Development of a Novel Biocatalyst for the Resolution of rac‐Pantolactone

A novel L ‐pantolactone hydrolase, Lph, from Agrobacterium tumefaciens Lu681 was characterized, which stereospecifically hydrolyses L ‐pantolactone to L ‐pantoic acid yielding D ‐pantolactone with > 95% enantiomeric excess. The enzyme was found to be a 30 kDa‐Zn²⁺‐hydrolase with a K_m for L ‐pantolactone of 7 mM and a V_max of 30 U/mg. The corresponding lph gene was identified as an 807 bp ORF and cloned into E. coli. It was overexpressed under control of P_tac and P_rha yielding enzyme activities of up to 600 U/g dry weight. Resolution of d,l ‐pantolactone in repeated batches with isolated Lph and enzyme recovery by membrane filtration gave D ‐pantolactone with 50% yield and 90–95% ee over 6 days. Covalent immobilization to EupergitC led to a stable biocatalyst easy to handle in a repeated batch production of D ‐pantolactone. Further improvements in the activity of Lph were achieved by directed evolution of the enzyme. Activities of mutants F62S, K197D and F100L were increased 2.3, 1.7, and 1.5 fold, respectively.     

Combinatorial Mutasynthesis of Scrambled Beauvericins,Cyclooligomer Depsipeptide Cell Migration Inhibitors from Beauveria bassiana

Fungal cyclooligomer depsipeptides such as beauvericin, bassianolide, and enniatins display antibiotic, antifungal, insecticidal, broad‐spectrum cancer cell antiproliferative, and cell migration inhibitory activities. We have identified a gene encoding a novel enzyme, ketoisovalerate reductase (KIVR), which is the sole provider of D ‐hydroxyisovalerate (D ‐Hiv), a common precursor for cyclooligomer depsipeptide biosynthesis in Beauveria bassiana. KIVR and related hypothetical oxidoreductases encoded in fungal genomes are similar to ketopantoate reductases but not to D ‐hydroxycarboxylate dehydrogenases. We demonstrate that a KIVR knockout B. bassiana strain can be used for the efficient mutasynthesis of unnatural beauvericin congeners. Simultaneous feeding of precursor analogues enabled the combinatorial mutasynthesis of scrambled beauvericins, some assembled entirely from unnatural precursors. The effects of the introduced structural changes on the antiproliferative and cell migration inhibitory activities of these analogues were evaluated.     

Impact of a Stereocentre Inversion in Cyclic Lipodepsipeptides from the Viscosin Group: A Comparative Study of the Viscosinamide and Pseudodesmin Conformation and Self‐Assembly

The viscosin group covers a series of cyclic lipodepsipeptides (CLPs) produced by Pseudomonas bacteria, with a range of biological functions and antimicrobial activities. Their oligopeptide moieties are composed of both L ‐ and D ‐amino acids. Remarkably, the Leu5 amino acid—centrally located in the nonapeptide sequence—is the sole residue found to possess either an L or D configuration, depending on the producing strain. The impact of this D /L switch on the solution conformation was investigated by NMR‐restrained molecular modelling of the epimers pseudodesmin A and viscosinamide A. Although the backbone fold remained unaffected, the D /L switch adjusted the segregation between hydrophobic and hydrophilic residues, and thus the amphipathicity. It also influenced the self‐assembly capacity in organic solvents. Additionally, several new minor variants of viscosinamide A from Pseudomonas fluorescens DR54 were identified, and an NMR assay is proposed to assess the presence of either an L ‐ or D ‐Leu5.     

Simple and Highly Convenient Two‐Step Practical Procedure for the Synthesis of Optically Pure Methyl D‐erythro‐2,3‐Dihydroxybutanoate

A synthesis of enantiopure methyl D ‐erythro‐2,3‐dihydroxybutanoate has been realized using two simple and consecutive reactions on D ‐erythronolactone as the starting material. The two reactions are lactone ring opening with hydrobromic acid in methanol and subsequent reductive debromination.     

