molecules Review Metabolic Engineering of Microbial Cell Factories for Biosynthesis of Flavonoids: A Review Hanghang Lou 1,†, Lifei Hu 2,†, Hongyun Lu 1, Tianyu Wei 1 and Qihe Chen 1,* 1 Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, China; [email protected] (H.L.); [email protected] (H.L.); [email protected] (T.W.) 2 Hubei Key Lab of Quality and Safety of Traditional Chinese Medicine & Health Food, Huangshi 435100, China; [email protected] * Correspondence: [email protected]; Tel.: +86-0571-8698-4316 † These authors are equally to this manuscript. Abstract: Flavonoids belong to a class of plant secondary metabolites that have a polyphenol structure. Flavonoids show extensive biological activity, such as antioxidative, anti-inflammatory, anti-mutagenic, anti-cancer, and antibacterial properties, so they are widely used in the food, phar- maceutical, and nutraceutical industries. However, traditional sources of flavonoids are no longer sufficient to meet current demands. In recent years, with the clarification of the biosynthetic pathway of flavonoids and the development of synthetic biology, it has become possible to use synthetic metabolic engineering methods with microorganisms as hosts to produce flavonoids. This article mainly reviews the biosynthetic pathways of flavonoids and the development of microbial expression systems for the production of flavonoids in order to provide a useful reference for further research on synthetic metabolic engineering of flavonoids. Meanwhile, the application of co-culture systems in the biosynthesis of flavonoids is emphasized in this review. Citation: Lou, H.; Hu, L.; Lu, H.; Wei, Keywords: flavonoids; metabolic engineering; co-culture system; biosynthesis; microbial cell factories T.; Chen, Q. Metabolic Engineering of Microbial Cell Factories for Biosynthesis of Flavonoids: A Review. Molecules 2021, 26, 4522. https:// 1. Introduction doi.org/10.3390/molecules26154522 Flavonoids are a kind of secondary metabolite produced by plants in the process of long-term natural selection. They are widely found in vegetables, fruits, and medicinal Academic Editor: Luciana Mosca plants. Structurally, they possess a C6–C3–C6 carbon framework, which is formed by con- necting two aromatic rings (rings A and B) through a heterocyclic ring that contains three Received: 5 July 2021 carbon atoms (ring C) [1] (Figure1). Thus far, about 10,000 flavonoids have been discovered, Accepted: 25 July 2021 which comprise one of the largest families of natural products [2]. There are two forms of Published: 27 July 2021 flavonoids in nature, one occurs in aglycone form in plants, and the other combines with sugar to form glycosides [3]. Common sugars attached to flavonoids include D-glucose, Publisher’s Note: MDPI stays neutral D-galactose, L-rhamnose, D-xylose, D-glucuronic acid, sophorose, rutinose, tangerine peel with regard to jurisdictional claims in published maps and institutional affil- sugar, and gentianose [3,4]. Flavonoids normally occur in plants as O-glycosylated deriva- iations. tives [5], and sugars are mostly attached to the hydroxyl groups at position 3 of the C-ring and positions 5 and 7 of the A-ring. However, some plants, such as maize, wheat, and rice, also produce C-glycosylated flavonoids [5]. These flavonoids play important roles in plants, animals, and humans. In plants, flavonoids are responsible for flower color and aroma and attract pollinators to disperse fruit and help plants propagate [6]. In addition, they also help Copyright: © 2021 by the authors. plants combat various biotic and abiotic stresses, including ultraviolet protection in leaves, Licensee MDPI, Basel, Switzerland. protection from microbial infections and herbivores, and defending from physical and This article is an open access article radical damage [7,8]. As a kind of phytochemicals, flavonoids cannot be synthesized by distributed under the terms and conditions of the Creative Commons humans or animals. Therefore humans generally absorb flavonoids from fruits, vegetables, Attribution (CC BY) license (https:// legumes, and other foods, and flavonoids are thought to have health-promoting properties. creativecommons.org/licenses/by/ Most studies have found that flavonoids have multiple physiological and pharmacological 4.0/). activities, such as antioxidative, anti-inflammatory, anti-mutagenic, anti-cancer, antiviral, Molecules 2021, 26, 4522. https://doi.org/10.3390/molecules26154522 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 18 and flavonoids are thought to have health-promoting properties. Most studies have found that flavonoids have multiple physiological and pharmacological activities, such Molecules 2021, 26, 4522 as antioxidative, anti-inflammatory, anti-mutagenic, anti-cancer, antiviral, antibacterial,2 