Biophysical and structural studies reveal marginal stability of a crucial hydrocarbon biosynthetic enzyme acyl ACP reductase.

Acyl-ACP reductase (AAR) is one of the two key cyanobacterial enzymes along with aldehyde deformylating oxygenase (ADO) involved in the synthesis of long-chain alkanes, a drop-in biofuel. The enzyme is prone to aggregation when expressed in Escherichia coli, leading to varying alkane levels. The present work attempts to investigate the crucial structural aspects of AAR protein associated with its stability and folding. Characterization by dynamic light scattering experiment and intact mass spectrometry revealed that recombinantly expressed AAR in E. coli existed in multiple-sized protein particles due to diverse lipidation. Interestingly, while thermal- and urea-based denaturation of AAR showed 2-state unfolding transition in circular dichroism and intrinsic fluorescent spectroscopy, the unfolding process of AAR was a 3-state pathway in GdnHCl solution suggesting that the protein milieu plays a significant role in dictating its folding. Apparent standard free energy [Formula: see text] of ~ 4.5 kcal/mol for the steady-state unfolding of AAR indicated borderline stability of the protein. Based on these evidences, we propose that the marginal stability of AAR are plausible contributing reasons for aggregation propensity and hence the low catalytic activity of the enzyme when expressed in E. coli for biofuel production. Our results show a path for building superior biocatalyst for higher biofuel production.

In vivo Quantification of Alkanes in Escherichia coli.

Microbial production of alkanes employing synthetic biology tools has gained tremendous attention owing to the high energy density and similarity of alkanes to existing petroleum fuels. One of the most commonly studied pathways includes the production of alkanes by AAR (acyl-ACP (acyl carrier protein) reductase)-ADO (aldehyde deformylating oxygenase) pathway. Here, the intermediates of fatty acid synthesis pathway are used as substrate by the AAR enzyme to make fatty aldehyde, which is then deformylated by ADO to make linear chain alkane. However, the variation in substrate availability to the first enzyme of the pathway, i.e., AAR, via fatty acid synthesis pathway and low turnover of the ADO enzyme make calculation of yields and titers under in vivo conditions extremely difficult. In vivo assay employing external addition of defined substrates for ADO enzyme into the medium helps to monitor the influx of substrate hence providing a more accurate measurement of the product yields. In this protocol, we include a detailed guide for implementing the in vivo assay for monitoring alkane production in E. coli.

Building cell factories for the production of advanced fuels.

Synthetic biology-based engineering strategies are being extensively employed for microbial production of advanced fuels. Advanced fuels, being comparable in energy efficiency and properties to conventional fuels, have been increasingly explored as they can be directly incorporated into the current fuel infrastructure without the need for reconstructing the pre-existing set-up rendering them economically viable. Multiple metabolic engineering approaches have been used for rewiring microbes to improve existing or develop newly programmed cells capable of efficient fuel production. The primary challenge in using these approaches is improving the product yield for the feasibility of the commercial processes. Some of the common roadblocks towards enhanced fuel production include - limited availability of flux towards precursors and desired pathways due to presence of competing pathways, limited cofactor and energy supply in cells, the low catalytic activity of pathway enzymes, obstructed product transport, and poor tolerance of host cells for end products. Consequently, despite extensive studies on the engineering of microbial hosts, the costs of industrial-scale production of most of these heterologously produced fuel compounds are still too high. Though considerable progress has been made towards successfully producing some of these biofuels, a substantial amount of work needs to be done for improving the titers of others. In this review, we have summarized the different engineering strategies that have been successfully used for engineering pathways into commercial hosts for the production of advanced fuels and different approaches implemented for tuning host strains and pathway enzymes for scaling up production levels.

A consensus-guided approach yields a heat-stable alkane-producing enzyme and identifies residues promoting thermostability.

Aldehyde-deformylating oxygenase (ADO) is an essential enzyme for production of long-chain alkanes as drop-in biofuels, which are compatible with existing fuel systems. The most active ADOs are present in mesophilic cyanobacteria, especially Nostoc punctiforme Given the potential applications of thermostable enzymes in biorefineries, here we generated a thermostable (Cts)-ADO based on a consensus of ADO sequences from several thermophilic cyanobacterial strains. Using an in silico design pipeline and a metagenome library containing 41 hot-spring microbial communities, we created Cts-ADO. Cts-ADO displayed a 3.8-fold increase in pentadecane production on raising the temperature from 30 to 42 °C, whereas ADO from N. punctiforme (Np-ADO) exhibited a 1.7-fold decline. 3D structure modeling and molecular dynamics simulations of Cts- and Np-ADO at different temperatures revealed differences between the two enzymes in residues clustered on exposed loops of these variants, which affected the conformation of helices involved in forming the ADO catalytic core. In Cts-ADO, this conformational change promoted ligand binding to its preferred iron, Fe2, in the di-iron cluster at higher temperature, but the reverse was observed in Np-ADO. Detailed mapping of residues conferring Cts-ADO thermostability identified four amino acids, which we substituted individually and together in Np-ADO. Among these substitution variants, A161E was remarkably similar to Cts-ADO in terms of activity optima, kinetic parameters, and structure at higher temperature. A161E was located in loop L6, which connects helices H5 and H6, and supported ligand binding to Fe2 at higher temperatures, thereby promoting optimal activity at these temperatures and explaining the increased thermostability of Cts-ADO.

