Bioconversion of resveratrol using resting cells of non–genetically modified Alternaria sp.

Abstract

Bioconversion of resveratrol is mainly achieved using plant cells and genetically modified microorganisms. We proposed a reaction system for resveratrol production using resting cells of a non-genetically modified strain, Alternaria sp. MG1, a resveratrol-producing endophytic fungus isolated from the grape. Effects of phenylalanine concentration, inoculum size, resting time, bioconversion medium, cell age, and pH on resveratrol production in the bioconversion process were investigated and their levels were optimized. The resulting optimal bioconversion medium was 0.2 mM phosphate buffer (pH 6.5), 0.1 g/L MgSO4 , 0.2 g/L CaSO4 , and 4.66 mM phenylalanine. Resting cells obtained from cultures of liquid potato-glucose medium after 4 days proved to be at the most suitable cell age for the bioconversion process with high resveratrol production and nonobvious cell growth. Highest resveratrol production (1.376 µg/L) was observed under the obtained optimal conditions of inoculum size, 12.16% (wet cell weight in 100 mL medium), and resting time, 21.3 H. The study provides a new way to produce resveratrol and establishes an essential reaction system for further study of the biosynthesis pathway of resveratrol in microorganisms, especially fungi.

A New Approach to Produce Resveratrol by Enzymatic Bioconversion

“…MG1 (224.40 g/l) was much higher than that found when resting cells of Alternaria sp. MG1 were used (1.376 g/l) [25] and a little lower than that when cell cultivation was used (422.04 g/l) [19], indicating a potential of this method in resveratrol production. Cell cultivation had the highest resveratrol production in the above three cases because there is consistent production of enzymes, substrates, and cofactors for resveratrol biosynthesis in this case, but not in the other two cases.…”

“…Extraction, identification, and determination of resveratrol produced in the enzymatic bioconversion systems were carried out according to previously developed methods [25], with slight modifications. After each reaction, the bioconversion system was extracted twice with ml of ethyl acetate for 10 h per extraction.…”

“…The absorption value at 306 nm was recorded and used for calculation of resveratrol concentration according to the stability of resveratrol [6]. The liquid systems and testing conditions used for the measurements were the same as that previously reported [25]. The negative-ion mode selected multi-reaction monitoring mode was used, and molecular ions with m/z = 226.5 -227.5 were the focus of analysis given the molecular weight of resveratrol (228.24).…”

Validation of reference genes for normalization of gene expression by qRT-PCR in a resveratrol-producing entophytic fungus (Alternaria sp. MG1)

“…Next, the collected cells were washed twice with sterile water and immediately stored in liquid nitrogen until further analysis. The resting cells were collected using the method reported by Zhang et al (2013a) as follows: after cultivation for 4 days, the rinsed cells were resuspended in 0.2 mol/L pH 7.0 phosphate buffer containing 0.1 g/L MgSO 4 , 0.2 g/L CaSO 4 and 4 mmol/L phenylalanine for 21 h. After that, cells were washed twice with sterile water and stored for further analysis.…”

“…were identified previously that are capable of independent resveratrol production (Shi et al 2012). Although fundamental physiological research has been performed (Zhang et al 2013a, b), the metabolic pathways and cellular processes remain to be elucidated.…”