Protocol to develop a synthetic biology toolkit for the non-model bacterium R. palustris.

Numerous biology tools are developed to work for model organisms, which, however, do not work effectively in non-model organisms. Here, we present a protocol for developing a synthetic biology toolkit for Rhodopseudomonas palustris CGA009, a non-model bacterium with unique metabolic properties. We describe steps for introducing and characterizing biological devices in non-model bacteria, such as the utilization of fluorescence markers and RT-qPCR.

Synthetic Biology Tool Development Advances Predictable Gene Expression in the Metabolically Versatile Soil Bacterium .

Harnessing the unique biochemical capabilities of non-model microorganisms would expand the array of biomanufacturing substrates, process conditions, and products. There are non-model microorganisms that fix nitrogen and carbon dioxide, derive energy from light, catabolize methane and lignin-derived aromatics, are tolerant to physiochemical stresses and harsh environmental conditions, store lipids in large quantities, and produce hydrogen. Model microorganisms often only break down simple sugars and require low stress conditions, but they have been engineered for the sustainable manufacture of numerous products, such as fragrances, pharmaceuticals, cosmetics, surfactants, and specialty chemicals, often by using tools from synthetic biology.

Heterologous phasin expression in CGA009 for bioplastic production from lignocellulosic biomass.

CGA009 is a metabolically robust microbe that can utilize lignin breakdown products to produce polyhydroxyalkanoates (PHAs), biopolymers with the potential to replace conventional plastics. Our recent efforts suggest PHA granule formation is a limiting factor for maximum production of the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by The Phap1 phasin () from the PHB-producing model bacterium H16 was expressed in with the aim of overproducing PHBV from the lignin breakdown product coumarate by fostering smaller and more abundant granules. Expression of yielded PHBV production from aerobically (0.

Synergistic experimental and computational approach identifies novel strategies for polyhydroxybutyrate overproduction.

Polyhydroxybutyrate (PHB) is a sustainable bioplastic produced by bacteria that is a potential replacement for conventional plastics. This study delivers an integrated experimental and computational modeling approach to decipher metabolic factors controlling PHB production and offers engineering design strategies to boost production. In the metabolically robust Rhodopseudomonas palustris CGA009, PHB production significantly increased when grown on the carbon- and electron-rich lignin breakdown product p-coumarate (CHO) compared to virtually no PHB titer from acetate (CHNaO).

One step forward, two steps back: Transcriptional advancements and fermentation phenomena in Actinobacillus succinogenes 130Z.

Within the field of bioproduction, non-model organisms offer promise as bio-platform candidates. Non-model organisms can possess natural abilities to consume complex feedstocks, produce industrially useful chemicals, and withstand extreme environments that can be ideal for product extraction. However, non-model organisms also come with unique challenges due to lack of characterization.

Cationic π-Conjugated Polyelectrolyte Shows Antimicrobial Activity by Causing Lipid Loss and Lowering Elastic Modulus of Bacteria.

Cationic, π-conjugated oligo-/polyelectrolytes (CCOEs/CCPEs) have shown great potential as antimicrobial materials to fight against antibiotic resistance. In this work, we treated wild-type and ampicillin-resistant (amp-resistant) () with a promising cationic, π-conjugated polyelectrolyte (P1) with a phenylene-based backbone and investigated the resulting morphological, mechanical, and compositional changes of the outer membrane of bacteria in great detail. The cationic quaternary amine groups of P1 led to electrostatic interactions with negatively charged moieties within the outer membrane of bacteria.

Mechanistic Understanding of the Interactions of Cationic Conjugated Oligo- and Polyelectrolytes with Wild-type and Ampicillin-resistant Escherichia coli.

An in-depth understanding of cell-drug binding modes and action mechanisms can potentially guide the future design of novel drugs and antimicrobial materials and help to combat antibiotic resistance. Light-harvesting π-conjugated molecules have been demonstrated for their antimicrobial effects, but their impact on bacterial outer cell envelope needs to be studied in detail. Here, we synthesized poly(phenylene) based model cationic conjugated oligo- (2QA-CCOE, 4QA-CCOE) and polyelectrolytes (CCPE), and systematically explored their interactions with the outer cell membrane of wild-type and ampicillin (amp)-resistant Gram-negative bacteria, Escherichia coli (E.

Modeling the Interplay between Photosynthesis, CO Fixation, and the Quinone Pool in a Purple Non-Sulfur Bacterium.

Rhodopseudomonas palustris CGA009 is a purple non-sulfur bacterium that can fix carbon dioxide (CO) and nitrogen or break down organic compounds for its carbon and nitrogen requirements. Light, inorganic, and organic compounds can all be used for its source of energy. Excess electrons produced during its metabolic processes can be exploited to produce hydrogen gas or biodegradable polyesters.

Synthetic Gene Regulation in Cyanobacteria.

Cyanobacteria are appealing hosts for green chemical synthesis due to their use of light and carbon dioxide. To optimize product yields and titers, specific and tunable regulation of the metabolic pathways is needed. Synthetic biology has increased and diversified the genetic tools available for biological process control.

Cyanobacterial carbon metabolism: Fluxome plasticity and oxygen dependence.

Synechocystis sp. strain PCC 6803 has been widely used as a photo-biorefinery chassis. Based on its genome annotation, this species contains a complete TCA cycle, an Embden-Meyerhof-Parnas pathway (EMPP), an oxidative pentose phosphate pathway (OPPP), and an Entner-Doudoroff pathway (EDP).

Physical, chemical, and metabolic state sensors expand the synthetic biology toolbox for Synechocystis sp. PCC 6803.

Many under-developed organisms possess important traits that can boost the effectiveness and sustainability of microbial biotechnology. Photoautotrophic cyanobacteria can utilize the energy captured from light to fix carbon dioxide for their metabolic needs while living in environments not suited for growing crops. Various value-added compounds have been produced by cyanobacteria in the laboratory; yet, the products' titers and yields are often not industrially relevant and lag behind what have been accomplished in heterotrophic microbes.

Oxygen-responsive genetic circuits constructed in Synechocystis sp. PCC 6803.

As photoautotrophic prokaryotes, cyanobacteria are promising platforms for producing value-added bioproducts. However, few regulatory genetic parts and devices (e.g.

Synthetic biology of cyanobacteria: unique challenges and opportunities.

Photosynthetic organisms, and especially cyanobacteria, hold great promise as sources of renewably-produced fuels, bulk and specialty chemicals, and nutritional products. Synthetic biology tools can help unlock cyanobacteria's potential for these functions, but unfortunately tool development for these organisms has lagged behind that for S. cerevisiae and E.

Microbial production of isoprenoids enabled by synthetic biology.

Microorganisms transform inexpensive carbon sources into highly functionalized compounds without toxic by-product generation or significant energy consumption. By redesigning the natural biosynthetic pathways in an industrially suited host, microbial cell factories can produce complex compounds for a variety of industries. Isoprenoids include many medically important compounds such as antioxidants and anticancer and antimalarial drugs, all of which have been produced microbially.