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, Picochlorum renovo. This microalga displays a rapid growth rate and high diel biomass productivity (34 g m⁻² day⁻¹), with a composition well-suited for downstream processing. P. renovo exhibits broad salinity tolerance (growth at 107.5 g L⁻¹ salinity) and thermotolerance (growth up to 40 °C), beneficial traits for outdoor cultivation. We report complete genome sequencing and analysis, and genetic tool development suitable for expression of transgenes inserted into the nuclear or chloroplast genomes. We further evaluate mechanisms of halotolerance via comparative transcriptomics, identifying novel genes differentially regulated in response to high salinity cultivation. These findings will enable basic science inquiries into control mechanisms governing Picochlorum biology and lay the foundation for development of a microalga with industrially relevant traits as a model photobiology platform.

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 clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene-editing system in Methylococcus capsulatus (Bath). Heterologous coexpression of the Streptococcus pyogenes Cas9 endonuclease and a synthetic single guide RNA (gRNA) showed efficient Cas9 DNA targeting and double-stranded DNA (dsDNA) cleavage that resulted in cell death. We demonstrated effective in vivo editing of plasmid DNA using both Cas9 and Cas9^D10A nickase to convert green fluorescent protein (GFP)- to blue fluorescent protein (BFP)-expressing cells with 71% efficiency. Further, we successfully introduced a premature stop codon into the soluble methane monooxygenase (sMMO) hydroxylase component-encoding mmoX gene with the Cas9^D10A nickase, disrupting sMMO function. These data provide proof of concept for CRISPR/Cas9-mediated gene editing in M. capsulatus. Given the broad-host-range replicons and conjugation capability of these CRISPR/Cas9 tools, they have potential utility in other methanotrophs and a wide array of Gram-negative microorganisms. IMPORTANCE In this study, we targeted the development and evaluation of broad-host-range CRISPR/Cas9 gene-editing tools in order to enhance the genetic-engineering capabilities of an industrially relevant methanotrophic biocatalyst. The CRISPR/Cas9 system developed in this study expands the genetic tools available to define molecular mechanisms in methanotrophic bacteria and has the potential to foster advances in the generation of novel biocatalysts to produce biofuels, platform chemicals, and high-value products from natural gas- and biogas-derived methane. Further, due to the broad-host-range applicability, these genetic tools may also enable innovative approaches to overcome the barriers associated with genetically engineering diverse, industrially promising nonmodel microorganisms.

Here, we demonstrate bioconversion of methane to muconic acid, a dicarboxylic acid that can be upgraded to an array of platform chemicals, by three gammaproteobacterial methanotrophs. All engineered methanotrophs expressing a heterologous dihydroxyshikimate dehydratase, protocatechuate decarboxylase, and catechol dioxygenase produced muconic acid from methane, with the highest titer (12.4 mg MA per L), yield (2.8 mg MA per g CH₄), and specific productivity (1.2 mg MA per g dcw, 48 hr) synthesized by Methylotuvimicrobium buryatense, Methylococcus capsulatus, and Methylotuvimicrobium alcaliphilium, respectively. Methylotuvimicrobium alcaliphilum genome-scale model-guided strain engineering predicted that disruption of the pyruvate dehydrogenase or shikimate dehydrogenase would significantly enhance flux to the heterologous muconic acid pathway in this organism. However, knock-out of these targets caused a growth defect, and coupled with similar muconic acid titers (∼1 mg L⁻¹), resulted in minimal flux enhancement to muconic acid in these genetically-modified strains. The shikimate dehydrogenase mutant's ability to grow without aromatic amino acid supplementation revealed that M. alcaliphilum likely encodes an unidentified enzyme or pathway with shikimate biosynthetic capacity, which prevents maximal flux through the synthetic muconic acid pathway. This study expands the suite of products that can be generated from methane using methanotrophic biocatalysts, lays the foundation for green production of muconic acid-derived polymers from methane, and highlights the need for further analysis of methanotroph biosynthetic potential to guide refinement of metabolic models and strain engineering.