Model-assisted metabolic engineering of Escherichia coli for long chain alkane and alcohol production.

Biologically-derived hydrocarbons are considered to have great potential as next-generation biofuels owing to the similarity of their chemical properties to contemporary diesel and jet fuels. However, the low yield of these hydrocarbons in biotechnological production is a major obstacle for commercialization. Several genetic and process engineering approaches have been adopted to increase the yield of hydrocarbon, but a model driven approach has not been implemented so far. Here, we applied a constraint-based metabolic modeling approach in which a variable demand for alkane biosynthesis was imposed, and co-varying reactions were considered as potential targets for further engineering of an E. coli strain already expressing cyanobacterial enzymes towards higher chain alkane production. The reactions that co-varied with the imposed alkane production were found to be mainly associated with the pentose phosphate pathway (PPP) and the lower half of glycolysis. An optimal modeling solution was achieved by imposing increased flux through the reaction catalyzed by glucose-6-phosphate dehydrogenase (zwf) and iteratively removing 7 reactions from the network, leading to an alkane yield of 94.2% of the theoretical maximum conversion determined by in silico analysis at a given biomass rate. To validate the in silico findings, we first performed pathway optimization of the cyanobacterial enzymes in E. coli via different dosages of genes, promoting substrate channelling through protein fusion and inducing substantial equivalent protein expression, which led to a 36-fold increase in alka(e)ne production from 2.8 mg/L to 102 mg/L. Further, engineering of E. coli based on in silico findings, including biomass constraint, led to an increase in the alka(e)ne titer to 425 mg/L (major components being 249 mg/L pentadecane and 160 mg/L heptadecene), a 148.6-fold improvement over the initial strain, respectively; with a yield of 34.2% of the theoretical maximum. The impact of model-assisted engineering was also tested for the production of long chain fatty alcohol, another commercially important molecule sharing the same pathway while differing only at the terminal reaction, and a titer of 1506 mg/L was achieved with a yield of 86.4% of the theoretical maximum. Moreover, the model assisted engineered strains had produced 2.54 g/L and 12.5 g/L of long chain alkane and fatty alcohol, respectively, in the bioreactor under fed-batch cultivation condition. Our study demonstrated successful implementation of a combined in silico modeling approach along with the pathway and process optimization in achieving the highest reported titers of long chain hydrocarbons in E. coli.

Heterogeneity of alkane chain length in freshwater and marine cyanobacteria.

The potential utilization of cyanobacteria for the biological production of alkanes represents an exceptional system for the next generation of biofuels. Here, we analyzed a diverse group of freshwater and marine cyanobacterial isolates from Indian culture collections for their ability to produce both alkanes and alkenes. Among the 50 cyanobacterial isolates screened, 32 isolates; 14 freshwater and 18 marine isolates; produced predominantly alkanes. The GC-MS/MS profiles revealed a higher percentage of pentadecane and heptadecane production for marine and freshwater strains, respectively. Oscillatoria species were found to be the highest producers of alkanes. Among the freshwater isolates, Oscillatoria CCC305 produced the maximum alkane level with 0.43 µg/mg dry cell weight, while Oscillatoria formosa BDU30603 was the highest producer among the marine isolates with 0.13 µg/mg dry cell weight. Culturing these strains under different media compositions showed that the alkane chain length was not influenced by the growth medium but was rather an inherent property of the strains. Analysis of the cellular fatty acid content indicated the presence of predominantly C16 chain length fatty acids in marine strains, while the proportion of C18 chain length fatty acids increased in the majority of freshwater strains. These results correlated with alkane chain length specificity of marine and freshwater isolates indicating that alkane chain lengths may be primarily determined by the fatty acid synthesis pathway. Moreover, the phylogenetic analysis showed clustering of pentadecane-producing marine strains that was distinct from heptadecane-producing freshwater strains strongly suggesting a close association between alkane chain length and the cyanobacteria habitat.