Common problems associated with the microbial productions of aromatic compounds and corresponding metabolic engineering strategies

Abstract

Recent progress in metabolic engineering and synthetic biology has enabled the production of valuable chemicals in microbial cell factories from renewable feedstocks. High value-added aromatic compounds, most of which are traditionally chemically synthesized from petroleum-derived feedstocks, represent a large class of chemicals with industrial significance. The microbial biosynthesis of aromatic compounds has been studied for decades, and varying yields have been achieved for different aromatic compounds. In this review, we describe the common problems associated with the microbial biosynthesis of diverse aromatic compounds and summarize the corresponding metabolic engineering strategies for resolving these problems. In addition, future perspectives on the microbial production of aromatic compounds are discussed.

Introduction

Aromatic compounds are a large class of chemicals with industrial applications, including the synthesis of polymers (e.g., polyhydroxyalkanoates (Yang et al., 2018a), styrene (Liu et al., 2018a)), food additives (L-tryptophan (Niu et al., 2019), L-phenylalanine (Liu et al., 2019a)), pharmaceuticals (e.g., salicylic acid (Ahmadi et al., 2016)), aromatic agents (e.g., vanillin (Hansen et al., 2009)) and biofuels (e.g., 2-phenylethanol (Zhang et al., 2014a)). The majority of aromatic compounds are currently produced via chemical conversion from petroleum-derived benzene, toluene, and xylene (BTX) (Lee and Wendisch, 2017). Considering the unsustainability of fossil resources and their associated environmental issues and, the development of strategies for microbial production of valuable chemicals from renewable carbohydrate feedstocks has become a promising alternative (Li et al., 2019). Therefore, the sustainable production of industrially significant aromatic compounds in microbial host cells has received much attention in recent years.

Cellular metabolism in all species requires aromatic compounds, and the shikimate pathway is the common pathway for the biosynthesis of aromatic compounds in bacteria, plants, fungi, algae and archaea (Fig. 1) (Mir et al., 2015). The shikimate pathway is absent in animals, and diet-dependent supplementation is required. The shikimate pathway involves seven enzymatic reactions that convert the precursors phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) to chorismate. The first step involves the condensation of PEP and E4P to 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) by DAHP synthetase (Fig. 1). One important intermediate, dehydroshikimate (DHS), is produced from DAHP via two reactions catalyzed by AroB and AroD, followed by the production of shikimate from DHS (Fig. 1). Shikimate is subsequently converted via three enzyme-catalyzed steps to chorismate, a branch point in the synthesis pathways for a variety of aromatic compounds (Fig. 1). All the enzymes involved in the shikimate pathway were summarized in detail in previous reviews (Herrmann and Weaver, 1999; Mir et al., 2015). The intermediates and end products of the shikimate pathway, which mainly include DHS and chorismate, respectively, are precursors for the production of various valuable aromatic compounds (Fig. 1). Specifically, from the last common precursor chorismate, the metabolic pathway branches off to produce three aromatic amino acids (AAAs; L-tryptophan, L-phenylalanine, and L-tyrosine) and their derivatives. Therefore, aromatic compounds can be categorized into dehydroshikimate derivatives, chorismate derivatives and AAAs derivatives (Fig. 1).

In previous studies, the shikimate pathway has been engineered to obtain microbial cell factories that can synthesize a broad range of aromatic compounds (Table 1), and high yields of some chemicals have been achieved. Engineered Corynebacterium glutamicum showed a shikimate titer of 141 g/L, with a dramatically high yield of 493 mg/g glucose, reaching 66% of the theoretical yield (Kogure et al., 2016). The corresponding productivity was approximately 3 g/(L×h); to the best of our knowledge, this is the highest reported productivity achieved by microbial synthesis to date (Kogure et al., 2016). These high yields indicate that the shikimate pathway is capable of high metabolic flux. However, some aromatic compounds, particularly some derivatives, are produced at very low levels, and research on the production of these compounds remains in the primary phase. Microbial synthesis of resveratrol, a potent antioxidant naturally produced by plants, has been studied, and a titer of 0.8 g/L and productivity of nearly 7 mg/(L×h) were achieved, the highest reported to date. The corresponding yield was only 9.23 mg/g glucose, reaching 0.32% of the theoretical yield. In another study on the production of styrene, an L-phenylalanine derivative, the highest yield was reported to be only 5.3 g/L, with a low productivity of 88 mg/(L×h), far less than that reported for shikimate (Lee et al., 2019). The relatively low productivity of these aromatic compounds can potentially be improved using appropriate engineering strategies. Because the production of various precursors involves the same metabolic pathway, the microbial production of aromatic compounds through metabolic engineering has some common problems and difficulties. Thus, a summary of the common problems associated with the microbial production of aromatic compounds and related strategies for addressing these problems would be instructive.

In this review, three common problems encountered during the engineering of strains for the production of aromatic compounds were categorized through an extensive literature assessment: precursor supplies, regulatory systems and product cytotoxicity. The related metabolic engineering strategies, including not only the strategies currently utilized for aromatic compound production but also the novel and promising strategies reported in recent years, were then evaluated. Furthermore, prospects and guidelines for the future engineering of cells for the production of aromatic compounds are discussed. This review does not address metabolic engineering strategies focused on the main shikimate pathway and the synthesis pathways for specific derivatives, which have been discussed elsewhere (Averesch and Kromer, 2018; Bilal et al., 2018; Cao et al., 2019; Huccetogullari et al., 2019; Noda and Kondo, 2017; Wang et al., 2018a). The difficulties associated with genetic engineering strategies for the synthesis of all types of compounds, such as stable gene expression in heterogeneous hosts and metabolic balance of multigene pathways, were discussed in previous reviews (Choi and Lee, 2016; Gu et al., 2017; Li et al., 2019) and are thus not discussed in this review