Recreating a Recombinant R.opacus Bacteria that Can Use Chitin

By Jackie Ramirez

 

Introduction

In 2022, roughly 119 million pounds of American lobster (Homarus americanus) were landed, and this catch was valued at around $515 million. With this gigantic haul of seafood, consumers will eat <50% of the animal, which makes up the lobster meat. The majority of lobster biomass is inedible and is discarded by homes, restaurants and other facilities, and the majority of that waste is lobster shell. The lobster shell contains three main constituents: minerals like calcium carbonate (CaCO3), proteins and chitin/chitosan polysaccharide. Of these shell components, chitin and chitosan have shown value in bio-based processes. Chitin and chitosan are carbohydrate polymers consisting of the amino sugars N-acetyl-D-glucosamine (glcNAc) and/or D-glucosamine (glcN) monomer units. Depending on the degree of acetylation of the polysaccharide, the polymer may be called chitin or chitosan, where the majority of the monomer concentration of chitosan is D-glucosamine. Chitin and chitosan are very attractive biomaterials with a range of household and industrial uses. Regardless, there remains a large percentage of lobster shells that are discarded or underutilized.

Chitin as a biomaterial for biofuel production is a promising and new area of research that will contribute to solving the global climate crisis. Chitinase ChiA, ChiB, and ChiC break down chitin into monomers of N-acetyl glucosamine (NAG). ChiA is an endochitinase that breaks down chitin within a polymer. ChiB and ChiC are exo-chitinases that cleave monomers at the end of a polymer. The monomers produced by these enzymes are used to produce triacylglycerols (TAG). From here, the triacyclglycerols can be trans-esterified into biodiesel. The bioengineering department here at UMass Dartmouth has looked to the surrounding South Coast of Massachusetts as a source of chitin for biofuel production. The shells of crustaceans comprise of 40% chitin by weight. Through research efforts at UMass Dartmouth, chitin has been derived and separated from the protein components of lobster shells (1). This ecofriendly extraction method has given researchers here the ability to utilize crustacean waste from human consumption to isolate chitin and use it for biofuels in conjunction with Rhodococcus Opacus (R. Opacus). R. Opacus, strain PD630, is a gram-positive microbe which will accumulate TAG in the presence of a steady carbon source. It’s high lipid storage ability and rapid turnover rate make it an excellent candidate for biofuel production (2). Chitin is a proposed carbon source for the bacterium. R. Opacus, which is unable to produce the chitinases necessary to break down chitin into its monomer counterparts for biofuel production, therefore this project will make a recombinant strain of R. Opacus to express and secrete chitinase enzymes.

Soon after receiving an OUR grant, my mentor and her collaborators changed the strategy to use the shells. R.opacus is a difficult bacteria to genetically manipulate, so they decided to get another bacteria that is easier to manipulate and has a better chance of taking up plasmids that have the chitinase genes on them. We switched to pseudomonas aeruginosa.

Methods

Genomic DNA isolation using the Promega gDNA isolation kit.

Design primers specific for ChiA from the bacteria S. marcescens and amplify the gene from the genomic DNA.

Initial PCR conditions: Using 5ul gDNA, 1ul of ChiA-For primer and 1ul ChiA-rev primer plus Taq Supermix. The reaction proceeded with standard PCR cycle parameters with annealing at 59 degrees Celsius and 45 cycles.

Second Attempt using a temperature gradient to see what temperature is ideal for primers to anneal to the template.

Figure 1: We tested 57 degrees upto 62 degrees. Each bar indicates the temperature in that well.

Third attempt PCR: We switched to using Q5 high-fidelity Taq DNA polymerase.

Figure 2: These are the PCR Parameters we used with High Fidelity Taq.

Results

Initial attempts to amplify the ChiA, ChiB and ChiC genes from s.marcescens gDNA (Figure 3). The faint bands at the bottom of each lane are primer dimers. We are expecting bands between 1.0kb and 1.5kb. After some research we decided to try using a Taq polymerase that had High Fidelity. The reason was because we are trying to find one gene in a genome of 5,241,455 bp, and we figured that a DNA polymerase that could stay associated with the template better might allow us to get the genes. The High fidelity Q5 DNA polymerase resulted in the expected products between 1kb and 1.5 kb (Figure 4).

                        

Figures 3 (L) and 4 (R): First attempts to amplify ChiA, B and C. 

Having figured out how to get the correct bands I focused on Chitinase B. I was able to amplify ChiB and gel purify only the correct sized band (Figure 5).

Figure 5: Gel purified ChiB genes

The project is being continued by another student. The next steps are to cut the ChiB insert with enzymes and insert it into the vector.