Team:MADRID UCM/Design

Cloning design - 4C_FUELS

Our project aims to engineer cyanobacteria in order to produce sustainable chemicals, starting with n-butanol production as an initial proof of concept. During this year our goal is to engineer a n-butanol overproducing cyanobacterial strain. To do so, we have designed multiple metabolic engineering strategies.
Within this page you will find all the information regarding the required metabolic modifications for efficient n-butanol production in cyanobacteria.

Our Metabolic Engineering Design

image/svg+xml MCG HspA Butanol Pathway SigB CO2 Acetyl-CoA Butanol AcrbV2
Use the mouse over the left image to explore our different metabolic engineering strategies

n-Butanol Biosynthesis Pathway

Implementing an oxygen-tolerant n-butanol biosynthesis pathway

The core of any metabolic engineering strategy are those modifications responsible for product biosynthesis. Then to create a n-butanol super producing strain, the first step should be the design and implementation of an efficient production pathway.

When thinking about biological butanol, immediately Clostridium acetobutylicum appears in the mind of most biofuel connoisseurs. This organism capable of performing the ABE fermentation (acetone, ethanol and butanol) is the most efficient n-butanol producer found in nature. This gram-positive bacillus is capable of fermenting a wide range of substrate reaching titers up to o 20 g*L-1 of butanol for some selected strains. Considering the above, looks reasonable to find the inspiration for pathway design within the metabolism of this amazing organism.

However, C. acetobutylicum is a strict-anaerobe and that’s not a small detail. Anaerobic organisms have evolved their metabolism in environments with the absence of oxygen. Actually some of the key enzymes for n-butanol biosynthesis within this organism are oxygen-sensitive. This way two strategies could be employed: guaranteeing an anaerobic environment for the pathway or adapting the pathway with oxygen-tolerant enzyme variants. Since cyanobacteria are well known for its ability to produce oxygen, we decided to take this second step.

Luckily there has been previous research in this topic, almost at the same time we were devising the project, a paper from Lindblad et al. came to our hands. In this paper a semi-synthetic oxygen tolerant n-butanol pathway was designed and optimized for a well known model cyanobacteria: Synechocystis PCC6803.

The design of a synthetic n-butanol pathway

Briefly, a n-butanol biosynthesis pathway consists of two steps. First, acetyl-CoA is condensed to form acetoacetyl-CoA, the four carbons metabolic precursor that provides the required four carbons skeleton.. Then the acetoacetyl-CoA backbone is furtherly modified via multiple reduction steps, which eventually ends with the release of CoA moiety and the final reduction of the organic backbone towards n-butanol.

We have conducted intensive research on the different possible modifications of the natural n-butanol biosynthesis pathway. We discovered which steps were critical, which not and observed how most of the stages of the pathway are indeed susceptible to be optimized for performance under aerobic conditions.

However, despite the existence of numerous publications on this subject, the pathway proposed by Peter Lindblad et al. implements almost all of these improvements. In the image below a general pathway overview is depicted.


The most critical steps are the initial thermodynamically unfavourable condensation of acetyl-CoA, as well as the final reduction steps. Likewise, the intermediate reduction reactions are performed by enzymes that are NADH dependent and have shown to perform poorly on phototrophic organisms, then adequate pathway redesign is required.

In general terms, the design guidelines for any metabolic engineering design has been used. Codon optimization of coding sequences, balancing pathway’s cofactor utilization, redirection of carbon flux towards the metabolic precursors and the generation of driving forces via irreversible reactions. Our team has written an extensive introduction to metabolic engineering applied to phototrophs. To know more about this visit the Phototrophs Synbio page.

A deep insight within n-butanol pathway

Initial Condensation


Regarding the first step, the biggest challenge is to provide enough metabolic precursor to the reactions downstream in the pathway. The abundance of acetyl-CoA pool as well as the condensation rate towards acetoacetyl-CoA are the critical factors that control the process.

However, homogeneous acetyl-CoA condensation catalyzed by acetyl transferases performing reversible reactions that are also not thermodynamically favourable.


