Difference between revisions of "Team:NPU-China/Background"

Introduction

In the past 100 years, the rapid development of the traditional chemical industry has greatly promoted the improvement of people’s material living standard. Our basic necessities of life are almost inseparable from the chemical synthesis goods. However, the environmental pollution and energy crises have also forced people to find new solutions. Synthetic biology instructs us that we can introduce new chemical reactions into biological cells, thus producing high quality chemical products in a greener way.

Then what does synthetic biology "synthesize"?

Biosynthesis of synthetic biology lies mainly in the biosynthesis of natural product and synthesis of bulk chemical. The former is represented by artemisinin, lycopene and carotene, etc., and the use of synthetic biology method to synthesize our daily necessities of traditional chemical products or raw materials can serve more people. Today, scientists have been able to use micro-organisms or modified industrial enzymes to synthesize bio-plastics, bio-fuels, chemical raw materials and other chemical products.


For example, DuPont has achieved the reality of micro-algae efficiently synthesizing isobutanol; Blupha, a well-known company to Chinese iGEM teams, has also mastered the biosynthetic method to get PHA production. However, most of the existing products are facing the dilemma as for the cost, making them outshined by the traditional chemical products, which in fact limits the industrial promotion of synthetic biology.

Background

This year, we focus mainly on an important synthetic organic chemical raw material——acrylic acid. We hope to build efficient cell factories to achieve "all green" production of acrylic acid.

What is acrylic acid?

Acrylic acid is an important synthetic organic chemical raw material. Acrylic acid and its ester compounds are widely used in adhesives, coatings, synthetic rubber, high absorbent resin and other chemical products.

The existing methods of producing acrylic acid

According to our current research carried out about the acrylic acid synthesis method, we list them as follows:

1.Traditional chemical synthesis

Acrylic acid two-step oxidation

The first step of two-step oxidation of acrylic acid is propylene reacts with oxygen, producing acrolein. And then, acrolein reacts with oxygen, producing acrylic acid. The conversion rate is up to 90%. Chemical Formula (1) and (2) are showed below. This method is widely used in industry production of acrylic acid.

H2C=CH-CH3+3/2O2→H2C=CH-CHO+H2O(1)
H2C=CH-CHO+ 1/2O2→H2C=CH-COOH(2)

The process of the oxidation method of the propylene is mentioned in the patent of the Japanese Catalyst Company in the 1880s. The reaction temperature of the first step is 320-330℃. If other additives such as W, Co, K, Si and O are added to the catalyst MoBiFe, the yield of acrolein can reach more than 90%. The reaction temperature of the second step is 210-255 ℃. The compositions of the catalyst are Mo, Bi, Fe, Co, K and O. The yield of acrylic acid can reach 97.5%. The yield of acrylic acid in the two-step oxidation process is much higher than that of the direct oxidation of propylene to produce acrylic acid. This is because the reaction temperature in the one-step process is 325-350 ° C, at which the conversion of propylene to acrolein is high. But acrolein and acrylic acid are further oxidized at that temperature. Therefore, to obtain higher yields of acrylic acid, control the reaction at different stages by controlling the temperature and changing the catalyst composition in the two-step method is more reasonable and more valuable. As the most widely used method in the industrial production of acrylic acid, oxidation of propylene has the advantages: high conversion efficiency and simple production process.

Propylene firstly reacts with oxygen to produce acrolein, whose deoxidation leads to the production of acrylic acid. The conversion rate is often up to 90%, so this method is applied in most industrial production of acrylic acid
Although this practice has many advantages, but the raw material depends heavily on the traditional fossil energy, bringing about heavy pollution, high energy consumption and a lack of sustainability. Therefore, it is imperative to develop renewable energy alternative to replace fossil energy to produce acrylic acid in a greener way.

2.Acrylic acid semi-biosynthesis
Acrylic acid semi-biosynthesis refers to the method of using micro-organisms to turn acrylonitrile, acrylamide and other petrochemical raw materials into acrylic acid.

Acrylonitrile conversion

So far, the researchers have found that certain microorganisms which contain nitrilase can catalyze the conversion of acrylonitrile to acrylic acid. Nitrilase can catalyze the nitrile compound directly to produce the corresponding products of carboxylic acid. Nitrilases are present in Rhodococcus, Pseudomonas, Nocardia and Bacillus.

There are many advantages of Hydrolysis of Acrylonitrile to produce acrylic acid, for example, specificity of nitrilases, high conversion rate of acrylonitrile, the moderate reaction conditions, and less by-products. However, acrylonitrile is a chemical product comes from fossils, and the price is higher than acrylic acid, so it is not suitable for the needs of sustainable development and does not meet the long-term development of society.

