Seven-enzyme in vitro cascade to (3R)-3-hydroxybutyryl-CoA

Abstract

Economical and environmentally-friendly routes to convert feedstock chemicals like acetate into valuable chiral products such as (R)-3-hydroxybutyrate are in demand. Here, seven enzymes (CoaA, CoaD, CoaE, ACS, BktB, PhaB, and GDH) are employed in a one-pot, in vitro, biocatalytic synthesis of (3R)-3-hydroxybutyryl-CoA, which was readily isolated. This platform generates not only chiral diketide building blocks but also desirable CoA derivatives.

ATP‐Independent and Cell‐Free Biosynthesis of β‐Hydroxy Acids Using Vinyl Esters as Smart Substrates

“…The 3‐HB titer decreased by 2‐fold compared to the reaction triggered with CoASH, reaching a Yield MTC of 6.2 % (Figure S15). Unlike the in vitro biosynthesis of 3‐hydroxybutyryl‐SNAC thioesters initiated by the ATP‐dependent acetyl‐CoA synthase and starting from acetate, [33] the abiotic thiolysis of VA with SNAC enables the formation of 3‐HB, indicating that all SNAC derivatives can be sequentially accepted by AsTHL5, TtHBDH and EcTesB. The low product yield achieved when starting with the truncated thiolated cofactor likely relies on the low affinity of these enzymes toward SNACs intermediates.…”

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).…”

“…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 of terpenes [13c] or 6‐deoxyerythronolide B (6‐DEB), [13b] the 4‐hydroxybutyryl‐CoA synthetase from Nitrosopumilus maritimus (UniProt ID: A9A1Y1) that is integrated into the CETCH cycle, [13,21] the succinyl‐CoA synthetase from Escherichia coli (UniProt ID: P0AGE9 and P0A836) involved in the synthesis of 6‐DEB [13b] or 5‐aminolevulinic acid [22] and the oxalyl‐CoA synthetase from Methylorubrum extorquens (UniProt ID: C5APP0) in the synthesis of α‐hydroxyacids [23] .…”

“…Notably, thiolases play a key role in the synthetic pathway to produce butanol from glucose, [10,31a] for the synthesis of polyhydroxyalkanoates (PHAs) [8a,16a–c,32] but also the synthesis of polyketide and terpene derivatives [20,29–30] . Within this enzyme family, thiolases from Escherichia coli (UniProt ID: A0A061KWL3 ( atoB )) and from Cupriavidus necator / Ralstonia eutropha (UniProt ID: P14611 ( phaA )) are the widest exploited ones, while thiolase from Cupriavidus necator (UniProt ID: Q0KBP1 ( bktB )) has been used to a lesser extent [8a,16a–c,18,20,30b,32,42] . The discovery of other thiolases from Saccharomyces cerevisiae (UniProt ID: P41338, Erg10 ) [45] and from Ascaris suum (UniProt ID: F1KZ13 ( Acat1 ), F1KYX0 ( Acat2 ), F1L3X9 ( Acat3 ), F1L0C5 ( Acat4 ), F1L3N8 ( Acat5 )) [46] combined with the protein engineering work reported on thiolases active sites, enlarge the possibilities towards the condensation of larger acyl‐CoA substrates to produce more complex linear or α‐branched ß‐ketoacyl‐CoA derivatives [45,47] .…”

“…combining a glyoxylate and pyruvate synthesis cycle (rGPS) with the malyl‐CoA‐glycerate pathway (MCG) [42] . A variety of enzymes were applied in cell‐free pathways for the acetoacetyl‐CoA reduction such as the ( S )‐selective Tt HBDH from Thermus thermophilus (UniProt ID: Q72J81 ( Hbd )) [10,39] and the ( R )‐selective reductases from Cupriavidus necator (UniProt ID: P14697 ( phaB )) [8a,16a–c,18,32] and Hbd1/Bhbd from Clostridium acetobutylicum (UniProt ID: P52051 ( Hbd )) [31a,42] …”