New Antimicrobial Hexapeptides: Synthesis,Antimicrobial Activities,Cytotoxicity, and Mechanistic Studies

Synthetic antimicrobial peptides have recently emerged as promising candidates against drug‐resistant pathogens. We identified a novel hexapeptide, Orn‐D ‐Trp‐D ‐Phe‐Ile‐D ‐Phe‐His(1‐Bzl)‐NH₂, which exhibits broad‐spectrum antifungal and antibacterial activity. A lead optimization was undertaken by conducting a full amino acid scan with various proteinogenic and non‐proteinogenic amino acids depending on the hydrophobic or positive‐charge character of residues at various positions along the sequence. The hexapeptide was also cyclized to study the correlation between the linear and cyclic structures and their respective antimicrobial activities. The synthesized peptides were found to be active against the fungus Candida albicans and Gram‐positive bacteria such as methicillin‐resistant Staphylococcus aureus and methicillin‐resistant Staphylococcus epidermidis, as well as the Gram‐negative bacterium Escherichia coli; MIC values for the most potent structures were in the range of 1–5 μg mL^?1 (IC₅₀ values in the range of 0.02–2 μg mL^?1). Most of the synthesized peptides showed no cytotoxic effects in an MTT assay up to the highest test concentration of 200 μg mL^?1. A tryptophan fluorescence quenching study was performed in the presence of negatively charged and zwitterionic model membranes, mimicking bacterial and mammalian membranes, respectively. The results of the fluorescence study demonstrate that the tested peptides are selective toward bacterial over mammalian cells; this is associated with a preferential interaction between the peptides and the negatively charged phospholipids of bacterial cells.     

Baceridin,a Cyclic Hexapeptide from an Epiphytic Bacillus Strain,Inhibits the Proteasome

A new cyclic hexapeptide, baceridin ( 1 ), was isolated from the culture medium of a plant‐associated Bacillus strain. The structure of 1 was elucidated by HR‐HPLC‐MS and 1D and 2D NMR experiments and confirmed by ESI MS/MS sequence analysis of the corresponding linear hexapeptide 2 . The absolute configurations of the amino acid residues were determined after derivatization by GC‐MS and Marfey's method. The cyclopeptide 1 consists partially of nonribosomal‐derived D ‐ and allo‐D ‐configured amino acids. The order of the D ‐ and L ‐leucine residues within the sequence cyclo(‐L ‐Trp‐D ‐Ala‐D ‐allo‐Ile‐L ‐Val‐D ‐Leu‐L ‐Leu‐) was assigned by total synthesis of the two possible stereoisomers. Baceridin ( 1 ) was tested for antimicrobial and cytotoxic activity and displayed moderate cytotoxicity (1–2 μg mL^?1) as well as weak activity against Staphylococcus aureus. However, it was identified to be a proteasome inhibitor that inhibits cell cycle progression and induces apoptosis in tumor cells by a p53‐independent pathway.     

Syntheses of poly(lactic acid‐co‐glycolic acid) serial biodegradable polymer materials via direct melt polycondensation and their characterization

The optimal synthetic conditions of poly(lactic acid‐co‐glycolic acid) (PLGA) via melt copolycondensation directly from L ‐lactic acid (L ‐LA) and glycolic acid (GA) with a feed molar ratio of 50/50 are discussed; the important drug‐delivery carrier PLGA50/50 is used as a special example. With reaction conditions of 165°C and 70 Pa and with 0.5 wt % SnCl₂ as the catalyst, 10 h of polymerization gave the L ‐PLGA50/50 with the biggest intrinsic viscosity ([η]), 0.1993 dL/g. The optimal synthetic conditions were verified by the synthesis of D,L ‐PLGA50/50 with D,L ‐lactic acid (D,L ‐LA) instead of L ‐LA, but the biggest [η] was 0.2382 dL/g. Under the same synthetic conditions with L ‐LA and D,L ‐LA as starting materials, serial PLGA with different molar feed ratios, including 100/0, 90/10, 70/30, 50/50, 30/70, 10/90, and 0/100, were synthesized via simple and practical direct melt copolycondensation, and their solubilities were investigated. When the glycolic acid feed molar percentage was equal to or more than 70%, solubilities in tetrahydrofuran and CHCl₃ became worse, and some samples were even wholly insoluble. These biodegradable polymers were also systematically characterized with gel permeation chromatography, Fourier transform infrared spectroscopy, ¹H‐NMR spectroscopy, differential scanning calorimetry, and X‐ray diffraction. PLGA synthesized from L ‐LA and D,L ‐LA had many differences in weight‐average molecular weight (M_w), glass‐transition temperature, crystallinity, and composition. When the molar feed ratio of LA to GA was 50/50, both the [η] and M_w values of D,L ‐PLGA were higher than those of L ‐PLGA. With D,L ‐LA as the starting material, the structure of the PLGA copolymer was relatively simple, and its properties were apt to be controlled by its GA chain segment. When the feed molar percentage of the monomer (LA or GA) was more than or equal to 90%, the copolymer was apt to be crystalline, and the aptness was more obvious for the L ‐LA monomer. The composition percentage of GA in PLGA was not only higher than the feed molar percentage of GA, but also, the GA percentage in D,L ‐PLGA was higher than in L ‐PLGA. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 99: 244–252, 2006     

