The Skin-Whitening and Antioxidant Effects of Protocatechuic Acid (PCA) Derivatives in Melanoma and Fibroblast Cell Lines.

The skin is the most voluminous organ of the human body and is exposed to the outer environment. Such exposed skin suffers from the effects of various intrinsic and extrinsic aging factors. Skin aging is characterized by features such as wrinkling, loss of elasticity, and skin pigmentation. Skin pigmentation occurs in skin aging and is caused by hyper-melanogenesis and oxidative stress. Protocatechuic acid (PCA) is a natural secondary metabolite from a plant-based source widely used as a cosmetic ingredient. We chemically designed and synthesized PCA derivatives conjugated with alkyl esters to develop effective chemicals that have skin-whitening and antioxidant effects and enhance the pharmacological activities of PCA. We identified that melanin biosynthesis in B16 melanoma cells treated with alpha-melanocyte-stimulating hormone (α-MSH) is decreased by PCA derivatives. We also found that PCA derivatives effectively have antioxidant effects in HS68 fibroblast cells. In this study, we suggest that our PCA derivatives are potent ingredients for developing cosmetics with skin-whitening and antioxidant effects.

Characterization of a non-phosphotransferase system for cis,cis-muconic acid production in Corynebacterium glutamicum.

Cis,cis-muconic acid (CCM) is a biochemical material that can be used for the production of various plastics and polymers and is particularly gaining attention as an adipic acid precursor for the synthesis of nylon-6,6. In the current study, the production of CCM was first attempted by introducing a newly developed protocatechuate (PCA) decarboxylase from Corynebacterium glutamicum 13032 to inha103, which completed the biosynthetic pathway therein. To improve CCM productivity, a phosphoenol pyruvate (PEP)-dependent phosphotransferase system (PTS) that consumed the existing glucose was developed, in the form of a strain with a non-PTS that did not consume PEP. To improve glucose uptake, we developed P25 strain, in which iolR (a transcriptional regulator gene) was additionally deleted. Strain P28, a P25 derivative expressing PCA decarboxylase, produced 4.01 g/L of CCM, which was 14% more than that produced by the parental strain. Moreover, strains P29 and P30, with an active pentose phosphate pathway and overexpressing important genes (qsuB) in the metabolic pathway, produced 4.36 and 4.5 g/L of CCM, respectively. Particularly, the yield per glucose in strain P30 was similar to that of the fed-batch culture of Escherichia coli, which has the highest reported yield of 22% (mol/mol). These results are underpinned by the characteristics of the non-PTS with increased PEP availability and a strain with deletion of the iolR gene, which greatly increased glucose uptake.

Corynebacterium Cell Factory Design and Culture Process Optimization for Muconic Acid Biosynthesis.

Muconic acid (MA) is a valuable compound for adipic acid production, which is a precursor for the synthesis of various polymers such as plastics, coatings, and nylons. Although MA biosynthesis has been previously reported in several bacteria, the engineered strains were not satisfactory owing to low MA titers. Here, we generated an engineered Corynebacterium cell factory to produce a high titer of MA through 3-dehydroshikimate (DHS) conversion to MA, with heterologous expression of foreign protocatechuate (PCA) decarboxylase genes. To accumulate key intermediates in the MA biosynthetic pathway, aroE (shikimate dehydrogenase gene), pcaG/H (PCA dioxygenase alpha/beta subunit genes) and catB (chloromuconate cycloisomerase gene) were disrupted. To accomplish the conversion of PCA to catechol (CA), a step that is absent in Corynebacterium, a codon-optimized heterologous PCA decarboxylase gene was expressed as a single operon under the strong promoter in a aroE-pcaG/H-catB triple knock-out Corynebacterium strain. This redesigned Corynebacterium, grown in an optimized medium, produced about 38 g/L MA and 54 g/L MA in 7-L and 50-L fed-batch fermentations, respectively. These results show highest levels of MA production demonstrated in Corynebacterium, suggesting that the rational cell factory design of MA biosynthesis could be an alternative way to complement petrochemical-based chemical processes.

Development of Miniaturized Culture Systems for Large Screening of Mycelial Fungal Cells of Aspergillus terreus Producing Itaconic Acid.

