Patrik Raymond Jones

Nitrogen homeostasis in heterocyst-forming filamentous diazotrophic cyanobacteria depends on the coordination between two cell types: heterocysts, which fix atmospheric N2, and vegetative cells, which carry out oxygenic photosynthesis. Yet the cell-type-specific contribution of glutamine synthetase (GS), the central enzyme of nitrogen assimilation, to this coordination remains poorly understood. We used cell-type-specific overexpression of IF7A, the GS posttranslational inhibitor, in both wild-type Anabaena sp. PCC 7120 and Δamt genetic backgrounds, the latter lacking the high-affinity ammonium transporters (Amt) responsible for NH4+ uptake, to dissect the contribution of each cell type to the nitrogen economy of the filament. Whole-filament GS inhibition caused photosynthetic impairment, disruption of the NtcA–PII–PipX signaling axis, and growth arrest, establishing GS in vegetative cells as rate-limiting for filament growth. Heterocyst-targeted GS inhibition triggered accumulation of the nitrogen storage polymer cyanophycin, revealing that heterocyst GS governs the routing of fixed nitrogen either toward distribution or storage. Both phenotypes were amplified in the Δamt background. Using pharmacological inhibition of GS with ammonium excretion measurements, we demonstrate that Amt transporters recover nitrogen lost by passive NH3 diffusion under diazotrophic conditions and that their absence renders the internal nitrogen economy chronically more vulnerable to GS inhibition. In natural nitrogen-limited environments, where diazotrophic cyanobacteria cannot compensate for passive nitrogen loss through external uptake, this recycling function is critical for nitrogen fixation efficiency. Overall, our findings redefine the architecture of intercellular nitrogen homeostasis in filamentous diazotrophic cyanobacteria, establishing GS and Amt transporters as interdependent determinants of intercellular nitrogen retention, distribution, and storage.

Quantitating intracellular oxidative damage caused by reactive oxygen species (ROS) is of interest in many fields of biological research. The current systems primarily rely on supplemented oxygen-sensitive substrates that penetrate the target cells, and react with ROS to produce signals that can be monitored with spectroscopic or imaging techniques. The objective here was to design a new non-invasive analytical strategy for measuring ROS-induced damage inside living cells by taking advantage of the native redox sensor system of E. coli. The developed plasmid-based sensor relies on an oxygen-sensitive transcriptional repressor IscR that controls the expression of a fluorescent marker in vivo. The system was shown to quantitatively respond to oxidative stress induced by supplemented H₂O₂ and lowered cultivation temperatures. Comparative analysis with fluorescence microscopy further demonstrated that the specificity of the reporter system was equivalent to the commercial chemical probe (CellROX). The strategy introduced here is not dependent on chemical probes, but instead uses a fluorescent expression system to detect enzyme-level oxidative damage in microbial cells. This provides a cheap and simple means for analysing enzyme-level oxidative damage in a biological context in E. coli.

Fatty acids and their derivatives are highly valuable chemicals that can be produced through chemical or enzymatic processes using plant lipids. This may compete with human food sources. Therefore, there has been an urge to create a new method for synthesizing these chemicals. One approach is to use microbial cells, specifically cyanobacteria, as a factory platform. Engineering may need to be implemented in order to allow a cost-competitive production and to enable a production of a variety of different fatty acids and derivatives. In this chapter, we explain in details the importance of fatty acids and their derivatives, including fatty aldehydes, fatty alcohols, hydrocarbons, fatty acid methyl esters, and hydroxy fatty acids. The production of these chemicals using cyanobacterial native metabolisms together with strategies to engineer them are also explained. Moreover, recent examples of fatty acid and fatty acid derivative production from engineered cyanobacteria are gathered and reported. Commercial opportunities to manufacture fatty acids and derivatives are also discussed in this chapter. Altogether, it is clear that fatty acids and their derivatives are important chemicals, and with recent advancements in genetic engineering, a cyanobacterial platform for bio-based production is feasible. However, there are regulations and guidelines in place for the use of genetically modified organisms (GMOs) and some further developments are still needed before commercialization can be reached. Graphical Abstract Keywords Fatty acids Derivatives Cyanobacteria Metabolic engineering Commercial opportunity