Stereocontrolled Synthesis and Pharmacological Evaluation of Azetidine‐2,3‐Dicarboxylic Acids at NMDA Receptors

The four stereoisomers of azetidine‐2,3‐dicaroxylic acid (L ‐trans‐ADC, L ‐cis‐ADC, D ‐trans‐ADC, and D ‐cis‐ADC) were synthesized in a stereocontrolled fashion following two distinct strategies: one providing the two cis‐ADC enantiomers and one giving access to the two trans‐ADC enantiomers. The four azetidinic amino acids were characterized in a radioligand binding assay ([³H]CGP39653) at native NMDA receptors: L ‐trans‐ADC showed the highest affinity (K_i=10 μM ) followed by the D ‐cis‐ADC stereoisomer (21 μM ). In contrast, the two analogues L ‐cis‐ADC and D ‐trans‐ADC were low‐affinity ligands (>100 and 90 μM , respectively). Electrophysiological characterization of the ADC compounds at the four NMDA receptor subtypes NR1/NR2A, NR1/NR2B, NR1/NR2C, and NR1/NR2D expressed in Xenopus oocytes showed that L ‐trans‐ADC displayed the highest agonist potency at NR1/NR2D (EC₅₀=50 μM ), which was 9.4‐, 3.4‐, and 1.9‐fold higher than the respective potencies at NR1/NR2A–C. D ‐cis‐ADC was shown to be a partial agonist at NR1/NR2C and NR1/NR2D with medium‐range micromolar potencies (EC₅₀=720 and 230 μM , respectively). A subsequent in silico ligand–protein docking study suggested an unusual binding mode for these amino acids in the agonist binding site.     

Identification and Characterization of D‐Succinylase,and a Proposed Enzymatic Method for D‐Amino Acid Synthesis

Chiral amino acids are important intermediates for the pharmaceutical industry. We have developed a novel one‐pot enzymatic method for D ‐amino acid synthesis by the dynamic kinetic resolution of N‐succinyl‐dl ‐amino acids using D ‐succinylase (DSA) and N‐succinylamino acid racemase (NSAR, EC 4.2.1.113). The DSA from Cupriavidus sp. P4‐10‐C, which hydrolyzes N‐succinyl‐D ‐amino acids enantioselectively to their corresponding D ‐amino acids, was identified for the first time by screening soil microorganisms. Subsequently, the DSA gene was cloned and overexpressed in Escherichia coli. DSA was shown to comprise two subunits with molecular masses of 26 kDa and 60 kDa. Additionally, the NSAR gene from Geobacillus stearothermphilus NCA1503, which racemizes N‐succinylamino acids, was also cloned and overexpressed in E. coli. The highly purified DSA and NSAR prepared from each recombinant E. coli were characterized and used for D ‐amino acid synthesis. A one‐pot enzymatic method converted 100 mM N‐succinyl‐dl ‐phenylalanine to D ‐phenylalanine in 91.1% conversion with 86.7% ee. This novel enzymatic method may be useful for the industrial production of many D ‐amino acids.

     

Plasticization of Poly‐L‐lactide with L‐lactide,D‐lactide,and D,L‐lactide monomers

Poly‐L ‐lactide (PLLA) is being widely considered for repair of damaged tissues, for controlled antibiotic release, and also as scaffolds for cultured cells. PLLA was blended with the lactide monomer in its two enantiomeric forms: D ‐lactide (D ‐la) and L ‐lactide (L ‐la) and with the cyclic dimmer D ,L ‐la, in order to enhance its flexibility and thereby overcome its inherent problem of brittleness. In this work, the crystallization, phase structure, and tensile properties of PLLA and PLLA plasticized with 5, 10, 15, and 20 wt% of D ‐la, L ‐la, and D ,L ‐la are explored. The three plasticizers used were effective in lowering the glass transition temperature (T_g) and the melting temperature (T_m) of PLLA, around 20°C for a plasticizer content of 20 wt%. The tensile strength and modulus of the blends decreased following the increasing content of plasticizers from approximately 58 MPa to values below 20 MPa, and from 1667 to 200 MPa, respectively. Aging the blends at storage ambient temperature revealed that the enhanced flexibility as well as the morphological stability was lost over time due to the migration of the plasticizer to the surface, this being less marked in the case of D ‐la as a result of interactions between the polymer and its enantiomeric monomer of complementary configuration. POLYM. ENG. SCI., 53:2073–2080, 2013. © 2013 Society of Plastics Engineers     