To overcome this limitation, an ATP-coupled heterogeneous acetoacetyl-CoA synthesis reaction is introduced which acts as the pathway's driving force. NphT7 is an active acetoacetyl-CoA Synthase from Streptomyces CL190 that catalyzes the irreversible condensation of acetyl-CoA and malonyl-CoA to form acetoacetyl-CoA and CoA, releasing a CO2 molecule. It has been proved that this condensation step can be comfortably tapped from the intracellular malonyl-CoA pool.

CoA-intermediates reduction reactions


In natural pathways usually β-oxidation related enzymes with multiple catalytic domains perform sequentially the initial reduction reactions. In the synthetic pathway, the subdivision of each reaction to more specific enzymes has shown to improve performance. Likewise, most of these multifunctional enzymes feature stereoselectivity for the S-OH-Butyryl-CoA, while the specific enzymes generate the opposite (S-OH-Butyryl-CoA), which for PHA forming organisms like many cyanobacteria could pose an advantage due to metabolic pool of S-OH-Butyryl-CoA produced by native enzymes. Although this is not the case in our chassis organisms, it is also an important consideration to easily export the pathway to other phototrophic organisms.


After the condensation step, the first reduction is performed by a PhaB, a PHA-synthase, derived from Cupriavidus necator. A study of directed evolution variants of PhaB enzymes discovered that the punctual mutation T173S improved substrate and NADPH affinity, leading to a higher activity.


The elimination of hydroxyl residue is performed by PhaJ from Aeromonas caviae. It is a 4 to 6 carbon specific enoyl-CoA reversible hydratase capable of generating a double crotonyl CoA via water elimination


The next step consists in the hydrogenation of the former double-bond. Natural pathways rely on the utilization of a multifunctional butyryl-CoA hydrogenase-dehydrogenase. Some authors propose the utilization of a specific trans-enoyl-CoA reductase, improving pathway performance. However most of these enzymes are NADH-dependent. In order to tap into the more available NADPH cofactor as hydrogen source, Ccr from Streptomyces coelicolor is utilized. It is a NADPH dependant specific irreversible trans-enoyl-CoA reductase, capable of unidirectionally performing the hydrogenation and allowing an efficient carbon channeling.

Final reduction reactions


In the naturally occuring biosynthesis pathways these last reduction-hydrogenation steps are performed by a single bifunctional aldehyde-alcohol dehydrogenase. The oxygen-sensitive nature of most of these enzymes, as well as their bidirectional activity (reconverting product backwards) are challenges to overcome. Last two enzymes of the pathway aim to solve these problems.


An oxygen-tolerant short chain aldehyde dehydrogenase from Salmonella enterica is utilized for the release of CoA moiety towards the generation of butyraldehyde, exploiting its reversible hydrogenase activity. PduP has shown to efficiently produce butyraldehyde and propionaldehyde in cyanobacteria which partially owes to its oxygen-tolerant nature.

Slr 1192

Final product generation is produced by the reduction of the carbonyl group towards the desired alcohol. This step can be performed for many different hydrogenase-dehydrogenase enzymes. In the proposed pathway Slr1192 (AdhA) is a wide-range aldehyde dehydrogenase from Synechocystis PCC6803. We have evaluated many other aldehyde dehydrogenases, looking for higher affinity towards butyraldehyde and higher specific activity. However, this enzyme utilized within the Lindblad et al. pathway featured the best combination of low Km and high specific activity when butyraldehyde was used as substrate.

Improving Pathway Performance

Expression Regulation

After defining the enzyme set required for the n-butanol production, the next step is to optimize the expression of each one of them. Once more, Lindblad et al. has already done intensive work in this aspect, selecting a combination of regulatory elements which lead to optimal pathway performance. We have taken all of this work into consideration and used a bioinformatic approach to estimate the relative expression levels of each enzyme in the pathway initially proposed by Lindblad. To do so, we employed the tools from DeNovoDNA Software to predict the expected expression rates of each enzyme. Eventually, we compared the results with the qualitative protein expression levels found within the SDS-PAGE results of Lindblad et al. paper.

Pathway expression regulation can be summarized in one main idea. Pathway enzymes can be grouped under two categories, attending to its relative expression requirements: high and moderate expression. The first four enzymes of the pathway should be expressed at a high rate, while the last ones require a more moderate expression level.

