Enhancement Of D-phenyllactic Acid Biosynthesis By Bifunctional Fusion Protein And Optimization Of Its Fermentation Process

Natural antimicrobial compounds(natural microflora and/or their anticterial products) have been part of biopreservation practices to extend shelf life and to enhance the safety of foods.With the “generally regarded as safe” status, Lactic acid bacteria(LAB) has been a major potential for using in biopreservation during the recent years. The only bacteriocins employed in food preservation are these produced by LAB, In this regard, 3-Phenyllactic acid(PLA),a by-product of LAB metabolism, has gained increasing interesting in decades for its broad antibacterial spectrum activity toward bacterial and fungi. Many studies have focused on the PLA biosynthesis utilizing various LAB strains, such as Lactobacillus plantarum, Enterococcus faecalis and Pediococcus acidilactici.PLA is a metabolite of pheylalanine(Phe) metabolism in LAB, in which pheylalanine was transaminated to phenylpyruvic acid(PPA) and PPA further reduced to PLA. L.plantarum D-LDH has already been characterized to produce D-PLA from PPA. As with other NADH-dependent dehydrogenases, L.plantarum D-LDH requires the regeneration of NADH for PLA production at every step. Given the high cost of the cofactors, engineering to generate the NAD(H) in a reaction has been performed by using enzymatic, chemical, electrochemical and photochemical methods. By far the most frequently applied strategy is the enzymatic regeneration using a second oxidoreductase in combination with a cheap co-substrate. In some circustances, these most common coexpression systems, due to their poor stability, need to be encapsulated and immobilized to achieve the successful of sequential reactions. As an alternative to using two separate enzymes, bifunctional fusion proteins can be constructed in which the production enzyme and the cofactor-regenerating enzyme are connected through a linker sequence to yield a single polypeptide exhibiting both catalytic activities. The general advantage of the fusion proteins is the inherent co-localization of the catalytic subunits. Then, the thereby achieved locally enhanced substrate concentrations and reduced diffusion distance enable a better performance of the fusion protein compared to the separated enzymes. In this study, we engineered a translational fusion of a mutant D-Lactate dehydrogenase(D-LDH) and formate dehydrogenase(FDH), the bifunctional enzyme catalyze the conversion of D-PLA in a sequential reaction, the formate dehydrogenase irreversible providing a strong driving force for NADH regeneration by using the formate as an innocuous substrate.The construct of the recombinant fusion protein D-LDH-Linker-FDH was based on the gene fusion technique. In this paper, four fusion proteins composed of D-LDH and FDH with different linkers were constructed. The fusion proteins were applied for the production of D-PLA. The main results were as follows:(1) The d-ldh and fdh genes were ligated to generate the fusion construct following an end-to-end fusion method using overlapping PCR, in which the coding sequence of D-LDH and FDH are directly apposed. We introduced four peptides to linker D-LDH and FDH to avoid interference of these polypeptides. Four bifunctional enzymes were verified correctly by sequencing. All the correct chimeric genes were inserted into the constitutive expression vector pET-28a(+). The recombinant E.coli BL21(DE3) bearing the fusion proteins were induced by IPTG and checked using SDS-PAGE. SDS-PAGE showed legible bands of the expect size for both unfused proteins and fused proteins. The molecular size of the fusion protein was approximately 78 kDa, since the native D-LDH existed mainly as a tetrameric form with a monomeric molecular weight of 37 kDa, while the FDH has a molecular weight of 41 kDa per Monomer. Our finding was in a good agreement with the theoretical calculation(about 78 kDa) of the complete length fusion protein. The specific activity of crude recombinant protein extracts was then examined. The result revealed that the fusion proteins all Vexhibited both D-LDH and FDH activity. The enzymatic action of the D-LDH and FDH was not affected by the fusion. However, the D-LDH exhibited only 34.25-47.47% of the enzymatic activity of the free enzyme in the system expressing D-LDH alone.(2) The feasibility of strains containing the fusion protein to synthesize the phenyllactic acid was verified by the experiment. Through the single factor and orthogonal experiments of the induction conditions, we optimized the induction conditions of the recombinant E. coli. The orthogonal experiments results showed that the strains of E.coli pET-28a-d-ldhY52V-s1-fdh, E.coli pET28a-d-ldhY52V-s2-fdh and E.coli pET28a-d-ldhY52V-e-fdh had a same induction conditions. The optimum induction conditions for fusion protein expression were IPTG 0.2 mmol/L and inducing for 12 h at 25 ℃ when cell density OD600 reached 1.0. The optimized induction conditions for E. coli pET28a-d-ldhY52V-g-fdh were IPTG 0.1 mmol/L and inducing for 12 h at 25℃ when cell density OD600 reached 1.0. The yield of the D-phenyllactic acid was increased 10.28%-21.1% by orthogonal optimization experiments.(3) We further optimized biotransformation conditions in flasks, results showed that the optimized batch reaction should be carried out in phosphate buffer(pH 7.0, 0.1 M) at 37℃,with 12 g/L PPA. Based on the above optimized conditions, the four forms of fusion protein system all showed statistically significant increase in D-PLA production, as compared to the nonfusion system. It was noteworthy that by inserting an α-helix peptide into the fusion protein, a highest enhancement of the D-PLA production was observed. The yield of D-PLA produced by the E.coli BL21(DE3) bearing the D-LDH-E-FDH protein was 1.48-fold of the D-LDH expressing alone system, and the highest D-PLA yield was 6.72 g/L.We set out to further research the rates of the D-PLA production by using the E. coli BL21(DE3) harbouring the protein D-LDH, and D-LDH-E-FDH. E. coli cells harbouring the chimaeric protein D-LDH-E-FDH exhibited a 1.8-fold higher initial reaction rate in the production of D-PLA than cells expressing the enzymes D-LDH alone. The Fed-batch incubation was executed for 4h was conducted by intermittent feeding PPA,glucose and sodium formate, the maximum D-PLA productivity reached to a level of 18.6 g/L, production intensity was 4.65 g/L/h the corresponding substrate molar conversion rates was 78.84%.