Structural Analysis Reveals the Substrate‐Binding Mechanism for the Expanded Substrate Specificity of Mutant meso‐Diaminopimelate Dehydrogenase

A meso‐diaminopimelate dehydrogenase (DAPDH) from Clostridium tetani E88 (CtDAPDH) was found to have low activity toward the D ‐amino acids other than its native substrate. Site‐directed mutagenesis similar to that carried out on the residues mutated by Vedha‐Peters et al. resulted in a mutant enzyme with highly improved catalytic ability for the synthesis of D ‐amino acids. The crystal structures of the CtDAPDH mutant in apo form and in complex with meso‐diaminopimelate (meso‐DAP), D ‐leucine (D ‐leu), and 4‐methyl‐2‐oxopentanoic acid (MOPA) were solved. meso‐DAP was found in an area outside the catalytic cavity; this suggested a possible two‐step substrate‐binding mechanism for meso‐DAP. D ‐leu and MOPA each bound both to Leu154 and to Gly155 in the open form of CtDAPDH, and structural analysis revealed the molecular basis for the expanded substrate specificity of the mutant meso‐diaminopimelate dehydrogenases.     

Biodegradations of statistical copolymers composed of D,L‐lactide and cyclic carbonates

Syntheses and biodegradation of statistical copolymers of D ,L ‐lactide (D ,L ‐LA) with trimethylene carbonate (TMC), rac‐1‐methyltrimethylene carbonate (1‐MTMC) and 2,2‐dimethyltrimethylene carbonate (2,2‐DTMC) were investigated at various monomer ratios using SmMe(C₅Me₅)₂THF as an initiator at 80 °C for 24 h in toluene. Biodegradations of poly(D ,L ‐LA‐co‐racemo‐1‐MTMC) (95/5) and poly(D ,L ‐LA‐co‐2,2‐DTMC) (98/2) with a compost at 60 °C proceed rapidly. Enzymatic degradations of these polymers were also performed using cholesterol esterase, lipoprotein lipase and proteinase K. Only poly(D ,L ‐LA‐co‐TMC) was biodegraded with cholesterol esterase, while poly(TMC), poly(1‐MTMC), poly(2,2‐DTMC) and poly(D ,L ‐LA) were barely degraded with these enzymes. Biodegradations of poly(D ,L ‐LA‐co‐TMC) (87/13) and poly(D ,L ‐LA‐co‐racemo‐1‐MTMC) (95/5) are rapid using proteinase K. Physical properties of these copolymers were also described. © 2003 Society of Chemical Industry     

Screening for Amidases: Isolation and Characterization of a Novel D‐Amidase from Variovorax paradoxus

Using racemic tert‐leucine amide as sole nitrogen source in minimal medium, 162 strains were isolated by enrichment techniques and shown to contain amidase activity. Among these isolates three D ‐amidase producers were found and identified as Variovorax paradoxus (two strains) and Klebsiella spec. The D ‐amidase from Variovorax paradoxus was purified to homogeneity by three chromatographic steps. With dl ‐Tle‐amide as substrate Michaelis Menten kinetics were observed with a K_M of 0.74 mM, a K_I of 640 mM and a V_max of 1.4 U/mg. The amidase has a broad pH‐optimum between 7 and 9.5 and a temperature optimum at 47–49 °C. The amidase hydrolyzed amino acid amides as well as carboxamides and 2‐hydroxy acid amides. The stereoselectivity of the reaction was variable, however. Hydrolyzing dl ‐Tle‐amide the enantiomeric ratio E was >200 resulting in D ‐Tle with an ee of >99% and up to 47% conversion. Similar results were obtained with dl ‐Leu‐amide and dl ‐Val‐amide while dl ‐Phe‐amide was hydrolyzed with an enantiomeric ratio E of only 5.     