The former “moderate-expression-requirement” may attend to the intrinsic reversible behavior of the PduP and Slr1192 dehydrogenases, since the last irreversible step reduction is performed by Ccr, it is very likely that butyryl-CoA pool needs to be high in order provide enough driving force for the downstream part of the pathway. This situation PduP and Slr1192 enzymes should feature lower activity than the upstream pathway.

Since there are no known irreversible hydrogenases, the only way to overcome this limitation is to achieve the highest expression possible of all enzymes while keeping the activity balance between the four first enzymes in respect with the last two. In addition, the implementation of product removal mechanisms will also ease n-butanol production, since equilibrium will be displaced towards product formation.


Enzyme coupling

When attempting to express metabolic pathways based on combinations of heterologous enzymes, performance problems may arise because the enzymes act in non-native environments. This can lead to loss of intermediate metabolites, which might be degraded, diffused or are taken up by other competing enzymes of the organism being used as a chassis. One solution is to physically bring the enzymes closer to their substrates in order to minimize intermediate metabolite wandering.

A simple experimental approach is to fuse consecutive enzyme’s ORFs to generate a multi-enzyme peptide. Enzymes end up being fused all together in an end-to-end fashion. However, in order to avoid folding issues, simple peptide linkers are included between enzymes. Several studies have shown how different linker properties such as flexibility or length affect the multi-enzyme complex performance.

Given the large number of enzymes we planned to express in our chassis we thought it might be a good idea to try this enzyme coupling strategy. Among all, we decided to couple the last two enzymes of the n-butanol biosynthesis pathway: PduP and Slr1992. The spatial organization of enzymes involved around a metabolic branch point has great potential for redirecting the flow of carbon to the desired product. As described previously, both PduP and Slr1992 catalyze reversible hydrogenations. By keeping them close to each other, we expect them to catalyze Butyryl-CoA to n-butanol in a single-step reaction fashion. Of course this will not be as simple, this is a concept simplification.

As reversible enzymes, they rely on the reagent:product concentration ratio to catalyze the reaction towards one or the other metabolite. For PduP, Butyryl-CoA is coming from a non-reversible reaction, so we expect this metabolite to accumulate. Thus, PduP might be prone to convert Butyryl-CoA to Butyraldehyde and not to do the reverse. On the other hand, Slr1992 converts this Butyraldehyde into n-butanol. However, Butyraldehyde is coming from a reversible reaction, and if not easily available for Slr1992, this hydrogenase enzyme might catalyze the reverse reaction consuming n-butanol to increase the Butyraldehyde pool. We aim to overcome this issue by coupling these enzymes, ensuring Butyraldehyde is readily available to Slr1992 and converted to n-butanol. We restrict the functionality of these enzymes only to Butyryl-CoA:n-butanol ratio, reducing the effect that Butyraldehyde wandering may cause.

When deciding how to link these enzymes, we found many linker examples in literature. We found no agreement or general guidelines for linker design. Linker properties seem to exert a wide effect upon the coupled enzyme’s performance, but also the intrinsic properties of the enzymes to be coupled are known to have a great impact. As we cannot predict the most efficient linker for our particular case of study, we end up choosing two linking strategies that were far apart from each other:

GSG Linker

FRRRF Linker

Optimizing Carbon Channeling

Pathway design and optimization is only laying the first stone for the generation of an overproducing strain. Once our organism is capable of generating the product of interest we have to move forward to remap the organism metabolism in order to unravel its real potential as a biomanufacturing platform. To do so, carbon utilization and channeling strategies can be implemented.

Acetyl-CoA:The key metabolite

In order to increase the productivity of a well optimized pathway the simplest strategy is to increase the availability of the metabolic precursor required. In the case of n-butanol production as well many other valuable compounds, this starting material is acetyl-CoA. However, many cellular functions rely also on the utilization of this precursor, so not only increasing the availability of this metabolite is required, but also pathway overexpression to efficiently drag carbon from the central metabolism.

Then, our objective should be to alter the metabolism of the organism in order to increase the availability of acetyl-CoA, without severely compromising their basic vital functions. To do so, a clear understanding of phototrophic metabolism is required.

