Secure biosystems design in establishes effective biocontainment strategies and mechanisms of escape.

The widespread application of recombinant DNA and synthetic biology approaches for microbial metabolic engineering pursuits has motivated the development of biocontainment strategies, targeting safe and secure deployment of genetically modified microorganisms (GMMs). However, the design rules and mechanistic drivers governing biocontainment efficacy, as well as impacts of biocontainment upon microbial fitness, remain to be comprehensively evaluated, hindering predictive design and application of these strategies. We have developed a platform for high-resolution analysis of a transactivated kill switch in laboratory and industrial strains of to assess modes of biocontainment escape and establish design rules for development of kill switch systems in diverse microbes.

Tailoring microbial fitness through computational steering and CRISPRi-driven robustness regulation.

The widespread application of genetically modified microorganisms (GMMs) across diverse sectors underscores the pressing need for robust strategies to mitigate the risks associated with their potential uncontrolled escape. This study merges computational modeling with CRISPR interference (CRISPRi) to refine GMM metabolic robustness. Utilizing ensemble modeling, we achieved high-throughput in silico screening for enzymatic targets susceptible to expression alterations.

Small cells with big photosynthetic productivities: biotechnological potential of the Picochlorum genus.

The Picochlorum genus is a distinctive eukaryotic green-algal clade that is the focus of several current biotechnological studies. It is capable of extremely rapid growth rates and has exceptional tolerances to high salinity, intense light, and elevated temperatures. Importantly, it has robust stability and high-biomass productivities in outdoor field trials in seawater.

Cyanobacteria newly isolated from marine volcanic seeps display rapid sinking and robust, high-density growth.

Unlabelled: Cyanobacteria are photosynthetic organisms that play important roles in carbon cycling and are promising bioproduction chassis. Here, we isolate two novel cyanobacteria with 4.6Mbp genomes, UTEX 3221 and UTEX 3222, from a unique marine environment with naturally elevated CO₂.

Polyphosphate kinase deletion increases laboratory productivity in cyanobacteria.

Identification and manipulation of cellular energy regulation mechanisms may be a strategy to increase productivity in photosynthetic organisms. This work tests the hypothesis that polyphosphate synthesis and degradation play a role in energy management by storing or dissipating energy in the form of ATP. A polyphosphate kinase () knock-out strain unable to synthesize polyphosphate was generated in the cyanobacterium sp.

Proximate biomass characterization of the high productivity marine microalga Picochlorum celeri TG2.

Microalgae are compelling renewable resources with applications including biofuels, bioplastics, nutrient supplements, and cosmetic products. Picochlorum celeri is an alga with high industrial interest due to exemplary outdoor areal biomass productivities in seawater. Detailed proximate analysis is needed in multiple environmental conditions to understand the dynamic biomass compositions of P.

Risk Assessment of Industrial Microbes Using a Terrestrial Mesocosm Platform.

Industrial microbes and bio-derived products have emerged as an integral component of the bioeconomy, with an array of agricultural, bioenergy, and biomedical applications. However, the rapid development of microbial biotechnology raises concerns related to environmental escape of laboratory microbes, detection and tracking thereof, and resultant impact upon native ecosystems. Indeed, though wild-type and genetically modified microbes are actively deployed in industrial bioprocesses, an understanding of microbial interactivity and impact upon the environment is severely lacking.

Heterologous expression of formate dehydrogenase enables photoformatotrophy in the emerging model microalga, .

Rising global greenhouse gas emissions and the impacts of resultant climate change necessitate development and deployment of carbon capture and conversion technologies. Amongst the myriad of bio-based conversion approaches under evaluation, a formate bio-economy has recently been proposed, wherein CO-derived formate serves as a substrate for concurrent carbon and energy delivery to microbial systems. To date, this approach has been explored in chemolithotrophic and heterotrophic organisms via native or engineered formatotrophy.

Metabolic engineering of Synechococcus elongatus 7942 for enhanced sucrose biosynthesis.

