One-pot synthesis of (R)- and (S)-phenylglycinol from bio-based l-phenylalanine by an artificial biocatalytic cascade

Chiral phenylglycinol is a very important chemical in the pharmaceutical manufacturing. Current methods for synthesis of chiral phenylglycinol often suffered from unsatisfied selectivity, low product yield and using the non-renewable resourced substrates, then the synthesis of chiral phenylglycinol remain a grand challenge. Design and construction of synthetic microbial consortia is a promising strategy to convert bio-based materials into high value-added chiral compounds. In this study, we reported a six-step artificial cascade biocatalysis system for conversion of bio-based l-phenylalanine into chiral phenylglycinol. This system was designed using a microbial consortium including two engineered recombinant Escherichia coli cell modules, one recombinant E. coli cell module co-expressed six different enzymes (phenylalanine ammonia lyase/ferulic acid decarboxylase/phenylacrylic acid decarboxylase/styrene monooxygenase/epoxide hydrolase/alcohol dehydrogenase) for efficient conversion of l-phenylalanine into 2-hydroxyacetophenone. The second recombinant E. coli cell module expressed an (R)-ω-transaminase or co-expressed the (S)-ω-transaminase, alanine dehydrogenase and glucose dehydrogenase for conversion of 2-hydroxyacetophenone into (S)- or (R)-phenylglycinol, respectively. Combining the two engineered E. coli cell modules, after the optimization of bioconversion conditions (including pH, temperature, glucose concentration, amine donor concentration and cell ratio), l-phenylalanine could be easily converted into (R)-phenylglycinol and (S)-phenylglycinol with up to 99% conversion and > 99% ee. Preparative scale biotransformation was also conducted on 100-mL scale, (S)-phenylglycinol and (R)-phenylglycinol could be obtained in 71.0% and 80.5% yields, > 99% ee, and 5.19 g/L d and 4.42 g/L d productivity, respectively. The salient features of this biocatalytic cascade system are good yields, excellent ee, mild reaction condition and no need for additional cofactor (NADH/NAD+), provide a practical biocatalytic method for sustainable synthesis of (S)-phenylglycinol and (R)-phenylglycinol from bio-based L-phenylalanine.

Directed evolution of prenylated FMN-dependent Fdc supports efficient in vivo isobutene production

Isobutene is a high value gaseous alkene used as fuel additive and a chemical building block. As an alternative to fossil fuel derived isobutene, we here develop a modified mevalonate pathway for the production of isobutene from glucose in vivo. The final step in the pathway consists of the decarboxylation of 3-methylcrotonic acid, catalysed by an evolved ferulic acid decarboxylase (Fdc) enzyme. Fdc belongs to the prFMN-dependent UbiD enzyme family that catalyses reversible decarboxylation of (hetero)aromatic acids or acrylic acids with extended conjugation. Following a screen of an Fdc library for inherent 3-methylcrotonic acid decarboxylase activity, directed evolution yields variants with up to an 80-fold increase in activity. Crystal structures of the evolved variants reveal that changes in the substrate binding pocket are responsible for increased selectivity. Solution and computational studies suggest that isobutene cycloelimination is rate limiting and strictly dependent on presence of the 3-methyl group.

One-pot Synthesis of (R)- and (S)-phenylglycinol From Bio-based L-phenylalanine by an Artificial Biocatalytic Cascade

Chiral phenylglycinol is a very important chemical in the pharmaceutical manufacturing. Current methods for synthesis of chiral phenylglycinol often suffered from unsatisfied selectivity, low product yield and using the non-renewable resourced substrates, then the synthesis of chiral phenylglycinol remain a grand challenge. Design and construction of synthetic microbial consortia is a promising strategy to convert bio-based materials to high value-added chiral compounds. In this study, we reported a six-step artificial cascade biocatalysis system for conversion of biobased L-phenylalanine to yield chiral phenylglycinol. The cascade biocatalysis system was conducted by a microbial consortium composed of two engineered recombinant Escherichia coli cells modules, one recombinant E. coli cell module co-expression of six different enzymes (phenylalanine ammonia lyase/ferulic acid decarboxylase/phenylacrylic acid decarboxylase/styrene monooxygenase/epoxide hydrolase/alcohol dehydrogenase) for efficient conversion of L-phenylalanine into 2-hydroxyacetophenone. The second recombinant E. coli cell module expression of an (R)-ω-transaminase or co-expression of the (S)-ω-transaminase, alanine dehydrogenase and glucose dehydrogenase for conversion of 2-hydroxyacetophenone to (S)- or (R)-phenylglycinol, respectively. Combining the two engineered E. coli cell modules, after the optimization of bioconversion conditions (including pH, temperature, glucose concentration, amine donor concentration and cell ratio), L-phenylalanine could be easily converted to (R)-phenylglycinol and (S)-phenylglycinol with up to 99% conversion and >99% ee. Preparative scale biotransformation was also conducted on 100 mL scale, (S)-phenylglycinol and (R)-phenylglycinol were obtained in 71.0% and 80.5% yield, >99% ee, and 5.19 g/L.d and 4.42 g/L.d productivity, respectively. The salient features of this biocatalytic cascade system are good yields, excellent ee, mild reaction conditions and no need for additional cofactor (NADH/NAD+), provide a practical biocatalytic method for sustainable synthesis of (S)-phenylglycinol and (R)-phenylglycinol from biobased L-phenylalanine.