Eventually it is important to consider that acetyl-CoA exhibits a “triangular” metabolism in the majority of cyanobacteria, where it is interconverted into acetyl-phosphate and acetate to keep metabolic balance. Then, knocking the conversion of Acetyl-CoA to acetate or even Acetyl-phosphate could be a potential strategy to minimize its utilization. However, we have considered other alternatives.

Within photoautotrophic organisms, the main carbon source is the gaseous CO2 and its solubilized form HCO3-, which is more abundant in the liquid phase in the range of standard growth conditions. This way, in cyanobacteria the vast majority of carbon supply is provided by the Calvin–Benson–Bassham (CBB) cycle. Because of that carbon flux optimization strategies can rely on the improvement of carbon fixation performance or in the redirection of carbon fluxes from CBB to the generation of acetyl-CoA.

Because of this, we have implemented two synthetic pathways which aim to rewrite carbon partitioning towards an increased acetyl-CoA pool. Both strategies will are combined under the PK-MCG system, which is detailed below.

Rewritting carbon partitioning towards acetyl-CoA: The PK-MCG system

The PK-MCG sysem is composed of two different artificial pathways which allow to rewire how carbon is routed across metabolic networks. A phosphoketolase bypass and a synthetic Malyl-CoA-Glycerate optimized carbon fixation pathway are implemented.

Phosphoketolase bypass

Since acetyl-CoA can interconvert with acetyl-phosphate, several researchers have designed synthetic pathways that look to increase acetyl-phosphate availability, in order to convert it to acetyl-CoA. Among all of them, phosphoketolases (PKs) are widely used. These enzymes are capable of converting a sugar phosphate into a smaller chain of sugar-phosphates and acetyl-phosphate. This way, PKs can be used to bypass natural metabolism for acetyl-CoA production. This bypass utilizes CBB sugar substrates, converting them to acetyl-phosphate by a phosphoketolase. After that, native or heterologous phosphate acetyltransferase (Pta) can act transforming acetyl-phosphate into acetyl-CoA. Considering the available literature, we decided to utilize a 2-enzyme bypass consisting of a phosphoketolase from Pseudomonas aeruginosa and the phosphate acetyltransferase from Bacillus subtilis. The first one can utilize the CBB generated Xylulose-5-phosphate or Fructose-6-Phosphate, generating byproducts that can be cycled back. The second one, is a small Pta that has shown higher activity than endogenous cyanobacterial variants.

Malyl-CoA-Glycerate-Fixation Pathway

We were concerned about the potential metabolic burden that our pathway may have. First because a strongly overexpressed pathway can consume high amounts of acetyl-CoA, compromising other essential functions. Secondly, because the initial condensation step of the pathway involves a decarboxylation, reducing overall metabolic carbon utilization efficiency.

MCG Image

To implement a solution for all of these limitations we have considered the implementation of a fully synthetic carbon fixation pathway. This pathway can be coupled with the CBB cycle to fix carbon towards the direct generation of acetyl-CoA from bicarbonate, with an efficiency close to 100% carbon conversion. This pathway has been demonstrated to enhance carbon fixation in cyanobacteria up to 70% with respect to the wild type. What’s even better, it can even reutilize toxic metabolites produced during photorespiration, minimizing carbon and energy losses derived from this process.

This pathway, proposed in 2018 by James C. Liao et al. consider is defined as a Malyl-CoA-Glycerate Cycle or MCG. It relies on the utilization of a highly active phosphoenolpyruvate-carboxylase, overcoming the limitations of RuBisCO via the utilization of CO2 solubilized as bicarbonate.

A deep insight within the MCG pathway

The MCG pathway is composed of eight enzymes, which drives the synthesis of acetyl-CoA from atmospheric CO2 with the expense of 5.5 ATP and 4 NADH at a 100% carbon efficiency. MCG pathways begin with the utilization of 2-phosphoglycerate (2-PG). A starting molecule is provided by a widely present phosphoglycerate mutase (Pgm) that converts the 3-phosphoglycerate (3PG) from CBB into 2-phosphoglycerate, then entering into the MCG pathway.”

Step 1: Carbon Fixation

2PG is converted into Phosphoenolpyruvate (PEP) which is the substrate of a phosphoenolpyruvate carboxylase (Ppc) from Corynebacterium Glutamicum. PEP is then converted into Oxaloacetate (OAA) and after this step, subsequent conversions to malate and Malyl-CoA are performed by a malate dehydrogenase (mdh) from E. Coli K12 and malate thiokinase (mtkAB) from Methylococcus Capsulatus respectively.

