Two-Phase Fermentation Systems for Microbial Production of Plant-Derived Terpenes

Abstract

Microbial cell factories, renowned for their economic and environmental benefits, have emerged as a key trend in academic and industrial areas, particularly in the fermentation of natural compounds. Among these, plant-derived terpenes stand out as a significant class of bioactive natural products. The large-scale production of such terpenes, exemplified by artemisinic acid-a crucial precursor to artemisinin-is now feasible through microbial cell factories. In the fermentation of terpenes, two-phase fermentation technology has been widely applied due to its unique advantages. It facilitates in situ product extraction or adsorption, effectively mitigating the detrimental impact of product accumulation on microbial cells, thereby significantly bolstering the efficiency of microbial production of plant-derived terpenes. This paper reviews the latest developments in two-phase fermentation system applications, focusing on microbial fermentation of plant-derived terpenes. It also discusses the mechanisms influencing microbial biosynthesis of terpenes. Moreover, we introduce some new two-phase fermentation techniques, currently unexplored in terpene fermentation, with the aim of providing more thoughts and explorations on the future applications of two-phase fermentation technology. Lastly, we discuss several challenges in the industrial application of two-phase fermentation systems, especially in downstream processing.

Keywords: biosynthesis; downstream processing; in situ extraction; microbial cell factory; plant-derived terpenes; two-phase fermentation.

Figure 1

A concept of different types of TPF in a bioreactor: (a) liquid–liquid TPF systems, where the orange color on the upper layer represents organic solvents or another aqueous solvent and the dark gray color on the lower layer represents the culture medium; (b–d) different types of liquid–solid TPF systems: (b) immobilized cells as the SP; (c) immobilized solvent as the SP; (d) solid adsorbents as the SP.

Figure 2

Scheme of aqueous–organic TPF and post-treatment, with main steps including fermentation, in situ extraction, post-fermentation stratification, and product collection; the orange color on the upper layer represents organic solvent or another aqueous solvent, the dark gray color on the lower layer represents the culture medium, and the light orange color of in situ extraction represents two phases mixed during the in situ extractive fermentation.

Figure 3

Illustration of a flowchart using immobilized cells as the solid phase in the liquid–solid TPF and downstream processing, with main steps including fermentation, in situ extraction, post-fermentation filtration, and product collection; the dark gray color represents the culture medium.

Figure 4

A conceptual scheme of using immobilized solvent as the solid phase in the liquid–solid TPF and downstream processing, with main steps including fermentation, in situ extraction, post-fermentation filtration, and product collection; the dark gray color represents the culture medium and the orange color represents solvents, like ILs.

Figure 5

A conceptual flowchart using solid adsorbents as the solid phase in the liquid–solid TPF and downstream processing, with main steps including fermentation, in situ extraction, post-fermentation filtration, and product collection; the dark gray color represents the culture medium.

Figure 6

Metabolic pathway of terpene biosynthesis. The terpenes’ skeletons are formed by the condensation of multiple units of IPP and its isomer, DMAPP. MEP biosynthetic pathway starts with pyruvate and G3P. Through a series of enzyme-catalyzed reactions, it ultimately produces IPP and DMAPP (shown on a light blue background). This process involves a variety of enzymes, DXS (1-deoxy-d-xylulose-5-phosphate synthase) and DXR (1-deoxy-d-xylulose-5-phosphate reductoisomerase), CMS (2-C-methyl-d-erythritol 4-phosphate cytidyltransferase), CMK (4-diphosphocytidyl-2-C-methyl-d-erythritol kinase), MDS (2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase), HDS (4-hydroxy-3-methylbut-2-enyl diphosphate synthase), and HDR (4-hydroxy-3-methylbut-2-enyl diphosphate reductase). The MVA biosynthetic pathway, which is another pathway for terpene biosynthesis, distinct from the MEP pathway, starts with acetyl-CoA. The primary enzymes involved in the MVA pathway are acetyl-CoA acetyltransferase (ACAT), hydroxymethylglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), mevalonate kinase (MVK), phosphomevalonate kinase (PMK), mevalonate-5-pyrophosphate decarboxylase (MVD), and isopentenyl-diphosphate delta-isomerase (IDI). Geranyl diphosphate synthase (GPPS), farnesyl diphosphate synthase (FPPS), geranylgeranyl diphosphate synthase (GGPPS), and farnesylgeranyl diphosphate synthase (GFPPS) TPs convert the basic terpene precursors, IPP and DMAPP, into various terpene compounds. Abbreviation of metabolites: DXP, 1-deoxy-d-xylulose-5-phosphate; MEP, 2-C-methyl-d-erythritol-4-phosphate; CDP-ME, 4-diphosphocytidyl-2-C-methyl-d-erythritol; CDP-MEP, 4-diphosphocytidyl-2-C-methyl-d-erythritol 2-phosphate; MEcPP, 2-C-methyl-d-erythritol-2,4-cyclodiphosphate; HMB-PP, 4-hydroxy-3-methylbut-2-enyl-diphosphate; AcAc-CoA, acetoacetyl-CoA; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; MVA, mevalonate; MVP, mevalonate-5-phosphate; MVPP, mevalonate-5-pyrophosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl diphosphate; GFPP, farnesylgeranyl diphosphate.