are ubiquitous in nature. Bacterial consortia live in and on our body and in our environment and more recently, biotechnology is applying microbial consortia for bioproduction. As part of our body, bacterial consortia influence us in health and disease. Microbial consortia function is determined by its composition, which in turn is driven by the interactions between species. Further understanding of microbial interactions will help us deciphering how consortia function in complex environments and may enable us to modify microbial consortia for health and environmental benefits.

Chimeric Fatty Acyl-Acyl Carrier Protein Thioesterases Provide Mechanistic Insight into Enzyme Specificity and Expression

ABSTRACT Medium-chain fatty acids are commodity chemicals. Increasing and modifying the activity of thioesterases (TEs) on medium-chain fatty acyl-acyl carrier protein (acyl-ACP) esters may enable a high-yield microbial production of these molecules. The plant Cuphea palustris harbors two distinct TEs: C. palustris FatB1 (CpFatB1) (C8 specificity, lower activity) and CpFatB2 (C14 specificity, higher activity) with 78% sequence identity. We combined structural features from these two enzymes to create several chimeric TEs, some of which showed nonnatural fatty acid production as measured by an enzymatic assay and gas chromatography-mass spectrometry (GC-MS). Notably, chimera 4 exhibited an increased C8 fatty acid production in correlation with improved microbial expression. This chimera led us to identify CpFatB2-specific amino acids between positions 219 and 272 that lead to higher protein levels. Chimera 7 produced a broad range of fatty acids and appeared to combine a fatty acid binding pocket with long-chain specificity and an ACP interaction site that may activate fatty acid extrusion. Using homology modeling and in silico docking with ACP, we identified a “positive patch” within amino acids 162 to 218, which may direct the ACP interaction and regulate access to short-chain fatty acids. On the basis of this modeling, we transplanted putative ACP interaction sequences from CpFatB1 into CpFatB2 and created a chimeric thioesterase that produced medium-chain as well as long-chain fatty acids. Thus, the engineering of chimeric enzymes and characterizing their microbial activity and chain-length specificity suggested mechanistic insights into TE functions and also generated thioesterases with potentially useful properties. These observations may inform a rational engineering of TEs to allow alkyl chain length control. IMPORTANCE Medium-chain fatty acids are important commodity chemicals. These molecules are used as plastic precursors and in shampoos and other detergents and could be used as biofuel precursors if production economics were favorable. Hydrocarbon-based liquid fuels must be optimized to have a desired boiling point, low freezing point, low viscosity, and other physical characteristics. Similarly, the solubility and harshness of detergents and the flexibility of plastic polymers can be modulated. The length and distribution of the carbon chains in the hydrophobic tails determine these properties. The biological synthesis of cell membranes and fatty acids produces chains of primarily 16 to 18 carbons, which give rise to current biofuels. The ultimate goal of the work presented here is to engineer metabolic pathways to produce designer molecules with the correct number of carbons in a chain, so that such molecules could be used directly as specialty commodity chemicals or as fuels after minimal processing.

Quorum Sensing Can Be Repurposed To Promote Information Transfer between Bacteria in the Mammalian Gut

The gut microbiome is intricately involved with establishing and maintaining the health of the host. Engineering of gut microbes aims to add new functions and expand the scope of control over the gut microbiome. To create systems that can perform increasingly complex tasks in the gut, it is necessary to harness the ability of the bacteria to communicate in the gut environment. Interestingly, acyl-homoserine lactone (acyl-HSL)-mediated Gram-negative bacterial quorum sensing, a widely used mode of intercellular signaling system in nature, has not been identified in normal healthy mammalian gut. It remains unknown whether the gut bacteria that do not natively use quorum sensing can be engineered to successfully signal to other bacteria using acyl-HSLs in the gut environment. Here, we repurposed quorum sensing to create an information transfer system between native gut Escherichia coli and attenuated Salmonella enterica serovar Typhimurium. Specifically, we functionalized one species with inducible signal production and the other with signal detection and recording using genomically integrated circuits. The information transfer system demonstrated successful intra- and interspecies signaling in the murine gut. This study provides a basis for further understanding of interbacterial interactions in an otherwise hard-to-study environment as well as a basis for further investigation of the potential of acyl-HSLs as intercellular signaling molecules of engineered gut consortia.