Acrylamide conversion

Acrylamide also relies on petrochemical resources to obtain the product. According to current report, Rhodococcus (Rhodococcus AJ270), Pseudomonas aeruginosa, Bacillus thuringiensis BR449 and other microorganisms can catalyze the conversion of acrylamide to acrylic acid. Acrylamide is converted to acrylic acid by amidase. Rhodococcus AJ270 can grow with acetamide as a carbon source and show high amidase activity during metabolism. The conversion of acrylamide has the advantages of moderate reaction conditions, high conversion rate, long reaction time and large yield, but it has the same shortcomings as the conversion of acrylonitrile. Rhodococcus AJ270 needs amidase as raw material to express acetamide, and the price of acetamide and acrylamide is high, thus limiting the industrial production using this method.

Acrylic acid semi-biological method, although possesses the high yield, its raw materials acrylonitrile and acrylamide cost even more than acrylic acid, which limits the industrialization of this method.

3.Acrylic acid complete biosynthesis
Acrylic acid complete biosynthesis method refers to the direct use of saccharides and other biomass fermentation to produce acrylic acid.

Lactate dehydration pathway

Just as its name implies, lactic acid dehydration pathway refers to a metabolic pathway using the dehydration of lactic acid to produce acrylic acid. Because the fermentation production process of lactic acid is very mature, so using the dehydration of lactic acid to produce acrylic acid has long been aroused the interest of the researchers.

At present, the method mainly uses Clostridium propionicum as the starting strain. Specific metabolic pathway is shown in the following picture. Although the route is short, but there are still many problems when used in producing acrylic acid.

As can be seen from the picture, metabolism of lactic acid is divided into the left side, the oxidation pathway and the right side, the reduction pathway. When Clostridium propionate uses lactic acid as a source of energy, one molecule of lactic acid produces one third of the molecule of acetic acid and 2/3 of the propionic acid. In the production process of acetic acid, there will be four electrons and 4 protons generate, as well as offering ATP for bacteria to grow. The acryloyl CoA in the reduction pathway receives electrons to produce propionyl CoA and finally generates propionic acid.

3-hydroxypropionic acid pathway

3-hydroxy propionic acid (3-HP) is an important chemical intermediate, there are a number of patents have reported the methods of sugar conversion to 3-HP. And 3-HP has two functional groups, carboxyl and hydroxyl. It is possible to synthesize a variety of important chemical substances such as 1,3-propanediol, succinic acid through oxidation, dehydration and esterification reaction,. Using 3-HP to develop downstream products, such as acrylic acid is also one of the hot spots.

Using 3-hydroxypropionic acid-related pathways to construct engineering bacteria to produce acrylic acid is a hotspot in recent years, but the yield is generally not high, partly because of the toxic side effects of acrylic acid on cells and, on the other hand, the pathway is long, and the reaction requires vitamin B12 to participate in, it is very difficult to achieve high yield of acrylic acid with this approach.

Propionic acid oxidation pathway

Propionic acid oxidation pathway refers to Clostridium propionate use propionic acid as a substrate to produce acrylic acid under the aerobic conditions. It is speculated that Propionic acid oxidation pathway is the reverse pathways from lactic acid to propionic acid in Clostridium propionate, namely propionic acid to propionyl CoA, and then to acryloyl CoA , finally converted to acrylic acid. Propionic acid oxidation pathway has the same problem as lactate dehydration pathways, that is, need to add exogenous electron acceptor to achieve coenzyme regeneration, so that the entire cell system remained stable.

DMSP pathway

Dimethylsulphoniopropionate (DMSP) is a sulfur metabolite synthesized in some higher plants in sea water, algae and marine microalgae. Some marine organisms such as bacteria, phytoplankton have DMSP lyase, which cleaves the DMSP into acrylate and dimethyl sulfide (DMS), which is also commonly found in Alcaligines faecalis, The route is shown in Figure 1.3. Extracellular DMSP is transported into the body by the carrier protein, be cleaved into acrylic acid and DMS by DMSP lyase. Acrylic acid is further reacted to form β-HP.

The DMSP pathway is the only biological pathway that can directly produce acrylic acid. The DMSP lyase is the key enzymes, but it is not clear at present that there is no definite sequence of its genes. Therefore, research report about making use of or modifying DMSP pathway is rarely seen.

Some shortcomings of the existing acrylic acid biosynthesis method include complexity of the synthetic pathway , obscuration of the synthesis mechanism and low efficiency of the synthesis. How to build a short and efficient acrylic acid biosynthetic pathway to achieve a highly efficient acrylic biosynthetic factory is the very key to success! And this is also the entry point of our project this year.

Why we choose Glycerol as cabon source？

Glycerol is a simple polyol compound, which presents as viscous liquid at the room temperature. It is colorless, tasteless and non-toxic. Glycerol is a by-product of the biodiesel manufacturing industry, which once was a relatively scarce chemical raw material. With the rapid development of bio-diesel manufacturing industry in recent years, the substantial increase of glycerol production has led to the significantly lower price. Therefore, the use of glycerol as a raw material for microbial cell factory to produce bulk chemicals has the advantage of being cheap and green, while it also allays the pressure of dealing with the by-products waste in the production of biodiesel. In addition, compared with glucose, xylose and other carbohydrate substrates, glycerol metabolism can produce higher reducing power, making it the ideal carbon source for the fermentation synthesis in cell factory.