Engineering Lactococcus lactis for the production of unusual anthocyanins using tea as substrate

Highlights

• Exploitation of L. lactis as a cell factory is an attractive alternative to extraction of anthocyanins from plants.

• Engineered L. lactis strains can quickly transform green tea infusion into a range of potentially valuable pigments.

• Cheap flavan-3-ol-rich sources can potentially be utilized as feedstock for the production of high-value colorants.

Abstract

Plant material rich in anthocyanins has been historically used in traditional medicines, but only recently have the specific pharmacological properties of these compounds been the target of extensive studies. In addition to their potential to modulate the development of various diseases, coloured anthocyanins are valuable natural alternatives commonly used to replace synthetic colourants in food industry. Exploitation of microbial hosts as cell factories is an attractive alternative to extraction of anthocyanins and other flavonoids from plant sources or chemical synthesis. In this study, we present the lactic acid bacterium Lactococcus lactis as an ideal host for the production of high-value plant-derived bioactive anthocyanins using green tea as substrate. Besides the anticipated red-purple compounds cyanidin and delphinidin, orange and yellow pyranoanthocyanidins with unexpected methylation patterns were produced from green tea by engineered L. lactis strains. The pyranoanthocyanins are currently attracting significant interest as one of the most important classes of anthocyanin derivatives and are mainly formed during the aging of wine, contributing to both colour and sensory experience.

Introduction

Various phytochemicals, such as flavonoids are increasingly valued for their health promoting activities (Rodriguez-Mateos et al., 2014; Atanasov et al., 2015; Perez-Vizcaino and Fraga, 2018; Foito et al., 2018). They are simple compounds present in fresh fruits and vegetables, or complex compounds present in bark, roots and leaves of plants. Flavonoids have become a topic of increasing interest not only because of their antioxidant properties but also due to their beneficial effects on health. Whilst they can act as free radical scavengers, metal chelators and singlet oxygen quenchers reducing lipid peroxidation, DNA damage and stimulating the expression of detoxification enzymes, these compounds demonstrate promising effects in the combat of cardiovascular disease, certain types of cancer, neurodegenerative diseases, diabetes and inflammation (Foito et al., 2018).

A group of flavonoids, known as anthocyanins, is responsible for the colour of many fruits, vegetables and flowers. Anthocyanins encompass a large group of compounds, with over 600 known molecular structures. However, this diversity is based on six naturally occurring anthocyanidins: pelargonidin, cyanidin, peonidin, delphinidin, petunidin and malvidin. They differ from each other by the hydroxylation and/or methoxylation pattern of ring B, which affects directly the hue and colour stability (de Freitas et al., 2017). Research activity into anthocyanins has increased in recent years, mainly driven by interest in their bioactive properties and colour properties (Khoo et al., 2017). Recently, there have been increasing efforts in reducing the use of synthetic colourants in the food industry and thus anthocyanins are increasingly being used as natural, healthier colour alternatives. Anthocyanins and other flavonoids are mainly extracted from plants and as result are subject to seasonality of raw material, variation of abundance in different species, and fluctuations in the abundance of these compounds driven by environmental variables. Additionally, the purification of a single chemical from complex plant matrices is often difficult due to the presence of structurally similar compounds. Plant cell cultures provide a promising strategy for the production of specific molecules, but to date they have had limited commercial success in food biotechnology applications as result of limited culture yields and/or poorly optimized production systems (Davies and Deroles, 2014; Appelhagen et al., 2018). Chemical synthesis of anthocyanins is complex and often produces large amounts of toxic waste. The exploitation of microbial hosts as cell factories for the production of various phytochemicals is an attractive environmentally-friendly and increasingly cost-effective alternative. The fast growth of bacteria allows short production times and generally chemically distinct structure of the product facilitates easy purification (Marienhagen and Bott, 2013; Lim et al., 2015; Stahlhut et al., 2015; Dudnik et al., 2017). Many plant pathways have been successfully reconstructed and expressed in microorganisms so far. However, almost all of them employed Escherichia coli and Saccharomyces cerevisiae as production hosts (Marienhagen and Bott, 2013; Milke et al., 2018). Lactic acid bacteria, and in particular L. lactis, provide an attractive alternative for production of plant high value chemicals. It has a long history of safe usage in food fermentations and has been granted a Generally Regarded As Safe (GRAS) status. Anthocyanins are a great target for heterologous production since their biosynthesis pathways are characterised and accumulation is quickly disclosed by the colour change of the production culture.

Anthocyanins are synthesized from flavanones in the plant cytosol, on the endoplasmic reticulum, and then transported into the vacuole. Anthocyanidin synthase (ANS) and UDP-glucose: anthocyanidin 3-O-glucosyltransferase (3GT) are the last two enzymes in the pathway responsible for the formation of a stable colourful product (Ferreyra et al., 2012). ANS belongs to the 2-oxoglutarate iron-dependent oxygenases and was cloned first from perilla (Perilla frutescens) (Saito et al., 1999). It uses ferrous iron as a cofactor and 2-oxoglutarate (2OG) as a co-substrate (Saito et al., 1999; Wilmouth et al., 2002). The enzyme requires an unusually high concentration of ascorbate for optimal turnover. ANS was postulated to catalyse the reaction from the colourless leucoanthocyanidins to the coloured anthocyanidins (Saito et al., 1999). Later in vitro activity studies revealed that the selectivities of 2OG-dependent oxygenases that are involved in flavonoid synthesis overlap. ANS was shown to have properties of a flavanol synthase FLS and catalyse oxidation of dihydroquercetin to quercetin (Wilmouth et al., 2002). ANS from Gerbera hybrida and Petunia hybrida accepted (+)-catechin as a substrate to form cyanidin, quercetin and an oxidized (+)-catechin dimer (Wellmann et al., 2006; Yan et al., 2008).

In this study, we engineered the food-grade bacterium L. lactis for anthocyanin production using flavan-3-ols as substrates. In order to demonstrate the feasibility of utilizing feedstocks naturally rich in flavan-3-ols we successfully converted green tea infusion into a variety of unusual and valuable pigments. We showed that the polysaccharides of the thick cell wall of the Gram-positive bacterium might retain polyphenols and investigated multiple options to overcome this barrier. Besides the classical anthocyanins, the engineered L. lactis strains were able to produce various red, orange and yellow cyanidin and delphinidin derivatives reaching total milligram-per-litre production titres. Some of the compounds belong to an intriguing class of methylpyranoanthocyanins that are known to form during ageing of wine. The approach used here offers a new environmentally friendly strategy for obtaining anthocyanin-rich fermentation products from various polyphenol-rich waste streams.