Difference between revisions of "Part:BBa K5255003"

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MLACS1

Long-chain acyl-coenzyme A (CoA) synthetases (LACSs) family play a critical roles in fatty acid metabolism by activate free fatty acids to acyl-CoA thioesters. LACSs uses DHA, CoA and ATP as substrates to synthesize DHA-CoA. Basic parts MLACS1 (BBa_K5255003) is a mutant of wild-type LACS1(BBa_K5255000)，a member of LACSs family. The MLACS1 sequence was obtained by mutating site 277 of LACS1 from histidine to methionine. In practice, MLACS1 was introduced into Saccharomyces cerevisiae YB525 , E.coli BL21 (DE3) and E.coli C43 (DE3) to achieve several goals. The activity of the enzyme expressed by MLACS1 was verified by 2 closely related E.coli and the catalyzed product DHA-CoA was verified by Saccharomyces cerevisiae to ensure the synthesis of DHA-CoA and the feasibility of subsequent synthesis of DHA-PC.

Usage & biology

The enzyme family of LACSs serves as an vital role in fatty acid metabolic pathway, the existence has been proven in a variety of species ranging from prokatyotes to eukaryotes. We first selected several subtypes of LACSs family in species with close relationship with our target fungus. Since the schizochytrium limacinum was identified as the protein expression chassis. the engineer object enzyme shoubld be a subtype of LACSs that has close phylogeny to target fungus. A set of characterized subtypes of LACS in protein database were screened out. After phylogenetic analysis, LACS1 was selected through prediction and experimental analysis.

LACS1 is derived from Arabidopsis thaliana(Mouse-ear cress) and can play a role in yeast (Pulsifer et al.,2012), promoting the uptake of a very long-chain fatty acid. It also can catalyze the following reaction: A long-chain fatty acid + ATP + CoA = a long-chain fatty acyl-CoA + AMP + diphosphate (Shockey et al., 2002). LACS1 is located in the endoplasmic reticulum (Hua, 2010) and has high synthase activity against VLCFAs (Very Long-Chain Fatty Acids) C(20)-C(30), among which it has the highest activity against C(30) fatty acids (Lu, 2009). In theory, LACS1 also has a catalytic effect on DHA (22:6), consequently modeling can be used to predict its mutation sites and improve the affinity of LACS1 to DHA substrates.

Nucleotide sequence optimization was first performed on LACS1. After obtaining the nucleotide sequence of LACS1 from protein database, we first parsed the sequence and intercepted the open reading frame to obtain cDNA, and while discarding the rest of the sequence. We then carried out codon optimization by increasing preferred codons, increasing C/G content, and reducing minor secondary structures including hairpins and internal loops. These modifications resulted in a significant increase in mRNA minimum free energy(MFE), and remained consistent with multiple estimation algorithms, proving a solid improvement in mRNA performance. To sum up, the methods of codon optimization aims to improve LACS1's efficiency of transcription and translation, which will further contribute to a better production of our target product DHA-PC.

Amino acid sequence optimization with protein engineering methods was then conducted. To find promising mutagenesis sites to modify, we first adopted several traditional semi-rational protein engineering methods on LACS1, including random mutagenesis, alanine scanning, conservation analysis and free energy grading. However, semi-rational approaches have its commonly acknowledged limited accuracy. In order to address that limitation and provide a more reasonable protein engineering solution, we further researched on the catalytic mechanism of LACS1, and performed rational engineering methods accordingly, including characterization and mutagenesis of motifs that are critical to the reaction process, modification of residues surrounding the substrate-binding pocket with respect to electric charge, hydrophobicity and volume. The semi-rational and rational methods stated above constitute our integrated protein engineering. Integrating our semi-rational and rational approaches, we then selected the most promising mutagenesis among all the candidating sites. We generated sites as candidates with some of the approaches, graded all candidates using other approaches, and finally selected the mutagenesis with the highest-ranking score. The semi-rational and rational approaches that we applied are all based on solid algorithms and principles, and the selected mutagenesis site is expected to yield improvement in the catalytic activity of LACS1.

The target gene of MLACS1 was connected to pYES2/CT vector plasmid, and then the plasmid containing the target gene was transfected into BL21 (DE3), C43 (DE3) and YB525 strains by chemical transformation method. Then colony PCR was used to verify whether the vector plasmid was successfully introduced.

C43(DE3) strain is more suitable for expressing membrane proteins compared to other strains like BL21(DE3), as it reduces toxicity and allows for higher yields of functional proteins.

NADH-coupled assay is performed to monitor NADH consumption over time, providing an indirect but rapid measurement of enzyme activity. It is based on linking the MLACS reaction to a subsequent reaction that consumes NADH, allowing for indirect measurement of MLACS activity by monitoring NADH depletion. In the reaction system, the hydrolysis of ATP is coupled with the oxidation of NADH, which has a characteristic absorption peak at 340nm, and its oxidation to NAD+ leads to a decrease in absorbance. By continuously monitoring the change in absorbance of NADH at 340nm, the hydrolysis rate of ATP can be tracked in real time and the enzyme activity of MLACS was indirectly verified.

Since the substrate is much larger than the enzyme concentration, the reaction rate reaches Vmax in a very short period of time, so the linear region is taken to fit the slope to obtain Vmax.

The samples used for LC-MS consisted of a reaction system of the purified enzymes LACS1 (BBa_K5255000) and MLACS1 (BBa_K5255003), which reacted for two minutes with the substrates DHA, ATP and coenzyme A, respectively, before the reaction was terminated. After extraction of the mixture, DHA is found in the bottom layer of the extract, while the product DHA-CoA is upper in the bottom layer of the extract. The aim of this experiment is to compare the activity of wild-type and mutant LACS1 (BBa_K5255000) by LC-MC detection of the upper phase of DHA-CoA.

Reference:

Shockey, J. M., Fulda, M. S., & Browse, J. A. (2002). Arabidopsis contains nine long-chain acyl-coenzyme a synthetase genes that participate in fatty acid and glycerolipid metabolism. Plant physiology, 129(4), 1710–1722. Available at: https://doi.org/10.1104/pp.003269

Pulsifer, I. P., Kluge, S., & Rowland, O. (2012). Arabidopsis long-chain acyl-CoA synthetase 1 (LACS1), LACS2, and LACS3 facilitate fatty acid uptake in yeast. Plant physiology and biochemistry : PPB, 51, 31–39. Available at: https://doi.org/10.1016/j.plaphy.2011.10.003

Lü, S., Song, T., Kosma, D. K., Parsons, E. P., Rowland, O., & Jenks, M. A. (2009). Arabidopsis CER8 encodes LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis. The Plant journal : for cell and molecular biology, 59(4), 553–564. https://doi.org/10.1111/j.1365-313X.2009.03892.x

Weng, H., Molina, I., Shockey, J., & Browse, J. (2010). Organ fusion and defective cuticle function in a lacs1 lacs2 double mutant of Arabidopsis. Planta, 231(5), 1089–1100. https://doi.org/10.1007/s00425-010-1110-4 </body>