The objective of this study was to implement direct sunlight-driven conversion of CO ₂ into a naturally excreted ready-to-use fuel. We engineered four different synthetic metabolic modules for biosynthesis of short-to medium-chain length hydrocarbons in the model cyanobacterium Synechocystis sp. PCC 6803. In module 1, the combination of a truncated clostridial n-butanol pathway with over-expression of the native cyanobacterial aldehyde deformylating oxygenase resulted in small quantities of propane when cultured under closed conditions. Direct conversion of CO ₂ into propane was only observed in strains with CRISPRi-mediated repression of three native putative aldehyde reductases. In module 2, three different pathways towards pentane were evaluated based on the polyunsaturated fatty acid linoleic acid as an intermediate. Through combinatorial evaluation of bioreaction ingredients it was concluded that linoleic acid undergoes a spontaneous non-enzymatic reaction to yield pentane and hexanal. When Synechocystis was added to the bioreaction, hexanal was converted into 1-hexanol, but there was no further stimulation of pentane biosynthesis. For modules 3 and 4, several different acyl-ACP thioesterases were evaluated in combination with two different decarboxylases. Small quantities of 1-heptene and 1-nonene were observed in strains expressing the desaturase-like enzyme UndB from Pseudomonas mendocina in combination with C8-C10 preferring thioestersaes. When UndB instead was combined with a C12-specific ‘ Uc FatB1 thioesterase, this resulted in ten-fold increase of alkene biosynthesis. When UndB was replaced with the light-dependent FAP decarboxylase, both undecane and tridecane accumulated, albeit with a 10-fold drop in productivity. Optimization of the RBS, promoter and gene order in these synthetic operons resulted in 1-alkene bioproductivity of 230 mg/L after 10 d with 15% carbon partitioning. In conclusion, the direct bioconversion of CO ₂ into secreted and ready-to-use hydrocarbon fuel was accomplished and optimal results were obtained with UndB and a C12 chain-length specific thioesterase. Highlights: Multiple repression of endogenous aldehyde reductases/dehydrogenases by CRISPRi enabled propane biosynthesis Biosynthesis of short-medium chain hydrocarbons (C7-C11) in a cyanobacterium was demonstrated for the first time The final enzymes of the hydrocarbon pathways influenced both productivity and product profile All volatile products were naturally secreted and accumulated outside of the cell

To enable a sustainable supply of chemicals, novel biotechnological solutions are required that replace the reliance on fossil resources. One potential solution is to utilize tailored biosynthetic modules for the metabolic conversion of CO₂ or organic waste to chemicals and fuel by microorganisms. Currently, it is challenging to commercialize biotechnological processes for renewable chemical biomanufacturing because of a lack of highly active and specific biocatalysts. As experimental methods to engineer biocatalysts are time- and cost-intensive, it is important to establish efficient and reliable computational tools that can speed up the identification or optimization of selective, highly active, and stable enzyme variants for utilization in the biotechnological industry. Here, we review and suggest combinations of effective state-of-the-art software and online tools available for computational enzyme engineering pipelines to optimize metabolic pathways for the biosynthesis of renewable chemicals. Using examples relevant for biotechnology, we explain the underlying principles of enzyme engineering and design and illuminate future directions for automated optimization of biocatalysts for the assembly of synthetic metabolic pathways.

1-Octanol has gained interest as a chemical precursor for both high and low value commodities including fuel, solvents, surfactants, and fragrances. By harnessing the power from sunlight and CO₂ as carbon source, cyanobacteria has recently been engineered for renewable production of 1-octanol. The productivity, however, remained low. In the present work, we report efforts to further improve the 1-octanol productivity. Different N-terminal truncations were evaluated on three thioesterases from different plant species, resulting in several candidate thioesterases with improved activity and selectivity toward octanoyl-ACP. The structure/function trials suggest that current knowledge and/or state-of-the art computational tools are insufficient to determine the most appropriate cleavage site for thioesterases in Synechocystis. Additionally, by tuning the inducer concentration and light intensity, we further improved the 1-octanol productivity, reaching up to 35% (w/w) carbon partitioning and a titer of 526 ± 5 mg/L 1-octanol in 12 days. Long-term cultivation experiments demonstrated that the improved strain can be stably maintained for at least 30 days and/or over ten times serial dilution. Surprisingly, the improved strain was genetically stable in contrast to earlier strains having lower productivity (and hence a reduced chance of reaching toxic product concentrations). Altogether, improved enzymes and environmental conditions (e.g., inducer concentration and light intensity) substantially increased the 1-octanol productivity. When cultured under continuous conditions, the bioproduction system reached an accumulative titer of >3.5 g/L 1-octanol over close to 180 days.

