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 (ppk) knock-out strain unable to synthesize polyphosphate was generated in the cyanobacterium Synechocystis sp. PCC 6803. This mutant strain demonstrated higher ATP levels and faster growth than the wildtype strain in high-carbon conditions and had a growth defect under multiple stress conditions. In a strain that combined ppk deletion with heterologous expression of ethylene-forming enzyme, higher ethylene productivity was observed than in the wildtype background. These results support the role of polyphosphate synthesis and degradation as an energy regulation mechanism and suggest that such mechanisms may be effective targets in biocontainment design.

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. celeri, and how these compositions might be leveraged in biotechnological applications. In this study, biomass characterization of P. celeri was performed under nutrient-replete, nitrogen-restricted, and hyper-saline conditions. Nutrient-replete cultivation of P. celeri resulted in protein-rich biomass (∼50% ash-free dry weight) with smaller carbohydrate (∼12% ash-free dry weight) and lipid (∼11% ash-free dry weight) partitions. Gradual nitrogen depletion elicited a shift from proteins to carbohydrates (∼50% ash-free dry weight, day 3) as cells transitioned into the production of storage metabolites. Importantly, dilutions in nitrogen-restricted 40 parts per million (1.43 mM nitrogen) media generated high-carbohydrate (∼50% ash-free dry weight) biomass without substantially compromising biomass productivity (36 g ash-free dry weight m-2 day-1) despite decreased chlorophyll (∼2% ash-free dry weight) content. This strategy for increasing carbohydrate content allowed for the targeted production of polysaccharides, which could potentially be utilized to produce fuels, oligosaccharides, and bioplastics. Cultivation at 2X sea salts resulted in a shift towards carbohydrates from protein, with significantly increased levels of the amino acid proline, which putatively acts as an osmolyte. A detailed understanding of the biomass composition of P. celeri in nutrient-replete, nitrogen-restricted, and hyper saline conditions informs how this strain can be useful in the production of biotechnological products.

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. In particular, the persistence and sustained ecosystem impact of industrial microbes following laboratory release or unintentional laboratory escape remains largely unexplored. Herein, we investigate the applicability of soil-sorghum mesocosms for the ecological risk assessment of the industrial microbe, Saccharomyces cerevisiae. We developed and applied a suite of diagnostic and bioinformatic analyses, including digital droplet PCR, microscopy, and phylogenomic analyses to assess the impacts of a terrestrial ecosystem perturbation event over a 30-day time course. The platform enables reproducible, high-sensitivity tracking of S. cerevisiae in a complex soil microbiome and analysis of the impact upon abiotic soil characteristics and soil microbiome population dynamics and diversity. The resultant data indicate that even though S. cerevisiae is relatively short-lived in the soil, a single perturbation event can have sustained impact upon mesocosm soil composition and underlying microbial populations in our system, underscoring the necessity for more comprehensive risk assessment and development of mitigation and biocontainment strategies in industrial bioprocesses.

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

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 CO2-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. However, utilization of this concept in phototrophic organisms has yet to be reported. Herein, we have taken the first steps to establish formate utilization in Picochlorum renovo, a recently characterized eukaryotic microalga with facile genetic tools and promising applied biotechnology traits. Plastidial heterologous expression of a formate dehydrogenase (FDH) enabled P. renovo growth on formate as a carbon and energy source. Further, FDH expression enhanced cultivation capacity on ambient CO2, underscoring the potential for bypass of conventional CO2 capture and concentration limitations. This work establishes a photoformatotrophic cultivation regime that leverages light energy-driven formate utilization. The resultant photosynthetic formate platform has widespread implications for applied phototrophic cultivation systems and the bio-economy at large.

Metabolic engineering of Synechococcus elongatus 7942 for enhanced sucrose biosynthesis.

The capability of cyanobacteria to produce sucrose from CO2 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. We used targeted metabolomics, 13C metabolic flux analysis (MFA), and genome-scale modeling (GSM) as complementary approaches to elucidate differences in cellular resource allocation by quantifying metabolic profiles of three cyanobacterial cultures - wild-type S. elongatus 7942 without salt stress (WT), wild-type with salt stress (WT/NaCl), and the cscB-overexpressing strain with salt stress (cscB/NaCl) - all under photoautotrophic conditions. We quantified the substantial rewiring of metabolic fluxes in WT/NaCl and cscB/NaCl cultures relative to WT and identified a metabolic bottleneck limiting carbon fixation and sucrose biosynthesis. This bottleneck was subsequently mitigated through heterologous overexpression of glyceraldehyde-3-phosphate dehydrogenase in an engineered sucrose-secreting strain. Our study also demonstrates that combining 13C-MFA and GSM is a useful strategy to both extend the coverage of MFA beyond central metabolism and to improve the accuracy of flux predictions provided by GSM.

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. The expanded reach of the method beyond currently targeted small RNA genes promotes the discovery and incorporation of gene synergy in natural and engineered processes such as biocontainment, the production of desired compounds, and previously uncharacterized fundamental biological mechanisms.