Chemical composition and structural feature of Populus gansuensis hemicellulosic polymers

Hemicellulosic polymers A and B were isolated from Populus gansuensis by extracting the chlorite‐delignified residue with 10% KOH. Fractional precipitation of hemicellulose B by graded ethanol resulted in six subfractions varying in yield, molecular size distribution, and sugar composition. Macromolecular and more linear hemicellulosic polymers with higher yields were preferentially precipitated in relatively lower ethanol concentrations, while more branched and complex hemicellulosic polymers with lower molecular weights were obtained in relatively higher ethanol concentrations. Chemical and spectral evidence suggested that H30 subfraction obtained by 30% ethanol precipitation was assumed to be 4‐O‐methyl‐D ‐glucurono‐D ‐xylans, with 4‐O‐methyl‐D ‐glucuronic acid linked to C‐2 of (1→4)‐β‐D ‐xylan. On average for every six D ‐xylopyranosyl residues, there is one 4‐O‐methyl‐D ‐glucuronic acid group. Hemicellulosic subfraction H75 precipitated at the ethanol concentration of 75% was more branched, and presumed to be heterogeneous mixture of arabinoglucurnoxylan and galactoglucomannan. In addition, the thermal stability of the linear large molecular hemicellulosic subfraction H30 appeared higher than the branched subfraction H75. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Redesigning the Active Site of Transaldolase TalB from Escherichia coli: New Variants with Improved Affinity towards Nonphosphorylated Substrates

Recently, we reported on a transaldolase B variant (TalB F178Y) that is able to use dihydroxyacetone (DHA) as donor in aldol reactions. In a second round of protein engineering, we aimed at improving the affinity of this variant towards nonphosphorylated acceptor aldehydes, that is, glyceraldehyde (GA). The anion binding site was identified in the X‐ray structure of TalB F178Y where a sulfate ion from the buffer was bound in the active site. Therefore, we performed site‐directed saturation mutagenesis at three residues forming the putative phosphate binding site, Arg181, Ser226 and Arg228. The focused libraries were screened for the formation of D ‐fructose from DHA and d,l ‐GA by using an adjusted colour assay. The best results with respect to the synthesis of D ‐fructose were achieved with the TalB F178Y/R181E variant, which exhibited an at least fivefold increase in affinity towards d,l ‐GA (K_M=24 mM ). We demonstrated that this double mutant can use D ‐GA, glycolaldehyde and the L ‐isomer, L ‐GA, as acceptor substrates. This resulted in preparative synthesis of D ‐fructose, D ‐xylulose and L ‐sorbose when DHA was used as donor. Hence, we engineered a DHA‐dependent aldolase that can synthesise the formation of polyhydroxylated compounds from simple and cheap substrates at preparative scale.     

Chiral separation with polyurea membrane consisting of L‐lysinyl residue: Proposal of facile method for prediction of permselectivity

Novel chiral polyureas were prepared from L ‐lysine‐4‐nitroanilide and 2,4‐toluene diisocyanate. The polyurea thus prepared gave a durable self‐standing membrane that can be directly converted into molecular recognition membranes by applying an alternative molecular imprinting. The Z‐D ‐glutamic acid (D ‐Glu) molecularly imprinted membrane adsorbed D ‐Glu in preference to L ‐glutamic acid (L ‐Glu) from racemic mixture of Glu and vice versa. Those two types of molecularly imprinted membrane showed optical resolution ability, adopting a concentration gradient as a driving force for membrane transport. A facile method to predict permselectivity of racemic mixture of charged permeant was proposed. From the observed membrane resistance, the permselectivity was predicted. The predicted permselectivity coincided with the observed one. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

One‐Pot Cascade Reactions using Fructose‐6‐phosphate Aldolase: Efficient Synthesis of D‐Arabinose 5‐Phosphate,D‐Fructose 6‐Phosphate and Analogues

One‐pot multienzymatic reactions have been performed for the synthesis of 1‐deoxy‐D ‐fructose 6‐phosphate, 1,2‐dideoxy‐D ‐arabino‐hept‐3‐ulose 7‐phosphate, D ‐fructose 6‐phosphate and D ‐arabinose 5‐phosphate. The whole synthetic strategy is based on an aldol addition reaction catalysed by fructose‐6‐phosphate aldolase (FSA) as a key step of a three or four enzymes‐catalysed cascade reaction. The four known donors for FSA – dihydroxyacetone (DHA), hydroxyacetone (HA), 1‐hydroxy‐2‐butanone (HB) and glycolaldehyde (GA) – were used with D ‐glyceraldehyde 3‐phosphate as acceptor substrate. The target phosphorylated sugars were obtained in good to excellent yields and high purity.     