Nitrogen sources are all converted into ammonium/ia as a first step of assimilation. It is reasonable to expect that molecular components involved in the transport of ammonium/ia across biological membranes connect with the regulation of both nitrogen and central metabolism. We applied both genetic (i.e., Δamt mutation) and environmental treatments to a target biological system, the cyanobacterium Anabaena sp PCC 7120. The aim was to both perturb nitrogen metabolism and induce multiple inner nitrogen states, respectively, followed by targeted quantification of key proteins, metabolites and enzyme activities. The absence of AMT transporters triggered a substantial whole‐system response, affecting enzyme activities and quantity of proteins and metabolites, spanning nitrogen and carbon metabolisms. Moreover, the Δamt strain displayed a molecular fingerprint indicating nitrogen deficiency even under nitrogen replete conditions. Contrasting with such dynamic adaptations was the striking near‐complete lack of an externally measurable altered phenotype. We conclude that this species evolved a highly robust and adaptable molecular network to maintain homeostasis, resulting in substantial internal but minimal external perturbations. This analysis provides evidence for a potential role of AMT transporters in the regulatory/signalling network of nitrogen metabolism and the existence of a novel fourth regulatory mechanism controlling glutamine synthetase activity.

In the growing field of metabolic engineering, where cells are treated as “factories” that synthesize industrial compounds, it is essential to consider the ability of the cells’ native metabolism to accommodate the demands of synthetic pathways, as these pathways will alter the homeostasis of cellular energy and electron metabolism. From the breakdown of substrate, microorganisms activate and reduce key co-factors such as ATP and NAD(P)H, which subsequently need to be hydrolysed and oxidized, respectively, in order to restore cellular balance. A balanced supply and consumption of such co-factors, here termed co-factor balance, will influence biotechnological performance. To aid the strain selection and design process, we used stoichiometric modelling (FBA, pFBA, FVA and MOMA) and the Escherichia coli (E.coli) core stoichiometric model to investigate the network-wide effect of butanol and butanol precursor production pathways differing in energy and electron demand on the final product yield. An FBA-based co-factor balance assessment (CBA) algorithm was developed to track and categorise how ATP and NAD(P)H pools are affected in the presence of a new pathway. CBA was compared to the balance calculations proposed by Dugar et al. (Nature Biotechnol. 29 (12), 1074–1078). Predicted solutions were compromised by excessively underdetermined systems, displaying greater flexibility in the range of reaction fluxes than experimentally measured by ¹³C-metabolic flux analysis (MFA) and the appearance of unrealistic futile co-factor cycles. With the assumption that futile cycles are tightly regulated in reality, the FBA models were manually constrained in a step-wise manner. Solutions with minimal futile cycling diverted surplus energy and electrons towards biomass formation. As an alternative, the use of loopless FBA or constraining the models with measured flux ranges were tried but did not prevent futile co-factor cycles. The results highlight the need to account for co-factor imbalance and confirm that better-balanced pathways with minimal diversion of surplus towards biomass formation present the highest theoretical yield. The analysis also suggests that ATP and NAD(P)H balancing cannot be assessed in isolation from each other, or even from the balance of additional co-factors such as AMP and ADP. We conclude that, through revealing the source of co-factor imbalance CBA can facilitate pathway and host selection when designing new biocatalysts for implementation by metabolic engineering. The chemicals industry is a major contributor to greenhouse gas emissions and desperately requires more sustainable alternatives. Genetically engineered microorganisms can be used as 'bio-factories' to manufacture chemicals, replacing those currently sourced from fossil fuels or unsustainable tropical plant agriculture. However, due to the complexity of biology, the features that render one bio-factory design more efficient than others are difficult to identify. Computational modelling of such designs can enable the selection of optimally performing designs, but it remains challenging as biology is complex and not fully understood. Microorganisms require energy for their own growth and maintenance, but also to convert molecules into desired target products. The supply and consumption of such energy is through co-factors, and the balance of such co-factors influences the performance of the engineered bio-factories. This study developed a computer-aided approach for quantification of the co-factor balance of bio-factories. Using the chemical n-butanol as a case study, our study explores the impact of variant bio-factory designs with differing co-factor balance on the potential efficiency of biomanufacturing. We provide insights into the relative balance of different designs and provide a computational framework to select the best-performing designs.