The capability of cyanobacteria to produce sucrose from CO and light has a remarkable societal and biotechnological impact since sucrose can serve as a carbon and energy source for a variety of heterotrophic organisms and can be converted into value-added products. However, most metabolic engineering efforts have focused on understanding local pathway alterations that drive sucrose biosynthesis and secretion in cyanobacteria rather than analyzing the global flux re-routing that occurs following induction of sucrose production by salt stress. Here, we investigated global metabolic flux alterations in a sucrose-secreting (cscB-overexpressing) strain relative to its wild-type Synechococcus elongatus 7942 parental strain.

Improved Combinatorial Assembly and Barcode Sequencing for Gene-Sized DNA Constructs.

Synergistic and supportive interactions among genes can be incorporated in engineering biology to enhance and stabilize the performance of biological systems, but combinatorial numerical explosion challenges the analysis of multigene interactions. The incorporation of DNA barcodes to mark genes coupled with next-generation sequencing offers a solution to this challenge. We describe improvements for a key method in this space, CombiGEM, to broaden its application to assembling typical gene-sized DNA fragments and to reduce the cost of sequencing for prevalent small-scale projects.

Biocontainment of Genetically Engineered Algae.

Algae (including eukaryotic microalgae and cyanobacteria) have been genetically engineered to convert light and carbon dioxide to many industrially and commercially relevant chemicals including biofuels, materials, and nutritional products. At industrial scale, genetically engineered algae may be cultivated outdoors in open ponds or in closed photobioreactors. In either case, industry would need to address a potential risk of the release of the engineered algae into the natural environment, resulting in potential negative impacts to the environment.

Quantification of Metabolic Products from Microbial Hosts in Complex Media Using Optically Diffracting Hydrogels.

We herein describe a highly versatile platform approach for the in situ and real-time screening of microbial biocatalysts for enhanced production of bioproducts using photonic crystal hydrogels. This approach was demonstrated by preparing optically diffracting films based on polymerized -isopropylacrylamide that contracted in the presence of alcohols and organic acids. The hydrogel films were prepared in a microwell plate format, which allows for high-throughput screening, and characterized optically using a microwell plate reader.

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RubisCO) Is Essential for Growth of the Methanotroph Methylococcus capsulatus Strain Bath.

The ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) enzyme found in plants, algae, and an array of autotrophic bacteria is also encoded by a subset of methanotrophs, but its role in these microbes has largely remained elusive. In this study, we showed that CO was requisite for RubisCO-encoding Methylococcus capsulatus strain Bath growth in a bioreactor with continuous influent and effluent gas flow. RNA sequencing identified active transcription of several carboxylating enzymes, including key enzymes of the Calvin and serine cycles, that could mediate CO assimilation during cultivation with both CH and CO as carbon sources.

Biotechnology for secure biocontainment designs in an emerging bioeconomy.

Genetically modified organisms (GMOs) have emerged as an integral component of a sustainable bioeconomy, with an array of applications in agriculture, bioenergy, and biomedicine. However, the rapid development of GMOs and associated synthetic biology approaches raises a number of biosecurity concerns related to environmental escape of GMOs, detection thereof, and impact upon native ecosystems. A myriad of genetic safeguards have been deployed in diverse microbial hosts, ranging from classical auxotrophies to global genome recoding.

A multi-omic characterization of temperature stress in a halotolerant Scenedesmus strain for algal biotechnology.

Microalgae efficiently convert sunlight into lipids and carbohydrates, offering bio-based alternatives for energy and chemical production. Improving algal productivity and robustness against abiotic stress requires a systems level characterization enabled by functional genomics. Here, we characterize a halotolerant microalga Scenedesmus sp.

Synthetic microbial communities of heterotrophs and phototrophs facilitate sustainable growth.

Microbial communities comprised of phototrophs and heterotrophs hold great promise for sustainable biotechnology. Successful application of these communities relies on the selection of appropriate partners. Here we construct four community metabolic models to guide strain selection, pairing phototrophic, sucrose-secreting Synechococcus elongatus with heterotrophic Escherichia coli K-12, Escherichia coli W, Yarrowia lipolytica, or Bacillus subtilis.

Methods for Algal Protein Isolation and Proteome Analysis.