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. Genetic biocontainment strategies are therefore under development to reduce the probability that these engineered bacteria can survive outside of the laboratory or industrial setting. These include active strategies that aim to kill the escaped cells by expression of toxic proteins, and passive strategies that use knockouts of native genes to reduce fitness outside of the controlled environment of labs and industrial cultivation systems. Several biocontainment strategies have demonstrated escape frequencies below detection limits. However, they have typically done so in carefully controlled experiments which may fail to capture mechanisms of escape that may arise in the more complex natural environment. The selection of biocontainment strategies that can effectively kill cells outside the lab, while maintaining maximum productivity inside the lab and without the need for relatively expensive chemicals will benefit from further attention.

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 N-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. While demonstrating the ability to detect a broad range of relevant alcohols and organic acids, we showed that the response of the films correlated strongly with the octanol-water partition coefficient (log P) of the analyte. Differences in the secretion of ethanol and succinic acid from strains of Zymomonas mobilis and Actinobacillus succinogenes, respectively, were further detected via optical characterization of the films. These differences, which in some cases were as low as ∼3 g/L, were confirmed by high-performance liquid chromatography, thereby demonstrating the sensitivity of this approach. Our findings highlight the potential utility of this multiplexed approach for the detection of small organic analytes in complex biological media, which overcomes a major challenge in conventional optical sensing methods.

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 CO2 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 CO2 assimilation during cultivation with both CH4 and CO2 as carbon sources. Marker exchange mutagenesis of M. capsulatus Bath genes encoding key enzymes of potential CO2-assimilating metabolic pathways indicated that a complete serine cycle is not required, whereas RubisCO is essential for growth of this bacterium. 13CO2 tracer analysis showed that CH4 and CO2 enter overlapping anaplerotic pathways and implicated RubisCO as the primary enzyme mediating CO2 assimilation in M. capsulatus Bath. Notably, we quantified the relative abundance of 3-phosphoglycerate and ribulose-1,5-bisphosphate 13C isotopes, which supported that RubisCO-produced 3-phosphoglycerate is primarily converted to ribulose-1-5-bisphosphate via the oxidative pentose phosphate pathway in M. capsulatus Bath. Collectively, our data establish that RubisCO and CO2 play essential roles in M. capsulatus Bath metabolism. This study expands the known capacity of methanotrophs to fix CO2 via RubisCO, which may play a more pivotal role in the Earth's biogeochemical carbon cycling and greenhouse gas regulation than previously recognized. Further, M. capsulatus Bath and other CO2-assimilating methanotrophs represent excellent candidates for use in the bioconversion of biogas waste streams that consist of both CH4 and CO2. IMPORTANCE The importance of RubisCO and CO2 in M. capsulatus Bath metabolism is unclear. In this study, we demonstrated that both CO2 and RubisCO are essential for M. capsulatus Bath growth. 13CO2 tracing experiments supported that RubisCO mediates CO2 fixation and that a noncanonical Calvin cycle is active in this organism. Our study provides insights into the expanding knowledge of methanotroph metabolism and implicates dually CH4/CO2-utilizing bacteria as more important players in the biogeochemical carbon cycle than previously appreciated. In addition, M. capsulatus and other methanotrophs with CO2 assimilation capacity represent candidate organisms for the development of biotechnologies to mitigate the two most abundant greenhouse gases, CH4 and CO2.

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. However, to realize the full potential of microbes as biocatalytic platforms in the bioeconomy, a deeper understanding of the fundamental principles governing microbial responsiveness to biocontainment constraints, and interactivity of GMOs with the environment, is required. Herein, we review recent analytical biotechnological advances and strategies to assess biocontainment and microbial bioproductivity, as well as opportunities for predictive systems biodesigns towards securing a viable bioeconomy.

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. NREL 46B-D3 demonstrating peak growth near 25 °C that reaches 30 g/m2/day and the highest biomass accumulation capacity post cell division reported to date for a halotolerant strain. Functional genomics analysis revealed that genes involved in lipid production, ion channels and antiporters are expanded and expressed. Exposure to temperature stress shifts fatty acid metabolism and increases amino acids synthesis. Co-expression analysis shows that many fatty acid biosynthesis genes are overexpressed with specific transcription factors under cold stress. These and other genes involved in the metabolic and regulatory response to temperature stress can be further explored for strain improvement.

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. Model simulations reveae metabolic exchanges that sustain the heterotrophs in minimal media devoid of any organic carbon source, pointing to S. elongatus-E. coli K-12 as the most active community. Experimental validation of flux predictions for this pair confirms metabolic interactions and potential production capabilities. Synthetic communities bypass member-specific metabolic bottlenecks (e.g. histidine- and transport-related reactions) and compensate for lethal genetic traits, achieving up to 27% recovery from lethal knockouts. The study provides a robust modelling framework for the rational design of synthetic communities with optimized growth sustainability using phototrophic partners.

