Cofactor recycling strategies for secondary metabolite production in cell-free protein expression systems

Abstract

Cell-free protein synthesis (CFPS) has emerged as an attractive platform for biotechnology and synthetic biology due to its numerous advantages to cell-based technologies for specific applications. CFPS can be faster, less sensitive to metabolite toxicity, and amenable to systems that are not easily genetically manipulated. Due to these advantages, a promising application of CFPS is to characterize biosynthetic gene clusters, particularly those harbored within the genomes of microorganisms that generate secondary metabolites, otherwise known as natural products. In the postgenomic era, genome sequencing has revealed an incredible wealth of metabolic diversity. However, far more of these pathways are termed "cryptic," i.e., unable to be produced under standard laboratory conditions than have been characterized. A major barrier to characterizing these cryptic natural products using CFPS is that many of these pathways require utilization of complex cofactors, many of which to date are not recycled efficiently or in an economically viable fashion. In this perspective, we outline strategies to regenerate cofactors relevant to secondary metabolite production in CFPS. This includes adenosine 5'-triphosphate, coenzyme A, redox cofactors (iron-sulfur clusters, nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide), all of which play a crucial role in important biosynthetic enzymes. Such advances in cofactor recycling enable continuous production of complex metabolites in CFPS and expand the utility of this emergent platform.

Keywords: Cell Free Protein Synthesis; Cofactor recycling strategies; Secondary metabolite production.

Fig. 1

Schematic diagram of secondary metabolites sources, classes, and biomanufacturing applications

Fig. 2

Mechanisms of different ATP regeneration systems. a, ATP regeneration using acetyl phosphate and acetate kinase (adapted from Bartzoka et al. 2022); b, glycolysis pathway and ATP regeneration using glucose-6-phosphate, phosphoenolpyruvate, and pyruvate as the energy sources (adapted from Kim and Kim ; Yan et al. 2014); c, ATP regeneration from AMP and poly(P) by the PAP-ADK system (adapted from Resnick and Zehnder 2000); d, ATP regeneration from AMP by the PAP-PPK system combined with the acetyl-CoA synthesis. ADK, adenylate kinase (adapted from Zhao and van der Donk 2003); AK, acetate kinase; PAP, polyphosphate AMP phosphotransferase; PPK, polyphosphate kinase; PPase, inorganic pyrophosphatase

Fig. 3

Schematic of CoA recycling in different pathways. a, the cell free pathway of n-butanol from glucose (adapted from Krutsakorn et al. 2013); b, the constructed biosynthetic pathway of n-butanol. Acetyl-CoA is produced from glycolysis pathway in E. coli, then utilized by CoA-dependent pathway in C. acetobutylicum to produce n-butanol (adapted from Karim and Jewett 2016); c, rosmarinic acid biosynthesis pathway with CoA and ATP regeneration (adapted from Yan et al. 2019); d, biosynthetic pathway for conversion of glucose to monoterpenes. PK, pyruvate kinase; PDC, pyruvate decarboxylase (adapted from Korman et al. 2017); ADDH, CoA-acylating aldehyde dehydrogenase; ACC, acetyl-CoA acetyltransferase; HBD, hydroxybutyryl-CoA dehydrogenase; HPD, 3-hydroxypropionyl-CoA dehydratase; NFO, NADH-dependent flavinoxidoreductase; HAD, 3-hydroxyacyl-CoA dehydrogenase; PDH, pyruvate dehydrogenase; AtoB, thiolase; CRT, crotonase; TER, butyryl-CoA dehydrogenase; ADHE, bifunctional acetaldehyde/alcohol dehydrogenase; 4CL, 4-coumarate: coenzyme A ligase; RAS, rosmarinic acid synthase; PPK, polyphosphate kinase; HMGS, hydroxymethylglutaryl-CoA synthase; HMGR, hydroxymethylglutaryl-CoA reductase; MVK, mevalonate-5-kinase; PMVK, phosphomevalonate kinase; MDC, mevalonate pyrophosphate decarboxylase; IDI, isopentenyl pyrophosphate isomerase; FPPS-S82F, farnesyl-pyrophosphate synthase-S82F mutant

Fig. 4

Schematic diagram of cell free mature [4Fe-4S] protein synthesis system. In vitro translation of mRNA was conducted with formate dehydrogenase (FDH), flavin reductase (FRE), and catalase (CAT). SUF pathway incorporates with [4Fe-4S] cluster to form the matured protein as the second step. Figure was adapted from (Wang et al. 2023)

Fig. 5

a, typical substrate-coupled approaches to regenerate NAD(P)H, ADH, alcohol dehydrogenase; FDH, formate dehydrogenase; GDH, glucose dehydrogenase; LDH, lactate dehydrogenase (adapted from Jia et al. ; Shi et al. 2018); b, chemoenzymatic approach with [Cp*Rh(bpy)(H₂O)]²⁺ to regenerate FADH₂ (adapted from Hollmann et al. 2003); c, electrochemical approach with artificial electrode to regenerate FADH₂ (adapted from Hollmann et al. 2005); d, enzymatic approach using formate dehydrogenase (FDH) and flavin reductase to regenerate FADH₂ (adapted from (Hofstetter et al. 2004)