Step 2: Acetyl-CoA Synthesis

The core of the pathway is the acetyl-CoA synthesis by a malyl-CoA lyase (mcl). This enzymatic activity is crucial for the adequate functioning of the pathway and the authors of the publication screened multiple variants till finding that mcl from Methylobacterium extorquens showed the best performance. Although it is an NADH dependent enzyme, this mcl features the advantage of accepting also NADPH as cofactor, which may explain its enhanced performance within phototrophic organisms. During this reaction, malyl-CoA is converted into acetyl-CoA and glyoxylate (GLX).

Step 3: Glyoxylate Recycling

After acetyl-CoA synthesis, glyoxylate can be decarboxylated by glyoxylate carboligase (gcl) , generating 2-hydroxy-3-oxopropanoate (2H3P) molecule that is furtherly reduced by a 2-hydroxy-3-oxopropionate reductase. Eventually, glycerate (GLY) is converted back into 2-PG via the activity of an glycerate-2-kinase, allowing the cycle to start again. The last conversion step from 2-PG to PEP is performed by an 2-PG enolase, a widely spread enzymatic functionality in the vast majority of cyanobacteria.

It is also important to note that during this step, metabolic excess of glyoxylate can be cycled up into the cycled, allowing to utilize one of the main toxic byproducts generated during photorespiration.

In this step, authors of the pathway screened the best variant for the glyoxylate carboligase, where gcl from Cuapriavidus nectator showed the best performance. However, for the last two enzymes an Escherichia Coli native version was used without codon optimization or variant screening. Although the demonstrated pathway performance was good in Synechococcus PCC7942, we considered that it could be furtherly upgraded via the codon optimization of these last two enzymes.

After exploring enzymes with a similar activity and catalytic performance, we selected the proposed glycerate-2-kinase garK from E. Coli, while for the 2-hydroxy-3-oxopropionate reductase, we decided to introduce a NADPH dependent variant. GarR is a NADPH dependant 2-hydroxy-3-oxopropionate reductase from E. Coli.

To sum up

The MCG pathway is composed of eight enzymes, each one of them with a crucial function for the fixation of carbon into acetyl-CoA.


Phosphoenolpyruvate Carboxylase

Phosphoenolpyruvate (PEP) carboxylase from Corynebacterium Glutamicum, a highly efficient carboxylase that allows the conversion of PEP to oxaloacetate via the incorporation of bicarbonate.

Malate Dehydrogenase

This Malate dehydrogenase comes from Escherichia coli K12It is a NADH-dependent enzyme that catalyzes the reduction of oxaloacetate to malate.

Malate Thiokinase

Malate thiokinase from Methylococcus Capsulatus. This enzyme catalyzes the ATP-mediated condensation of CoA with malate, forming Malyl-CoA. The enzyme is composed of two subunits; alpha (CDS mtkA) and beta (CDS mtkB).

Malyl-CoA Lyase

Malyl-CoA lyase which catalyzes the conversion to glyoxylate and Acetyl-CoA from (S) Malyl-CoA. It is one of the enzymes whose activity is most critical in the pathway, catalyzing also a reversible reaction. Screening of possible variants has already been performed in the reference paper, considering the variant from Methylobacterium extorquens as the more active one.

Glyoxilate Carboglyase

This glyoxylate carboglase from Cuapriavidus necator catalyzes the decarboxylation of glyoxylate to tartronate semialdehyde. The variant from Cuapriavidus N. shows higher activity in Synechococcus than the homologous enzyme from E. coli.

2-Hydroxy-3-oxopropionate reductase

GarR is a Native NADPH-coupled reductase of E. coli that catalyzes the conversion of tartronate semialdehyde to glycerate. This enyme has been replaced by the studied NADH dependent reducatse (glxR) in order to tap into the more abundant NADPH pool of cyanobacteria.


GarK is an ATP-coupled glycerate-2-kinase from EEscherichica coli that catalyzes the substrate phosphorylation of glycerate to form 2-phosphoglycerate (2-PG).


