Production increase and control module

As the major part of our project, this module is aimed to transform the normal E.coli into a powerful E.coli factory for FFA (free fatty acids) production. Specifically, we expect this factory to produce higher amounts of FFAs than usual. Also, like a real factory providing products to meet various demands of different clients, its production process would be highly controllable so that we could obtain the desired fatty acid whenever we want it. To achieve this goal, we target several genes in the fatty acids biosynthetic pathway in E.coli.

What are the key genes

First of all, we cast our eyes at genes fabA and fabB so as to control the saturation levels of FFAs. In fact, among all the reactions of FFAs biosynthesis in E.coli, there are only two unique reactions specifically required to produce unsaturated fatty acids. And they are catalyzed by enzymes FabA and FabB, respectively. Therefore, we are lucky that we could just regulate the expression of these two genes when we want to change the saturation levels of final products.

While in terms of controlling the carbon chain length of FFAs, it is another story since it is far more complicated than we thought. We first focused on the rate-limiting reaction among the whole synthesized process, which was catalyzed by the enzyme AccABCD (acetyl-CoA carboxylasea). We thought maybe we could increase the FFAs production by overexpressing this key enzyme. There was, however, a huge problem in this method. As the concentration of acyl-ACP (acylated derivatives of acyl carrier protein, the end-product of fatty acids biosynthesis) gets higher, its feedback inhibition to AccABCD as well as another two enzymes essential for fatty acids synthesis also gets stronger.

In fact, at the final step of fatty acids biosynthesis, the enzyme TesA (the major thioesterase in E.coli) would remove the fatty acyl moiety from the acyl-ACPs, releasing them as FFAs and relieving their inhibited effects on the whole pathway. While in wide-type E.coli, the enzyme activity of TesA is very low with the substrate acyl-ACPs. In this case, the E.coli can switch off the fatty acids synthesis when fatty acids are sufficient to support their life.

This negative feedback inhibition is no doubt the nature of an organism. And now, we have to break this natural rule. In other words, if we can enhance the activity of TesA by gene modification or other means, acytl-ACP’s inhibited effects on rate-determing step can be automatically released, thereby continuously boosting the production of FFAs. More surprisedly, after reading a vast number of reports and papers, we found that thioesterases from different organisms varied with each other in substrate specificities, targeting acyl-ACPs with different length of carbon chain. And some researches showed that the overexpression of thioesterases from other species could indeed overproduce FFAs in E.coli. So by just introducing this one gene into E.coli, we could realize both the production increase and carbon chain length control. And now, what we have to do is to find out the exact thioesterase genes that we want.

After devouring all kinds of researches, we found out several promising thioesterase genes from plants or bacteria source (see table below). As reported, they all have the superiority of FFAs accumulation or a novel substrate specificity.

Among these genes, we want the one with perfect balance between the high efficiency and specificity. In addition, the C12/14 and C16/18 fatty acids are the most widely used feedstocks for the production of surfactants, detergents and other products worldwide. Thus, we decided to use bte gene and AtfatA gene in order to improve the function of wide-type E.coli.

Make it controllable

Next, what we need to do is to construct a plasmid with all the genes we want, introduce the circuit into E.coli and let it overexpress these genes. This being said, it is enough to just add a strong promoter at their upper stream? Obviously the answer is no. We do not want to produce FFAs in an indiscriminate manner. Instead, we are determined to make it controllable. Technically, when given a specific signal, the engineered E.coli should be able to produce the corresponding FFAs. Considering this criteria, we think the operon would be an ideal controlled switch.

As we know, operon is composed of several structural genes, a single promoter enabling them to be transcribed and an operator to regulate their transcription. When a repressor or activator binds to the operator, the transcription of structural genes would be repressed or activated. While some specific signals exist, the situation could be reversed. Inspired by this, we decided to insert the promoter and the corresponding operator structures at the upstream, which could help to control the transcription of our targeting genes. Now, there were tons of operons, which one should we choose?

Since we were using E.coli as our biofactory, we narrowed down the scope into three most common operons in E.coli: the lactose operon, the tryprophan operon and the arabinose operon. And we would construct our circuit as showed in picture below. The lac operon would be responsible for the transcription of Atfata gene, the ara operon for bte gene and the trp operon for fabA gene and fabB gene.

How the module works

In summary, the whole module would work as following:

Both the lac operon and the ara operon work as negative inducible operons in our module. Normally, there is a regulatory repressor bound to the operator and preventing the transcription of downstream genes. So when we want to obtain C16/C18 and C12/C14 FFAs, we would place lactose/ IPTG and arabinose into the culture medium, respectively. And this would allow the expression of enzymes AtFatA and BTE, which leads to the further production of desirable FFAs.

While trp operon is a typical negative repressible operon. When no tryptophan is added to the culture medium, the concentration of tryptophan is low in E.coli. As a result, the repressor cannot bind to the operator and gene fabB and fabA would be transcribed. What we finally obtain are therefore the high-level unsaturated fatty acids. Otherwise, we could increase the saturation levels by using culture with tryptophan.