The task of improving a fungal strain is highly time-consuming due to the requirement of a large number of flasks in order to obtain a library with enough diversity. In addition, fermentations (particularly those for fungal cells) are typically performed in high-volume (100-250 ml) shake-flasks. In this study, for large and rapid screening of itaconic acid (IA) high-yielding mutants of Aspergillus terreus, a miniaturized culture method was developed using 12-well and 24-well microtiter plates (MTPs, working volume = 1-2 ml). These miniaturized MTP fermentations were successful, only when highly filamentous forms were induced in the growth cultures. Under these conditions, loose-pelleted morphologies of optimum sizes (less than 0.5 mm in diameter) were casually induced in the MTP production cultures, which turned out to be the prerequisite for the active IA biosynthesis by the mutated strains in the miniaturized fermentations. Another crucial factor for successful MTP fermentation was to supply an optimal amount of dissolved oxygen into the fermentation broth through increasing the agitation speed (240 rpm) and reducing the working volume (1 ml) of each 24-well microtiter plate. Notably, almost identical fermentation physiologies resulted in the 250 ml shake-flasks, as well as in the 12-well and 24-well MTP cultures conducted under the respective optimum conditions, as expressed in terms of the distribution of IA productivity of each mutant. These results reveal that MTP cultures could be considered as viable alternatives for the labor-intensive shake-flask fermentations even for filamentous fungal cells, leading to the rapid development of IA high-yield mutant strains.

Enhanced Production of Itaconic Acid through Development of Transformed Fungal Strains of Aspergillus terreus.

Metabolic engineering with a high-yielding mutant, A. terreus AN37, was performed to enhance the production of itaconic acid (IA). Reportedly, the gene cluster for IA biosynthesis is composed of four genes: reg (regulator), mtt (mitochondrial transporter), cad (cis-aconitate decarboxylase), and mfs (membrane transporter). By overexpressing each gene of the IA gene cluster in A. terreus AN37 transformed by the restriction enzyme-mediated integration method, several transformants showing high productivity of IA were successfully obtained. One of the AN37/cad transformants could produce a very high amount of IA (75 g/l) in shake-flask cultivations, showing an average of 5% higher IA titer compared with the high-yielding control strain. Notably, in the case of the mfs transformants, a maximal increase of 18.3% in IA production was observed relative to the control strain under the identical fermentation conditions. Meanwhile, the overexpression of reg and mtt genes showed no significant improvements in IA production. In summary, the overexpressed cis-aconitate decarboxylase (CAD) and putative membrane transporter (MFS) appeared to have positive influences on the enhanced IA productivity of the respective transformant. The maximal increases of 13.6~18.3% in IA productivity of the transformed strains should be noted, since the parallel mother strain used in this study is indeed a very high-performance mutant that has been obtained through intensive rational screening programs in our laboratory.

Application of scale-up criterion of constant oxygen mass transfer coefficient (kLa) for production of itaconic acid in a 50 L pilot-scale fermentor by fungal cells of Aspergillus terreus.

The scale-up criterion of constant oxygen mass transfer coefficient (kLa) was applied for the production of itaconic acid (IA) in a 50 L pilot-scale fermentor by the fungal cells of Aspergillus terreus. Various operating conditions were examined to collect as many kLa data as possible by adjusting the stirring speed and aeration rate in both 5 L and 50 L fermentor systems. In the fermentations performed with the 5 L fermentor, the highest IA production was obtained under the operating conditions of 200 rpm and 1.5 vvm. Accordingly, we intended to find out parallel agitation and aeration rates in the 50 L fermentor system, under which the kLa value measured was almost identical to that (0.02 sec(-1)) of the 5 L system. The conditions of 180 rpm and 0.5 vvm in the 50 L system turned out to be optimal for providing almost the same volumetric amount of dissolved oxygen (DO) into the fermentor, without causing shear damage to the producing cells due to excessive agitation. Practically identical fermentation physiologies were observed in both fermentations performed under those respective operating conditions, as demonstrated by nearly the same values of volumetric (Qp) and specific (qp) IA production rates, IA production yield (Yp/s), and specific growth rate (µ). Specifically, the negligible difference of the specific growth rate (µ) between the two cultures (i.e., 0.029 h(-1) vs. 0.031 h(-1)) was notable, considering the fact that µ normally has a significant influence on qp in the biosynthesis of secondary metabolites such as itaconic acid.