2-PG is converted to 2-phosphoenolpyruvate by the action of an endogenous enolase, regenerating the starting molecule of the cycle.

MCG Image

n-Butanol Tolerance & Secretion

Previous strategies have a common objective: produce n-butanol and maximize its production. However, like many other alcohols, butanol is toxic to cells. As efficient as biosynthesis pathways may be, a production phenotype is always going to be limited by cell viability. Here we find a huge limitation: if we give the cells the tools to produce too much, the product might severely impair their growth. This is why many studies have been carried out to identify new strategies that help overcome this bottleneck.

Enhancing n-Butanol Tolerance

To overcome toxicity, many strategies are focused on increasing the host’s tolerance to the produced molecule. In our specific case, that molecule is butanol, a well-known toxic compound. As other soluble polar toxic molecules, it is known to cause protein misfolding issues and generate Reactive Oxygen Species (ROS). Another of the toxic effects associated with butanol is to cause membrane instability. Butanol destabilizes membranes causing ‘leakiness’ and thus compromising cell regulation at multiple levels. This effect is even more pronounced in photosynthetic organisms, as their whole photosynthetic apparatus is located within membranes. All of these effects generate stress for cells, compromising their growth and global performance.

We identified two main strategies to cope with butanol-derived stress:

  • Overexpression of heat-shock proteins (chaperones) has been shown to improve general tolerance of cells in the presence of alcohols like butanol. They prevent misfolding, protect membranes and protein complexes from butanol destabilization and deal with generated oxidative radicals.
  • Up- or downexpress certain regulators that operate on a larger scale. When exposing cells to alcohol stress there are some regulators that get up- and downregulated. Their effects are more difficult to predict because one single regulator controls the expression of many different genes which could be related or not with enhanced solvent tolerance.

Small heat shock protein HspA

We selected the well characterized heat shock protein HspA. The gene that codes this protein has been found to be upregulated almost 5-fold in the presence of 40 mg/L of butanol in Syechocystis. It deals with ROS derived from n-butanol stress and stabilizes misfolded proteins. It also associates with thylakoid membranes to help modulate membrane fluidity during heat shock, and is able to bind to photosystems and phycobilisomes in oxidative stress conditions. We definitely wanted to include this protein to our tolerance engineering strategy.

HspA Icon

Regulation factor SigB

SigB Icon

As a secondary approach, we wanted to include one of the regulation factors that many authors have found to be upregulated in butanol-stress conditions. This is SigB regulation factor, a cyanobacteria stress-related regulator protein. As a regulation factor, their direct targets are not entirely understood. However, an overexpression of SigB has demonstrated an improved n-butanol tolerance together with an enhanced high temperature tolerance. It also has been proved to reduce ROS derived from butanol-stress.

Implementing an efficient n-butanol secretion systrem

Another line of strategies to deal with toxic compounds is secretion. Instead of handling the toxic effects inside, we can avoid toxicity by secreting the toxic molecule outside the cell. We found that Resistance-nodulation-division (RND) efflux pumps are widely studied for secretion of target molecules. These transport machines are exclusive of Gram-negative bacteria, and have a typical three-component structure: an general outer membrane channel, a substrate specific inner membrane channel, and an intermediate periplasmic component that joins both inner and outer membrane subunits. These pumps rely on the proton gradient power rather than ATP. They are able to secrete substances directly from the cytoplasm, inner membrane and periplasm to the extracellular space. For the particular case of n-butanol secretion, a modified version of E. coli RND-efflux pump AcrAB-TolC system capable of selectively secreting n-butanol was found in the bibliography. Among all the proteins of the complex, AcrB protein is the main responsible of substrate recognition. By directed evolution, an n-butanol specific AcrB protein was achieved, termed AcrBv2.


acrAB-TolC Scheme

We found this protein a quite interesting approach to test in our secretion engineering strategy. Despite the fact that the functional RND complex comprises three components, cyanobacteria are known to have homolog proteins to AcrA and TolC. As these proteins can accommodate in a modular way, the system might work by only introducing AcrBv2, since analog proteins like Sll0180 or Slr1270 (analogs to AcrA and TolC, respectively) are already in the cyanobacteria genome.

