MLACS1

Introduction

Long-chain acyl-coenzyme A (CoA) synthetases (LACSs) family play critical roles in fatty acid metabolism by activating free fatty acids to acyl-CoA thioesters. LACSs uses DHA, CoA, and ATP as substrates to synthesize DHA-CoA. Basic part 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 two 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.

Figure 1 | MLACS1 generated using homology protein modeling with SWISS-MODEL. The model shows a large N-terminal domain and a small C-terminal domain linked by a highlighted cyan linker (Asp513-Leu518). Ligand ATP and cofactor Mg2+ reside between the two domains.

The enzyme family of LACSs serves a vital role in the fatty acid metabolic pathway, and its existence has been proven in a variety of species ranging from prokaryotes to eukaryotes. We first selected several subtypes of the LACSs family in species with close relationships with our target fungus. Since Schizochytrium limacinum was identified as the protein expression chassis, the engineered enzyme should be a subtype of LACSs that has close phylogeny to the target fungus. A set of characterized subtypes of LACS in the 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 very long-chain fatty acids. 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), and 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 a protein database, we first parsed the sequence and intercepted the open reading frame to obtain cDNA, 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), which remained consistent across multiple estimation algorithms, proving a solid improvement in mRNA performance. To sum up, the methods of codon optimization aim 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 their commonly acknowledged limited accuracy. In order to address that limitation and provide a more reasonable protein engineering solution, we further researched 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 approach. We generated candidates with some of the approaches, graded all candidates using other approaches, and finally selected the mutagenesis with the highest-ranking score. The selected mutagenesis site is expected to yield improvement in the catalytic activity of LACS1.

The target gene of MLACS1 was connected to the 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. Colony PCR was used to verify the successful introduction of the vector plasmid.

Figure 2 | The target gene of MLACS1 was connected to the pYES2/CT vector plasmid, and the plasmid containing the target gene was transfected into BL21 (DE3), C43 (DE3), and YB525 strains by chemical transformation method. 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.

Figure 3 | This is a western blot of the induced expression of MLACS1. The whole cell before ultrasonic lysis, the supernatant, and lower precipitation after ultrasonic lysis were respectively taken for western blot. The results show that there are obvious target bands in lower precipitation, and the size is 77.7kDa. In addition, there are fewer impurity bands.

NADH-coupled assay was 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.

Figure 4 | This diagram depicts the enzymatic activity of MLACS1 after NADH enzyme coupling. The activity of MLACS was calculated by calculating the rate of absorbance change at 340nm.

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

Figure 5 | Change in concentration over time when the protein is saturated (substrate excess) 30 seconds before the reaction. Vmax = 0.0048 M/s (under 0.5 mM DHA).

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 was found in the bottom layer of the extract, while the product DHA-CoA was found in the upper layer. The aim of this experiment was to compare the activity of wild-type and mutant LACS1 (BBa_K5255000) by LC-MS detection of the upper phase of DHA-CoA.

Figure 6 | This is the result of testing the product with LC-MS and searching for molecular weight. The blue peaks are the result of the MLACS1 sample and the red peaks are the result of the LACS1 sample. Below the baseline is the result of the blank without enzyme added. The peak area represents the relative amount of the corresponding material. The results show that the catalytic activity of the mutant protein (MLACS1) is significantly higher than that of the wild-type protein (LACS1).

Other Parts

Basic Parts

Code Name Length Description
BBa_K5255000 LACS1+his 1980 For more than 24 carbon has a certain catalytic capacity, including DHA
BBa_K5255001 Czlacs5+his 2145 The activity of [C16:1] [C18:3n3] [EPA] [DHA] [C18:1] was significantly decreased from left to right
BBa_K5255002 LACS6+his 2763 It has certain catalytic activity for DHA
BBa_K5255003 MLACS1 1982 Mutant LACS1 has higher catalytic activity on DHA
BBa_K5255004 pAR-Ec633 7602 A nuclear plasmid encoding an error-prone mutant TP-DNAP1 (L477V, L640Y, I777K, W814N) for OrthoRep
BBa_K5255005 ΔFAS1 379 Used to knock out the FAS1 gene of INVSC1
BBa_K5255006 ΔFAS1 ΔFAA4 547 Used to knock out the FAS1 and FAA4 genes of INVSC1
BBa_K5255007 ΔFAS1 ΔFAA4 ΔFAA1 715 Used to knock out the FAS1, FAA4, and FAA1 genes of INVSC1
BBa_K5255008 FAS1 homologous repair fragment 182 Used to repair FAS1 gene knocked out by CRISPR
BBa_K5255009 FAA1 homologous repair fragment 182 Used to repair FAA1 gene knocked out by CRISPR
BBa_K5255010 FAA4 homologous repair fragment 182 Used to repair FAA4 gene knocked out by CRISPR
BBa_K5255011 pGKL1-LACS1 7051 Linearized plasmid for homologous recombination with pccl-LACS1
BBa_K5255012 pGKL1-Czlacs5 7216 Linearized plasmid for homologous recombination with pccl-Czlacs5
BBa_K5255013 pGRKL1-LACS6 7726 Linearized plasmid for homologous recombination with pccl-LACS6

Composite Parts

Code Name Length Description
BBa_K5255015 gal+LACS1+his 2326 LACS1-his fusion protein regulated by galactose promoter
BBa_K5255016 gal+Czlacs5+his 2491 Czlacs5-his fusion protein regulated by galactose promoter
BBa_K5255017 gal+LACS6+his 8917 LACS6-his fusion protein regulated by galactose promoter
BBa_K5255018 T7+LACS1+his 2005 LACS1-his fusion protein regulated by IPTG
BBa_K5255019 T7+Czlacs5+his 2170 Czlacs5-his fusion protein regulated by IPTG
BBa_K5255020 T7+LACS6+his 2680 LACS6-his fusion protein regulated by IPTG
BBa_K5255021 T7+MLACS1+his 2007 MLACS1-his fusion protein regulated by IPTG

References

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