of 17 and protecting the heart and cerebrovascular vessels [6,9,10]. Therefore, flavonoids show extensive applications in functional foods and some medicine. In fact, some flavonoid extracts,antibacterial, such as andMasquelier’s protecting grape the heartseed extr andact, cerebrovascular have been sold vessels as dietary [6,9 ,10supplements]. Therefore, [11].flavonoids show extensive applications in functional foods and some medicine. In fact, some flavonoid extracts, such as Masquelier’s grape seed extract, have been sold as dietary supplements [11]. Figure 1. General structure of flavonoids. Figure 1. General structure of flavonoids. The wide application of flavonoids has led to their increasing demand. At present, flavonoids are generally extracted from plants. However, plants have a long culture period, The wide application of flavonoids has led to their increasing demand. At present, and their growth is also limited by the temperature and season. Additionally, the process flavonoids are generally extracted from plants. However, plants have a long culture pe- of extraction and purification could increase the cost of production and lead to the loss of riod, and their growth is also limited by the temperature and season. Additionally, the the product and the reduction of product activity [12]. Therefore, it is a huge challenge process of extraction and purification could increase the cost of production and lead to to produce these compounds on a large scale through plant extraction to meet medical the loss of the product and the reduction of product activity [12]. Therefore, it is a huge and nutritional needs [13]. The total synthesis of some flavonoids has been realized by challenge to produce these compounds on a large scale through plant extraction to meet chemical approaches; however, this method required some extreme reaction conditions, medical and nutritional needs [13]. The total synthesis of some flavonoids has been real- such as high temperature, strong acid, strong base, and the use of heavy metal, which is izedunfriendly by chemical to the approaches; environment however, and hindered this method its scale-up required and commercial some extreme application. reaction In conditions,recent years, such the as productionhigh temperature, of flavonoids strong by acid, microbial strong cell base, factories and the has use attracted of heavy wide metal,attention which with is unfriendly the development to the environm of metabolicent engineeringand hindered tools, its scale-up the in-depth and commercial studies on the application.synthesis pathwayIn recent ofyears, flavonoids the production and available of flavonoids substrates by [7, 12microbial,14]. Engineering cell factories the wholehas attractedbiosynthetic wide pathway attention to producewith the flavonoids developm inent microorganisms of metabolic hasengineering many advantages, tools, the such in-depthas a short studies production on the cycle, synthesis low waste pathway production, of flavonoids low energy and requirements, available substrates and mass [7,12,14].production. Engineering Some microorganisms, the whole biosynthetic such as Escherichia pathway to coli produce(E. coli), flavonoidsSaccharomyces in micro- cerevisiae organisms(S. cerevisiae has), manyYarrowia advantages, lipolytica (Y. such lipolytica) as a sh,ort grow production faster, and cycle, various low genetic waste manipulationsproduction, lowof energy this species requirements, and metabolic and modificationsmass production. have Some been microorganisms, studied in detail, such so it hasas Esche- become richiapossible coli (E. to coli use), theseSaccharomyces microorganisms cerevisiae as ( hostsS. cerevisiae for the), productionYarrowia lipolytica of flavonoids (Y. lipolytica) [2,15–17, ]. growIn a faster, previous and study, various the genetic production manipulations of flavonoids of this by geneticallyspecies and engineered metabolic bacteriamodifica- had tionsnot have been been achieved, studied and in one detail, of the so barriersit has become to produce possible these to compounds use these microorganisms was the difficulty as inhosts the for expression the production of active of C4H.flavonoids In 2003, [2,15– a 4CL17]. (4-coumarin-CoA In a previous study, ligase) the production was discovered of flavonoidsin the gram-positive by genetically filamentous engineered bacterium bacteria hadStreptomyces not been achieved, coelicolor [and18]. one The of enzyme the bar- not riersonly to activatedproduce these cinnamic compounds acid to was cinnamoyl-CoA the difficulty but in the also expression activated of 4-coumaric active C4H. acid In to 2003,4-coumaroyl-CoA. a 4CL (4-coumarin-CoA The use of ligase) the enzyme was woulddiscovered