Team:Amsterdam/Project/Eng rom/Photosyn car

Engineering Stable Carbon Production

Sharing is Caring

Overview

Background

The driving force of our consortium's romance is the phototrophic carbon-sharing module: an engineered Synechocystis that fixes CO2 and produces compounds that can be used by a chemotroph to create desired end-products like biofuels.

Aim

Productive relationships need to stand the test of time. Having experienced the perils of instability firsthand, we sought to engineer carbon production in way that would last.

Approach

Using genetic engineering strategies guided by modelling results, we used a combination of gene knock-outs and over-expressions to target a pathway that would result in growth-coupled acetate production.

Results

• We engineered growth-coupled acetate production by knocking out the acs gene, showing that this indeed leads to stable acetate production
• We over-expressed the Pta and AckA1 proteins to increase the flux towards acetate formation, but found that the burden this puts on growth comes at a cost.

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Connections

• Stable Romance: Measure stability.
• Engineering Romance: Using software tools to select targets
• Measuring Romance: Turbidostat experiments.

Figure 1. - Growth and Q_p of strain Δacs over 900 hours of continuous culture showing a constant production over the duration of the experiment.

Figure 2. - Acetate concentrations over time in the variety of engineered Synechocystis strains.

Change of plans

Different carbon compounds produced by Synechocystis could serve as fuel for E. coli in our consortium: glucose, lactate, and glycerol for example, are all products that an engineered cyanobacteria can produce.

Figure 3. - Potential carbon compounds that Synechocystis can produce.

Although glucose seems like a great compound to drive sustainable bio-production with, our initial plan was to develop Synechocystis strains that produce all of these products, and to compare their performances in a consortium with E. coli generating different products.

After the initial results of lactate production came in, however, we decided to make genetic stability the central focus of our carbon fixation efforts: engineering a Synechocystis that produces a carbon compound in the most stable way possible such that its relationships with other consortia-species last more than one or a few nights. Based on the results of our computational tools, which parsed the genome-scale metabolic map to search for compounds that could be produced in a growth-coupled way, we decided to focus on acetate production.

Methods

As the genetic engineering of carbon production in Synechocystis involved relatively simple constructs, standard restriction-digestion cloning protocols were used for most engineering efforts. Synechocystis knock-outs were created using the specific markerless knock-out method described here. This two-round knock-out approach allowed for the cultivation of knock-out strains without the need of antibiotics and thus increased flexibility of implementation in a potential consortium.

Acetate production involved the knock-out of the native acs gene and heterologous over-expression of the ackA1 gene from Lactococcus lactis and pta gene from Synechocystis.

Figure 4. - Schematic overview of the targeted acetate pathway. The acs gene represents the recycling reaction that is knocked-out to enable growth-coupled production, while ackA1 and pta were targeted to further increase acetate production.

The acs gene encodes for acetyl-CoA synthase, an enzyme that converts acetate into acetyl-CoA. This is the acetate recycling reaction in the biomass formation pathway. The reactions that actually lead to the formation of acetate as a side-product represent a two-step process and are governed by pta, encoding for the phosphate acetyl-transferase enzyme that converts acetyl-CoA into acetyl-phosphate, and ack, encoding for an acetate kinase that removes acetyl-phosphate's phosphate group to produce acetate (technically functioning as a phosphatase). Although over-expressing ackA1 and pta via gene insertions is prone to the fixation of mutations, we still wanted to see its impact on overall acetate production. The following constructs were used for the knocking out of acs and insertion of ackA1, pta, or fused ackA1/pta:

Figure 5. - Constructs used to knock-out acs and over-express ackA1 and pta. All plasmids contained homologous sequences of neutral sites in the Synechocystis genome that enabled homologous recombination to take place in order to insert or eliminate target genes. AckA1 and Pta were expressed via the constitutive Pcpc promoter.

After the genetic engineering steps, batch and turbidostat cultivation experiments were conducted to measure acetate production levels and stability. Acetate concentrations were measured using the Megazyme acetate assay kit.

Results

Stable acetate production

By knocking out the acs gene, we expected to eliminate the acetate recycling reaction in the biomass formation pathway, which would lead to acetate accumulation in growth-coupled fashion. Although this is what the model indicated should happen, modelling results hardly ever translate perfectly to real-world scenarios. In order to test the Synechocystis acs mutant's acetate production (only acs was knocked out for this strain, ack and pta were not yet overexpressed), we conducted the turbidostat experiments described in more detail on this page.

Figure 6. - Turbidostat results showing stable production of acetate by a Synechocystis acs mutant over long incubation periods.

We indeed observed stable acetate production over time, maintained well over 900 hours until the end of the experiment. The contrast this result offers to the lactate results shows that knocking out the acetate recycling reaction in the biomass-formation pathway represents a succesful strategy for inducing stable production.

Ramping up production

In an attempt to further increase acetate production and increase the production capacity of our consortium, we took the acs mutant described above and over-expressed the ackA1 and pta genes - encoding the two reactions that lead to acetate formation in the central carbon metabolism pathway. During the engineering of the pta strain, we discovered several mutations had occurred in the Pta encoding fragment, resulting in a frame-shift mutation that removed the stop codon and extended the amino acid chain. We therefore created a second strain from scratch, but included the first strain to investigate the impact of the frameshift mutations. The three resulting strains, ackA1, pta mutated, and pta, were cultivated in a batch reactor with OD-dependent light intensity over a period of four days.

Figure 7. - Total acetate concentrations over the course of tcultivation. Although acetate concentrations reach their highest values for the pta strain, the yields and Q_p's are lower than the acs mutant strain during the period of growth. Only when growth - and thus growth-coupled production - stops can pta's acetate concentrations accumulate beyond the acs mutant strain's.

The results depicted in figure 7 show that although acetate concentrations were higher in the pta strain compared to the acs mutant strain (again, all engineered pta and ack strains also had the acs KO background). Nevertheless, both the total yields and Q_p's are higher for the acs mutant during the period that both strains grow. More than that, the pta strain has a lower growth rate than the acs mutant during this period, providing evidence that any non-growth coupled production strategy is ultimately out-competed during growth phases. We show that this statement holds even when the non-growth coupled strategy is implemented in a strain that already makes a product in a growth coupled fashion. The take-away here is that in order to stabilize non-growth coupled production strategies, we need synthetic gene-circuits that conditionally activate production during non-growth period. In this case, a circuit activating pta under dark conditions only, i.e. when Synechocystis does not grow, would help improve production, as at that stage, it would not produce acetate otherwise via growth-coupled production.

Table 1. - Estimated Growth and Q_p for acs, ackA1, and pta Synechocystis strains.