Microalgae present promising feedstocks to produce renewable fuel and chemical intermediates, in part due to high storage carbon flux capacity to triacylglycerides or storage carbohydrates upon nutrient deprivation. However, the mechanism(s) governing deprivation-mediated carbon partitioning remain to be fully elucidated, limiting targeted strain engineering strategies in algal biocatalysts. Though genomic and transcriptomic analyses offer key insights into these mechanisms, active post-transcriptional regulatory mechanisms, ubiquitous in many microalgae, necessitate proteomic and post-translational (e.

Development of a high-productivity, halophilic, thermotolerant microalga .

Microalgae are promising biocatalysts for applications in sustainable fuel, food, and chemical production. Here, we describe culture collection screening, down-selection, and development of a high-productivity, halophilic, thermotolerant microalga, . This microalga displays a rapid growth rate and high diel biomass productivity (34 g m day), with a composition well-suited for downstream processing.

Development of a CRISPR/Cas9 System for Methylococcus capsulatus Gene Editing.

Methanotrophic bacteria play a crucial role in the Earth's biogeochemical cycle and have the potential to be employed in industrial biomanufacturing processes due to their capacity to use natural gas- and biogas-derived methane as a sole carbon and energy source. Advanced gene-editing systems have the potential to enable rapid, high-throughput methanotrophic genetics and biocatalyst development. To this end, we employed a series of broad-host-range expression plasmids to construct a conjugatable lustered egularly nterspaced hort alindromic epeats (CRISPR)/Cas9 gene-editing system in (Bath).

Direct Writing of Tunable Living Inks for Bioprocess Intensification.

Critical to the success of three-dimensional (3D) printing of living materials with high performance is the development of new ink materials and 3D geometries that favor long-term cell functionality. Here we report the use of freeze-dried live cells as the solid filler to enable a new living material system for direct ink writing of catalytically active microorganisms with tunable densities and various self-supporting porous 3D geometries. Baker's yeast was used as an exemplary live whole-cell biocatalyst, and the printed structures displayed high resolution, large scale, high catalytic activity and long-term viability.

Down-Selection and Outdoor Evaluation of Novel, Halotolerant Algal Strains for Winter Cultivation.

Algae offer promising feedstocks for the production of renewable fuel and chemical intermediates. However, poor outdoor winter cultivation capacity currently limits deployment potential. In this study, 300 distinct algal strains were screened in saline medium to determine their cultivation suitability during winter conditions in Mesa, Arizona.

Biogas Biocatalysis: Methanotrophic Bacterial Cultivation, Metabolite Profiling, and Bioconversion to Lactic Acid.

Anaerobic digestion (AD) of waste substrates, and renewable biomass and crop residues offers a means to generate energy-rich biogas. However, at present, AD-derived biogas is primarily flared or used for combined heat and power (CHP), in part due to inefficient gas-to-liquid conversion technologies. Methanotrophic bacteria are capable of utilizing methane as a sole carbon and energy source, offering promising potential for biological gas-to-liquid conversion of AD-derived biogas.

Combined Effects of Carbon and Nitrogen Source to Optimize Growth of Proteobacterial Methanotrophs.

Methane, a potent greenhouse gas, and methanol, commonly called wood alcohol, are common by-products of modern industrial processes. They can, however, be consumed as a feedstock by bacteria known as methanotrophs, which can serve as useful vectors for biotransformation and bioproduction. Successful implementation in industrial settings relies upon efficient growth and bioconversion, and the optimization of culturing conditions for these bacteria remains an ongoing effort, complicated by the wide variety of characteristics present in the methanotroph culture collection.

Different Behaviors of a Substrate in P450 Decarboxylase and Hydroxylase Reveal Reactivity-Enabling Actors.

Biological routes to the production of fuels from renewable feedstocks hold significant promise in our efforts towards a sustainable future. The fatty acid decarboxylase enzyme (OleT) is a cytochrome P450 enzyme that converts long and medium chain fatty acids to terminal alkenes and shares significant similarities in terms of structure, substrate scope and mechanism with the hydroxylase cytochrome P450 (P450). Recent reports have demonstrated that catalytic pathways in these enzymes bifurcate when the heme is in its iron-hydroxo (compound II) state.