The metabolic processes that enable the replication of intracellular Salmonella under nitrosative stress conditions engendered in the innate response of macrophages are poorly understood. A screen of Salmonella transposon mutants identified the ABC-type high-affinity zinc uptake system ZnuABC as a critical determinant of the adaptation of Salmonella to the nitrosative stress generated by the enzymatic activity of inducible nitric oxide (NO) synthase of mononuclear phagocytic cells. NO limits the virulence of a znuB mutant in an acute murine model of salmonellosis. The ZnuABC transporter is crucial for the glycolytic function of fructose bisphosphate aldolase, thereby fueling growth of Salmonella during nitrosative stress produced in the innate response of macrophages. Our investigations demonstrate that glycolysis mediates resistance of Salmonella to the antimicrobial activity of NO produced in an acute model of infection. The ATP synthesized by substrate-level phosphorylation at the payoff phase of glycolysis and acetate fermentation powers the replication of Salmonella experiencing high levels of nitrosative stress. In contrast, despite its high potential for ATP synthesis, oxidative phosphorylation is a major target of inhibition by NO and contributes little to the antinitrosative defenses of intracellular Salmonella. Our investigations have uncovered a previously unsuspected conjunction between zinc homeostasis, glucose metabolism and cellular energetics in the adaptation of intracellular Salmonella to the reactive nitrogen species synthesized in the innate host response. Microbial pathogens are exposed to multiple antimicrobial defenses during their associations with host cells. Nitric oxide generated in the innate response exerts widespread antimicrobial activity against a variety of pathogenic microorganisms. Nitric oxide has high affinity for metal groups of terminal cytochromes of the respiratory chain, and thus nitrosative stress exerts extreme deleterious actions against the cellular energetics that rely on oxidative phosphorylation. Intracellular Salmonella have resolved this dilemma by satisfying a significant portion of their energetic demands via substrate level phosphorylation in the payoff phase of glycolysis and acetate fermentation. A high affinity zinc uptake system promotes antinitrosative defense of intracellular Salmonella by in great part supporting the enzymatic activity of an essential enzyme in the preparatory phase of glycolysis. Our research provides novel insights into the metabolic and energetic adaptations that allow a bacterial pathogen to thrive in the midst of the innate host response of vertebrate cells.

The importance of RubisCO and CO ₂ in M. capsulatus Bath metabolism is unclear. In this study, we demonstrated that both CO ₂ and RubisCO are essential for M. capsulatus Bath growth. ¹³ CO ₂ tracing experiments supported that RubisCO mediates CO ₂ fixation and that a noncanonical Calvin cycle is active in this organism.

The genus Salmonella is responsible for many illnesses in humans and other vertebrate animals. We report here that Salmonella enterica serovar Typhimurium harbors three transketolases that support the non-oxidative branch of the pentose phosphate pathway. BLAST analysis identified two genes, STM14_2885 and STM14_2886, that together encode a putative transketolase (TktC) with 46–47% similarity to the known TktA and TktB isoforms. Assessing the mRNA and protein expression for each of the three transketolases, we determined that all are expressed in WT cells and regulated to varying extents by the alternative sigma factor RpoS. Enzyme assays with lysates from WT and transketolase-knockout strains established that TktA is responsible for >88% of the transketolase activity in WT cells. We purified recombinant forms of each isoenzyme to assess the kinetics for canonical transketolase reactions. TktA and TktB had comparable values for V_max (539–1362 μm NADH consumed/s), K_m (80–739 μm), and catalytic efficiency (1.02 × 10⁸-1.06 × 10⁹ m⁻¹/s) for each substrate tested. The recombinant form of TktC had lower K_m values (23–120 μm), whereas the V_max (7.8–16 μm NADH consumed/s) and catalytic efficiency (5.58 × 10⁶ to 6.07 × 10⁸ m⁻¹/s) were 10–100-fold lower. Using a murine model of Salmonella infection, we showed that a strain lacking all three transketolases is avirulent in C57BL/6 mice. These data provide evidence that S. Typhimurium possesses three transketolases that contribute to pathogenesis.