Optical Resolution Membranes from Polysulfones Bearing Alanine Derivatives as Chiral Selectors

Polysulfones bearing a derivative of alanyl residue employed as chiral selectors were prepared by polymer modification. The specific rotation ([α]_D) of the polysulfone with a derivative of D ‐alanyl residue (PSf‐Ac‐D ‐Ala) was determined to be 2.87 deg · cm² · g^?1 (c = 1.00 g · dL^?1 in DMF) and that with L ‐alanyl residue (PSf‐Ac‐L ‐Ala) to be ‐2.36 deg · cm² · g^?1 (c = 1.00 g · dL^?1 in DMF). The membrane from PSf‐Ac‐D ‐Ala preferentially adsorbed the D ‐isomer of Glu from racemic mixture of Glu and vice versa. Chiral separation ability was studied by applying a potential difference as a driving force for membrane transport. The permselectivity of PSf‐Ac‐D ‐Ala toward D ‐Glu (α_D/L) was determined to be 1.40, and that of PSf‐Ac‐L ‐Ala toward the L ‐isomer (α_L/D) to be 1.48 at 18.0 V, reflecting their adsorption selectivity.

     

Syntheses of poly(lactic acid)‐poly(ethylene glycol) serial biodegradable polymer materials via direct melt polycondensation and their characterization

Directly starting from lactic acid (LA) and poly(ethylene glycol) (PEG), biodegradable material polylactic acid‐polyethylene glycol (PLEG) was synthesized via melt copolycondensation. The optimal synthetic conditions, including prepolymerization method, catalyst kinds and quantity, copolymerization temperature and time, LA stereochemical configuration, feed weight ratio m_LA/m_PEG and M_n of PEG, were all discussed in detail. When D ,L ‐LA and PEG (M_n = 1000 Da) prepolymerized together as feed weight ratio m_{D ,l‐LA}/m_PEG = 90/10, 15 h copolycondensation under 165°C and 70 Pa, and 0.5 wt % SnO as catalyst, gave D ,L ‐PLEG1000 with the highest [η] of 0.40 dL/g, and the corresponding MW was 41,700 Da. Using L ‐LA instead of D ,L ‐LA, 10 h polymerization under 165°C and 70 Pa, and 0.5 wt % SnO as catalyst, gave L ‐PLEG1000 with the highest [η] of 0.21 dL/g and MW of 15,600 Da. Serial D ,L ‐PLEG with different feed weight ratio and M_n of PEG were synthesized via the simple and practical direct melt copolycondensation, and characterized with FTIR, ¹H NMR, GPC, DSC, XRD, and contact angle testing. D ,L ‐PELG not only had higher MW than PDLLA, PLLA and L ‐PELG, but also better hydrophilicity than PDLLA. The novel one‐step method could be an alternative route to the synthesis of hydrophilic drug delivery carrier PLEG instead of the traditional two‐step method using lactide as intermediate. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 577–587, 2006     

Activated α,β‐Unsaturated Aldehydes as Substrate of Dihydroxyacetone Phosphate (DHAP)‐Dependent Aldolases in the Context of a Multienzyme System

The utility for carbon‐carbon bond formation of a multienzyme system composed of recombinant dihydroxyacetone kinase (DHAK) from Citrobacter freundii, the fructose bisphosphate aldolase from rabbit muscle (RAMA) and acetate kinase (AK) for adenosine triphosphate (ATP) regeneration has been studied. Several aldehydes with great structural diversity, including three α,β‐unsaturated aldehydes, have been analysed as acceptor substrates. It was found that α,β‐unsaturated aldehydes bearing an electron‐withdrawing group in the β position to the double bond with a trans configuration are good acceptors for RAMA in this multienzyme system. The aldol reaction proceeds with excellent D ‐threo enantioselectivity and the aldol adduct is obtained in good overall yield. The L ‐threo and D ‐erythro enantiomers are also accessible from rhamnulose 1‐phosphate aldolase (Rha‐1PA) and fuculose 1‐phosphate aldolase (Fuc‐1PA) catalysed reactions, respectively.