The authors that created AcrBv2 also reported that uncontrolled overexpression of this transporter causes a detrimental effect for the cells. Transporter proteins have to be handled with care, as they can cause quinone extrusion from membranes. This same effect is produced due to membrane ‘leakiness’ induced by butanol-stress. This drawback can be overcome with a tight regulation of AcrBv2 expression. For this purpose, we will employ inducible and self-regulating promoters like Pgntk, which self-inhibits upon membrane stress, to tightly control the transporter expression.

acrAB-TolC Scheme

Implementing a secretion strategy has benefits regarding tolerance, as we remove the toxic compound from the cells avoiding its accumulation and reducing toxic effects. However, secretion has other important advantages for our project:

  • If we secrete the product, it does not accumulate inside the cell. This enhances the metabolic flux towards the product. By product removal we pull the carbon flux towards butanol synthesis.
  • Current cyanobacteria industry is mainly focused on intracellular products such as carotenoids or lipids. Once they have cultured enough biomass, they harvest and extract the compounds from the cells. This is a highly expensive and inefficient process. In the case of n-butanol we are able to retrieve the product directly from the culture media. This is cheap and does not rely on biomass, allowing treating the cells as photobiocatalysts. fMCGThe implementation of active secretion systems can furtherly ease n-butanol recovery.

Unraveling the potential of cyanobacteria

Finally, the last ace up the sleeve in any metabolic engineering strategy is the chassis. Any current metabolic design is eventually limited by the capacities of its expression host. Until we develop precise tools for complex pathway designs at genome-scale, the chassis where the metabolic pathway is implemented is still a crucial aspect of the overall pathway performance.

Even when closely related strains harboring a similar set of metabolites and metabolic reactions are used, high differences can be observed in pathway expression. With this in mind, the last piece for the generation of a truly cellular biofactory could be the utilization of a robust chassis whose metabolic capacities allows to unravel the full potential of the designed metabolic engineering approach.

Then we have decided to move forward from the conventional laboratory strains and utilize a recently discovered fast-growing cyanobacteria as chassis. This cyanobacteria, Synechcococcus elongatus PCC11801, exhibits a fast- growing phenotype at atmospheric CO2 concentrations4and double times compared with the fastest known cyanobacteria: Synechococcus UTEX2973 . To know more about the great potential of emerging phototrophic visit the Phototrophs SynBio page.

Eventually, we proceed to carefully design our genetic constructions that will harbor all the required information for the implementation of our metabolic engineering strategy. During this process we performed codon optimization of all the constructs for Synechococcus elongatus PCC7942. The vast majority of emerging phototrophics chassis shares a highly similar codon usage and even metabolic reactions , then the core of our genetic constructions could be easily transferred from one to another cyanobacterial strains.

n-butanol pathway

Liu, X., Miao, R., Lindberg, P., Lindblad, P., 2019. Modular engineering for efficient photosynthetic biosynthesis of 1-butanol from CO2 in cyanobacteria. Energy Environ. Sci. 12, 2765–2777.

Lan, E.I., Ro, S.Y., Liao, J.C., 2013. Oxygen-tolerant coenzyme A-acylating aldehyde dehydrogenase facilitates efficient photosynthetic n-butanol biosynthesis in cyanobacteria. Energy Environ. Sci. 6, 2672–2681.

Anfelt, J., Kaczmarzyk, D., Shabestary, K., Renberg, B., Rockberg, J., Nielsen, J., Uhlén, M., Hudson, E.P., 2015. Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production. Microb. Cell Factories 2015 141 14, 1–12.

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Lan, E.I., Ro, S.Y., Liao, J.C., 2013. Oxygen-tolerant coenzyme A-acylating aldehyde dehydrogenase facilitates efficient photosynthetic n-butanol biosynthesis in cyanobacteria. Energy Environ. Sci. 6, 2672–2681.

Shen, C.R., Lan, E.I., Dekishima, Y., Baez, A., Cho, K.M., Liao, J.C., 2011. Driving Forces Enable High-Titer Anaerobic 1-Butanol Synthesis in Escherichia coli. Appl. Environ. Microbiol. 77, 2905.

Lan, E.I., Liao, J.C., 2012. ATP drives direct photosynthetic production of 1-butanol in cyanobacteria. Proc. Natl. Acad. Sci. 109, 6018–6023.

