Introduction

Due to the competition time limitation, and the experiment progress has been influenced by COVID19, some of our ideas for the project didn't get carried out in time. Nonetheless, if conditions allow, we hope to continue refining and verifying our project in the future.

In preliminary work, we found there is an abundance of evidence to show that short-chain fatty acids (SCFAs) play an important role in the maintenance of health and the development of disease. SCFAs are a subset of fatty acids that are produced by the gut microbiota during the fermentation of partially and nondigestible polysaccharides. The highest levels of SCFAs are found in the proximal colon, where they are used locally by enterocytes or transported across the gut epithelium into the bloodstream. Two major SCFA signaling mechanisms have been identified, inhibition of histone deacetylases (HDACs) and activation of G-protein-coupled receptors (GPCRs). Since HDACs regulate gene expression, inhibition of HDACs has a vast array of downstream consequences. Our understanding of SCFA-mediated inhibition of HDACs is still in its infancy. GPCRs, particularly GPR43, GPR41, and GPR109A, have been identified as receptors for SCFAs. Studies have implicated a major role for these GPCRs in the regulation of metabolism, inflammation, and disease. SCFAs have been shown to alter chemotaxis and phagocytosis; induce reactive oxygen species (ROS); change cell proliferation and function; have anti-inflammatory, antitumorigenic, and antimicrobial effects; and alter gut integrity. These findings highlight the role of SCFAs as a major player in maintenance of gut and immune homeostasis.

Therefore, for SCFAS having those beneficial influence for our gut homeostasis, we designed pathways as well as experiment, aiming to conduct our engineering bacteria to produce SCFAs.

Four kinds of SCFAs were included: Acetate, Propanoate, Butyrate, and Beta-hydroxybutyrate (BHB). These four short-chain fatty acids can be feasible to be produced by synthetic biology and our meal replacement machine. We designed production pathways of these four SCFAs in lactic acid bacteria.

Acetate

Pathway: we designed to add citrate as substrate, target gene citE regulates the expression of enzyme citrate lyase beta chain which decompose citrate and produce acetate.

To test its feasibility, we plan to inserting the target gene into the plasmid pET-28a(+), adding fluorescent protein in the whole sequence. By detecting the fluorescent protein , we can reveal the expression of the protein: RFU unit (or OD )

More about the functionality of the product:

Acetate can be characterized in multiple ways, simply combined with acid and test the pH can be feasible. Besides, the special pungent smell can be characterized as well.

Plasmid:

Propanoate

Pathway: adding pyruvate as substrate, target gene ldhA regulates the expression of D-lactate dehydrogenase which decompose pyruvate and produce (R)-lactate. Gene Me-pct regulates the expression of propanoyl-CoA transferase, gene Ap-pct regulates the expression of propionate CoA-transferase. and propionate CoA-transferase decompose lactate and produce our target production propanoate.

For testing procedures: we insert two target genes into the plasmid pET-28a(+), adding fluorescent protein in the whole sequence. Then detect the fluorescent protein to reveal the expression of the protein。

More about functionality of the product: Propanoate can be characterized in multiple ways, simply combined with acid and test the pH can be feasible. Besides, the fruity odor of it can be characterized easily.

Butyrate

Pathway: ethanol degradation I is a degradation progress happens in our engineered bacteria. The degradation produces acetyl-CoA. Gene fadA regulates the expression of coenzyme A. Coenzyme A oxidizes acetyl-CoA to acetoacetyl-CoA. Gene yafH regulates the expression of acetyl-CoA which oxidizes crotonyl-CoA to butanoyl-CoA and finally leads to butryate.

Plasmid:

Beta-hydroxybutyrate

β-hydroxybutyrate (BHB)is synthesized in the liver from fatty acids and represents an essential carrier of energy from the liver to peripheral tissues when the supply of glucose is too low for the body’s energetic needs. Now, some researches indicate that BHB have other functions except from being as a convenient carrier of energy from adipocytes to peripheral tissues during fasting or exercise. BHB have important implications for the pathogenesis and treatment of metabolic diseases including type 2 diabetes. In small studies, provision of BHB precursor molecules improves cognition in an Alzheimer’smouse model and in a patient with Alzheimer’s disease. Further exploration of the links between BHB signaling, epilepsy, and dementia may prove fruitful in generating new translational therapies.

As a result, it will be of great benefits if we can produce BHB easily with synthetic biology skills. Luckily, we find a pathway feasibly.

Pathway:

1. Acetoacetyl-CoA: the oxidation of fatty acid β produces acetoacetyl-CoA, or acetoCoA is synthesized under the action of thiolase.

2. HMG-CoA: Acetylacetyl-CoA and acetyl-CoA form HMG-CoA under the catalysis of HMG-CoA synthase.

3. Acetoacetate: HMG-CoA form Acetoacetate under the catalysis of HMG-CoA lyase.

4. D-β-Hydroxybutyrate: Acetoacetate formed D-β-hydroxybutyrate under the catalysis of D-β-Hydroxybutyrate dehydrogenase.