Direct 1,3-butadiene biosynthesis in Escherichia coli via a tailored ferulic acid decarboxylase mutant

The C4 unsaturated compound 1,3-butadiene is an important monomer in synthetic rubber and engineering plastic production. However, microorganisms cannot directly produce 1,3-butadiene when glucose is used as a renewable carbon source via biological processes. In this study, we construct an artificial metabolic pathway for 1,3-butadiene production from glucose in Escherichia coli by combining the cis,cis-muconic acid (ccMA)-producing pathway together with tailored ferulic acid decarboxylase mutations. The rational design of the substrate-binding site of the enzyme by computational simulations improves ccMA decarboxylation and thus 1,3-butadiene production. We find that changing dissolved oxygen (DO) levels and controlling the pH are important factors for 1,3-butadiene production. Using DO–stat fed-batch fermentation, we produce 2.13 ± 0.17 g L−1 1,3-butadiene. The results indicate that we can produce unnatural/nonbiological compounds from glucose as a renewable carbon source via a rational enzyme design strategy.

Generation of 4-vinylguaiacol through a novel high-affinity ferulic acid decarboxylase to obtain smoke flavours without carcinogenic contaminants

Traditional smoke flavours bear the risk of containing a multitude of contaminating carcinogenic side-products. Enzymatic decarboxylation of ferulic acid released from agro-industrial side-streams by ferulic acid esterases (FAE) enables the sustainable generation of pure, food grade 4-vinylguaiacol (4-VG), the impact compound of smoke flavour. The first basidiomycetous ferulic acid decarboxylase (FAD) was isolated from Schizophyllum commune (ScoFAD) and heterologously produced by Komagataella phaffii. It showed a molecular mass of 21 kDa, catalytic optima at pH 5.5 and 35°C, and a sequence identity of 63.6% to its next relative, a FAD from the ascomycete Cordyceps farinosa. The ScoFAD exhibited a high affinity to its only known substrate ferulic acid (FA) of 0.16 mmol L-1 and a turnover number of 750 s-1. The resulting catalytic efficiency kcat KM-1 of 4,779 L s-1 mmol-1 exceeded the next best known enzyme by more than a factor of 50. Immobilised on AminoLink Plus Agarose, ScoFAD maintained its activity for several days. The combination with FAEs and agro-industrial side-streams paves the way for a new generation of sustainable, clean, and safe smoke flavours.

Fluorescent enzyme-coupled activity assay for phenylalanine ammonia-lyases

Phenylalanine ammonia-lyases (PALs) catalyse the non-oxidative deamination of l-phenylalanine to trans-cinnamic acid, while in the presence of high ammonia concentration the reverse reaction occurs. PALs have been intensively studied, however, their industrial applications for amino acids synthesis remained limited, mainly due to their decreased operational stability or limited substrate specificity. The application of extensive directed evolution procedures to improve their stability, activity or selectivity, is hindered by the lack of reliable activity assays allowing facile screening of PAL-activity within large-sized mutant libraries. Herein, we describe the development of an enzyme-coupled fluorescent assay applicable for PAL-activity screens at whole cell level, involving decarboxylation of trans-cinnamic acid (the product of the PAL reaction) by ferulic acid decarboxylase (FDC1) and a photochemical reaction of the produced styrene with a diaryltetrazole, that generates a detectable, fluorescent pyrazoline product. The general applicability of the fluorescent assay for PALs of different origin, as well as its versatility for the detection of tyrosine ammonia-lyase (TAL) activity have been also demonstrated. Accordingly, the developed procedure provides a facile tool for the efficient activity screens of large mutant libraries of PALs in presence of non-natural substrates of interest, being essential for the substrate-specificity modifications/tailoring of PALs through directed evolution-based protein engineering.

Direct 1,3-butadiene biosynthesis in Escherichia coli via a tailored ferulic acid decarboxylase mutant

The C4 unsaturated compound 1,3-butadiene is an important monomer in synthetic rubber and engineering plastic production. However, microorganisms cannot directly produce 1,3-butadiene using glucose as a renewable carbon source via biological processes. This study constructed a novel artificial metabolic pathway for 1,3-butadiene production from glucose in Escherichia coli by combining the cis,cis-muconic acid (ccMA)-producing pathway and tailored ferulic acid decarboxylase mutants. The rational enzyme design of the substrate-binding site by computational simulation improved 1,3-butadiene-producing ccMA decarboxylation with a small mutant library. We found that changing dissolved oxygen and controlling pH were important factors for 1,3-butadiene production. Using dissolved oxygen–stat fed-batch fermentation in a 1-L jar fermenter, we could produce 2.1 g L− 1 of 1,3-butadiene. These results indicated that using a rational enzyme design, we can produce unnatural/nonbiological compounds (those that are made from petroleum and cannot be produced by microorganisms) using glucose as a renewable carbon source.