An in vitro synthetic biosystem based on acetate for production of phloroglucinol

Abstract

BackgroundPhloroglucinol is an important chemical, and the biosynthesis processes which can convert glucose to phloroglucinol have been established. However, due to approximate 80% of the glucose being transformed into undesirable by-products and biomass, this biosynthesis process only shows a low yield with the highest value of about 0.20 g/g. The industrial applications are usually hindered by the low current productivity and yield and also by the high costs. Generally, several different aspects limit the development of phloroglucinol biosynthesis. The yield of phloroglucinol is one of the most important parameters for its bioconversion especially from economic and ecological points of view. The in vitro biosynthesis of bio-based chemicals, is a flexible alternative with potentially high-yield to in vivo biosynthetic technology.ResultsBy comparing the activity of acetyl-CoA synthetase (ACS) from Escherichia coli and Acetobacter pasteurianus, the highly active ACS2 was identified in A. pasteurianus. Acetyl-CoA carboxylase (ACC) from Acinetobacter calcoaceticus and phloroglucinol synthase (PhlD) from Pseudomonas fluorescens pf-5 were expressed and purified. Acetate was successfully transformed into phloroglucinol by the combined activity of above-mentioned enzymes and required cofactor. After optimization of the in vitro reaction system, phloroglucinol was then produced with a yield of nearly 0.64 g phloroglucinol/g acetic acid, which was equal to 91.43% of the theoretically possible maximum.ConclusionsIn this work, a novel in vitro synthetic system for a highly efficient production of phloroglucinol from acetate was demonstrated. The system’s performance suggests that in vitro synthesis of phloroglucinol has some advantages and is potential to become a feasible industrial alternative. Based on the results presented herewith, it is believed that in vitro biosystem will provide a feasible option for production of important industrial chemicals from acetate, which could work as a versatile biosynthetic platform.

Efficient conversion of acetate into phloroglucinol by recombinant Escherichia coli

“…[4][5][6][7] Zhang et al successfully produced phloroglucinol from acetate by in vitro reaction. 8 However, the costings and nonrecycling of the enzymes used in vitro were the main obstacles. It is still an attractive avenue to convert acetate into phloroglucinol in vivo.…”

Yeast Surface Display for In Vitro Biosynthetic Pathway Reconstruction

“…It was probably because degeneration and diffusive loss of intermediates, co-factors, and group donors limited the on-going occurrence of several catalytic reactions in longer time. 48 Nevertheless, when the synthesis of geraniol was integrated with all functional modules (configuration IV), it was significantly maximized (Figure 6c). Thus, the in vitro multienzymatic system should be optimized systematically based on the entire reconstituted metabolic pathway.…”

“…Currently, the utilization of acetate as a nontraditional carbon source is becoming popular in industrial biotechnology due to its low cost and competition with food supplies being avoided. 48 In this study, the synthetic module of acetyl-coA was integrated with the heterozygous MVA pathway that fulfilled the geraniol production from acetate. Acetyl-coA recycling under the catalysis of E-Acs2 was an important factor to reduce the cost and operation.…”

“…Even if various functional modules were integrated together, the productivity was below expectation. It was probably because degeneration and diffusive loss of intermediates, co-factors, and group donors limited the on-going occurrence of several catalytic reactions in longer time . Nevertheless, when the synthesis of geraniol was integrated with all functional modules (configuration IV), it was significantly maximized (Figure c).…”

Coenzyme A Thioester Intermediates as Platform Molecules in Cell‐Free Chemical Biomanufacturing

“…Among the substrates, acetic acid is the most widespread and its transformation into acetyl-CoA is performed by acetyl-CoA synthetases, usually the one from Escherichia coli (UniProt ID: P27550) or the commercially available one from Baker's yeast. ACSs have been successfully integrated into different cell-free systems to synthesize poly(hydroxybutyric acid) (PHB), [16] phloroglucinol, [17] (3R)-hydroxybutyryl-CoA, [18] or phospholipids. [19] Other relevant acyl-CoA synthetases are the medium-chain acyl-CoA synthetase from Cannabis sativa (UniProt ID: H9A1V5), which was used for the synthesis of cannabinoids, [20] the malyl-CoA synthetase from Methylorubrum extorquens (UniProt ID: P53594) for the synthesis CoA-propanoylating propanal dehydrogenase 1.2.1.87 [35] Carboxylic acid reductase (adenylation domain) [24] Cofactors: ATP, Adenosine 5'-triphosphate; AMP, Adenosine 5'-monophosphate; CoA, Coenzyme A; NAD(P)H, β-Nicotinamide adenine dinucleotide (2'phosphate).…”