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.g., phospho- and nitroso-proteomic) analyses to more completely evaluate algal responsiveness to nutrient deprivation. Herein, we describe methods for isolating total algal protein and conducting proteomic, phosphoproteomic, and nitrosoproteomic analyses. We focus on methods deployed for the chlorophyte, Chlorella vulgaris, a model oleaginous alga with high flux to renewable fuel and chemical precursors.

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

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.

Development of a CRISPR/Cas9 System for Methylococcus capsulatus In Vivo 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 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 Cas9D10A 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 Cas9D10A 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.

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. An unprecedented high cell loading was achieved, and cell inks showed unique thixotropic behavior. In the presence of glucose, printed bioscaffolds exhibited increased ethanol production compared to bulk counterparts due largely to improved mass transfer through engineered porous structures. The new living materials developed in this work could serve as a versatile platform for process intensification of an array of bioconversion processes utilizing diverse microbial biocatalysts for production of high-value products or bioremediation applications.

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. Here, we report cultivation of three phylogenetically diverse methanotrophic bacteria on biogas streams derived from AD of a series of energy crop residues. Strains maintained comparable central metabolic activity and displayed minimal growth inhibition when cultivated under batch configuration on AD biogas streams relative to pure methane, although metabolite analysis suggested biogas streams increase cellular oxidative stress. In contrast to batch cultivation, growth arrest was observed under continuous cultivation configuration, concurrent with increased biosynthesis and excretion of lactate. We examined the potential for enhanced lactate production via the employ of a pyruvate dehydrogenase mutant strain, ultimately achieving 0.027 g lactate/g DCW/h, the highest reported lactate specific productivity from biogas to date.

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. Three strains, from the genera Micractinium, Chlorella, and Scenedesmus, were chosen following laboratory evaluations and grown outdoors in 1000 L raceway ponds during the winter. Strains were down-selected based on doubling time, lipid and carbohydrate amount, final biomass accumulation capacity, cell size and phylogenetic diversity. Algal biomass productivity and compositional analysis for lipids and carbohydrates show successful outdoor deployment and cultivation under winter conditions for these strains. Outdoor harvest-yield biomass productivities ranged from 2.9 to 4.0 g/m2/day over an 18 days winter cultivation trial, with maximum productivities ranging from 4.0 to 6.5 g/m2/day, the highest productivities reported to date for algal winter strains grown in saline media in open raceway ponds. Peak fatty acid levels ranged from 9 to 26% percent of biomass, and peak carbohydrate levels ranged from 13 to 34% depending on the strain. Changes in the lipid and carbohydrate profile throughout outdoor growth are reported. This study demonstrates that algal strain screening under simulated outdoor environmental conditions in the laboratory enables identification of strains with robust biomass productivity and biofuel precursor composition. The strains isolated here represent promising winter deployment candidates for seasonal algal biomass production when using crop rotation strategies.

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. Here, we demonstrate the variable growth outcomes of five diverse methanotrophic strains - Methylocystis sp. Rockwell, Methylocystis sp. WRRC1, Methylosinus trichosporium OB3b, Methylomicrobium album BG8, and Methylomonas denitrificans FJG1 - grown on either methane or methanol, at three different concentrations, with either ammonium or nitrate provided as nitrogen source. Maximum optical density (OD), growth rate, and biomass yield were assessed for each condition. Further metabolite and fatty acid methyl ester (FAME) analyses were completed for Methylocystis sp. Rockwell and M. album BG8. The results indicate differential response to these growth conditions, with a general preference for ammonium-based growth over nitrate, except for M. denitrificans FJG1. Methane is also preferred by most strains, with methanol resulting in unreliable or inhibited growth in all but M. album BG8. Metabolite analysis points to monitoring of excreted formic acid as a potential indicator of adverse growth conditions, while the magnitude of FAME variation between conditions may point to strains with broader substrate tolerance. These findings suggest that methanotroph strains must be carefully evaluated before use in industry, both to identify optimal conditions and to ensure the strain selected is appropriate for the process of interest. Much work remains in addressing the optimization of growth strategies for these promising microorganisms since disregarding these important steps in process development could ultimately lead to inefficient or failed bioprocesses.

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 (OleTJE) 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 (P450BSß). Recent reports have demonstrated that catalytic pathways in these enzymes bifurcate when the heme is in its iron-hydroxo (compound II) state. In spite of significant similarities, the fundamental underpinnings of their different characteristic wild-type reactivities remain ambiguous. Here, we develop point charges, modified parameters and report molecular simulations of this crucial intermediate step. Water occupancies and substrate mobility at the active site are observed to be vital differentiating aspects between the two enzymes in the compound II state and corroborate recent experimental hypotheses. Apart from increased substrate mobility in the hydroxylase, which could have implications for enabling the rebound mechanism for hydroxylation, OleTJE is characterized by much stronger binding of the substrate carboxylate group to the active site arginine, implicating it as an important enabling actor for decarboxylation.