Enzyme compartimentalization

Meng, D., Wang, J., You, C., 2020. The properties of the linker in a mini-scaffoldin influence the catalytic efficiency of scaffoldin-mediated enzyme complexes. Enzyme Microb. Technol. 133, 109460.

L, A., Y, C., LS, B., S, R., J, M., S, B., J, N., UH, M., 2011. Diversion of flux toward sesquiterpene production in Saccharomyces cerevisiae by fusion of host and heterologous enzymes. Appl. Environ. Microbiol. 77, 1033–1040.

Yang, J., & Zhang, Y. (2015). Protein Structure and Function Prediction Using I-TASSER. Current Protocols in Bioinformatics / Editoral Board, Andreas D. Baxevanis et Al], 52(1), 5.8.1.

Carbon Flux Rewritting

Yu, H., Li, X., Duchoud, F., Chuang, D.S., Liao, J.C., 2018. Augmenting the Calvin–Benson–Bassham cycle by a synthetic malyl-CoA-glycerate carbon fixation pathway. Nat. Commun. 9, 2008.

Hirokawa, Y., Kubo, T., Soma, Y., Saruta, F., Hanai, T., 2020. Enhancement of acetyl-CoA flux for photosynthetic chemical production by pyruvate dehydrogenase complex overexpression in Synechococcus elongatus PCC 7942. Metab. Eng. 57, 23–30.

Wang, B., Wang, P., Zheng, E., Chen, X., Zhao, H., Song, P., Su, R., Li, X., Zhu, G., 2011. Biochemical properties and physiological roles of NADP-dependent malic enzyme in Escherichia coli. J. Microbiol. 49, 797–802.

Claassens, N.J., 2017. A warm welcome for alternative CO 2 fixation pathways in microbial biotechnology. Microb. Biotechnol. 10, 31.

Zhou, J., Zhu, T., Cai, Z., Li, Y., 2016. From cyanochemicals to cyanofactories: a review and perspective. Microb. Cell Fact. 15.

n-butanol Tolerance

Anfelt, J., Hallström, B., Nielsen, J., Uhlén, M., Hudson, E.P., 2013. Using Transcriptomics To Improve Butanol Tolerance of Synechocystis sp. Strain PCC 6803. Appl. Environ. Microbiol. 79, 7419.

Srivastava, A., Varshney, R.K., Shukla, P., 2021. Sigma Factor Modulation for Cyanobacterial Metabolic Engineering. Trends Microbiol. 29, 266–277.

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Jaiswal, D., Sengupta, A., Sohoni, S., Sengupta, S., Phadnavis, A.G., Pakrasi, H.B., Wangikar, P.P., 2018. Genome Features and Biochemical Characteristics of a Robust, Fast Growing and Naturally Transformable Cyanobacterium Synechococcus elongatus PCC 11801 Isolated from India. Sci. Rep. 8.

Novel Photoautotrophic Chassis

Durall, C., Kukil, K., Hawkes, J. A., Albergati, A., Lindblad, P., & Lindberg, P. (2021). Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt. Microbial Cell Factories 2021 20:1, 20(1), 1–14.

Sengupta, S., Jaiswal, D., Sengupta, A., Shah, S., Gadagkar, S., & Wangikar, P. P. (2020). Metabolic engineering of a fast-growing cyanobacterium Synechococcus elongatus PCC 11801 for photoautotrophic production of succinic acid. Biotechnology for Biofuels 2020 13:1, 13(1), 1–18.

Jaiswal, D., Sengupta, A., Sohoni, S., Sengupta, S., Phadnavis, A. G., Pakrasi, H. B., Wangikar, P. P. (2018). Genome Features and Biochemical Characteristics of a Robust, Fast Growing and Naturally Transformable Cyanobacterium Synechococcus elongatus PCC 11801 Isolated from India. Scientific Reports, 8(1).

Jaiswal, D., Sengupta, A., Sengupta, S., Madhu, S., Pakrasi, H. B., & Wangikar, P. P. (2020). A Novel Cyanobacterium Synechococcus elongatus PCC 11802 has Distinct Genomic and Metabolomic Characteristics Compared to its Neighbor PCC 11801. Scientific Reports 2020 10:1, 10(1), 1–15.