RELATED COMPOUNDS

FIELD OF INVENTION

This invention relates to the bioproduction of useful and valuable phenethyl alcohol, aldehyde, acid, amine, and related compounds using novel biocatalysts. More particularly, the present invention provides methods of bioproduction of substituted or unsubstituted phenylacetaldehyde, 2-phenylethanol, phenylacetic acid or phenylethylamine by one or more recombinant microbial cells genetically engineered to overexpress, relative to a wild type cell, at least one enzyme, which method comprises subjecting a starting material to a plurality of enzyme-catalyzed chemical transformations in a one-pot reaction system, wherein the starting material is selected from a group comprising glucose, L-phenylalanine or substituted L-phenylalanine, styrene or substituted styrene. BACKGROUND

2-Phenylethanol (2-PE), phenylacetaldehyde (PA), phenylacetic acid (PAA), and phenylethylamine (PEA) are widely used in cosmetic, perfume, and food industries. The current industrial methods to produce these compounds depend on traditional chemocatalysis of toxic and "dirty" petro-based chemicals, such as benzene. For food and cosmetic applications, natural 2-PE, PA, PAA, and PEA are preferred by customers. For instance, the natural 2-PE sells at about USD 1000/kg, while the traditional chemical synthesized 2-PE sells at only about USD 5/kg. However, the production process of natural 2-PE, PA, PAA, and PEA, extraction from botanical sources cannot meet the large market demand. Bioproduction of 2-PE, PA, PAA, and PEA from natural bioresources is regarded as a promising alternative way, yet the efficiency of existing bioproduction is limited due to the low efficiency of natural synthesis pathway and the toxicity of products.

2-Phenylethanol (2-PE) is a rose-like fragrance (FEMA-GRAS 2858) with an annual production of 10,000 tonnes and is mainly produced by chemical synthesis from benzene or styrene [Etschmann, M., Bluemke, W., et al., J. Appl. Microbiol. Biotechnol. 59: 1-8; (2002); Hua, D., Xu, P. Biotechnol. Adv. 29: 654-660 (201 1)]. Phenylacetaldehyde (PA) (FEMA-GRAS 2874) is a valuable aroma for food and cosmetic application, and the natural PA is more preferred in these applications. Phenylacetic acid (PAA) (FEMA-GRAS 2878) possesses a honey-like odor in low concentration and thus is used in some perfumes. Phenylethylamine (PEA) is a natural monoamine alkaloid with psychoactive and stimulant effects. It has been widely used as food supplement and nutrition supplement to boost mood and mental performance. To provide enough supply of natural 2-PE or other related compounds, biotechnological methods from natural origin were developed and the products can be considered natural. Microbial production of 2-PE has been very attractive for producing a "natural" product with high value. The natural Ehrlich pathway in some yeast was employed for microbial production (e.g., by some yeasts, fungi, and very few bacteria) of these natural compounds [Etschmann, M., Bluemke, W., et al., J. Appl. Microbiol. Biotechnol. 59: 1-8; (2002); Hua, D., Xu, P. Biotechnol. Adv. 29: 654-660 (2011)]. However, these methods only produced the desired products in low to moderate concentration. For example, baker's yeast Saccharomyces cerevisiae was recently engineered to produce 4.8g/L of 2-PE from 10g/L of Phe via the traditional Ehrlich pathway [Kim, B., Cho, B. R., Hahn, J. S. Biotechnol. Bioeng. 1 11 : 1 15-124 (2014)].

There is a need for improved methods to produce useful and valuable "natural" phenethyl alcohol, aldehyde, acid, amine, and related compounds. SUM MARY OF INVENTION

In this invention, novel and efficient biosynthesis pathways were engineered into microbial cells for bioproduction of 2-PE, PA, PAA, and PEA from styrene, natural products L-phenylalanine (L-Phe) and glucose, respectively. The metabolic engineering approach disclosed in the present invention can also be applied to the production of other biochemicals in E. coli and other microbial strains. The novel synthetic route involves 1) upstream shikimate pathway to produce L-Phe from glucose; 2) midstream deamination- decarboxylation module to convert L-Phe to styrene; 3) downstream modules to functionalize styrene to 2-PE, PA, PAA, and PEA, respectively. The downstream modules alone could be used for conversion of styrene to 2-PE, PA, PAA, and PEA. The midstream and downstream modules could be combined in one recombinant strain to directly convert biobased L-Phe to these products. By further integrated with the upstream pathway, the recombinant biocatalysts enable the fermentative production of these products from biobased glucose. In addition, ring-substituted derivatives of 2-PE, PA, PAA, and PEA were also produced from the corresponding substituted styrenes.

Thus, in a first aspect of the invention, there is provided a method for bioproduction of substituted or unsubstituted phenylacetaldehyde, 2-phenylethanol, phenylacetic acid or phenylethylamine by one or more recombinant microbial cells genetically engineered to overexpress, relative to a wild type cell, at least one enzyme, which method comprises subjecting a starting material to a plurality of enzyme-catalyzed chemical transformations in a one-pot reaction system, wherein the starting material is selected from a group comprising glucose, L-phenylalanine or substituted L-phenylalanine, styrene or substituted styrene. In some embodiments of the first aspect of the invention, the genetically engineered cells:

(i) overexpress styrene monooxygenase and styrene oxide isomerase for generating substituted or unsubstituted phenylacetaldehyde from styrene or substituted styrene;

(ii) overexpress styrene monooxygenase, styrene oxide isomerase and an aldehyde dehydrogenase for generating substituted or unsubstituted phenylacetic acid from styrene or substituted styrene;

(iii) overexpress styrene monooxygenase, styrene oxide isomerase, an aldehyde reductase and/ or an alcohol dehydrogenase for generating substituted or unsubstituted 2-phenylethanol from styrene or substituted styrene; or

(iv) overexpress styrene monooxygenase, styrene oxide isomerase and a transaminase for generating substituted or unsubstituted phenylethylamine from styrene or substituted styrene.

In some embodiments, the styrene monooxygenase comprises an amino acid sequence set forth in SEQ ID NO: 1 and 2, variants, mutants, or fragments thereof; styrene oxide isomerase comprises an amino acid sequence set forth in SEQ ID NO: 3, variants, mutants, or fragments thereof; the aldehyde dehydrogenase comprises an amino acid sequence set forth in SEQ ID NO: 4, variants, mutants, or fragments thereof; the alcohol dehydrogenase comprises an amino acid sequence set forth in SEQ ID NO: 5, variants, mutants, or fragments thereof and the transaminase is ω-transaminase comprises an amino acid sequence set forth in SEQ ID NO: 6, variants, mutants, or fragments thereof.

In some embodiments, the styrene monooxygenase is from Pseudomonas sp. VLB120 or its mutants, styrene oxide isomerase is from Pseudomonas sp. VLB120 or its mutants, the aldehyde dehydrogenase is from Escherichia coli or its mutants, the aldehyde reductase is from Solanum lycopersicum or its mutants or is YqhD from Escherichia coli or its mutants; the alcohol dehydrogenase is from Saccharomyces cerevisiae and the transaminase is ω-transaminase from Chromobacterium violaceum or its mutants or Vibrio fluvialis or its mutants.

In some embodiments, the styrene monooxygenase is encoded by a nucleic acid sequence set forth in SEQ ID NOs: 7 and 8; styrene oxide isomerase is encoded by a nucleic acid sequence set forth in SEQ ID NO: 9; the aldehyde dehydrogenase is encoded by a nucleic acid sequence set forth in SEQ ID NO: 10; the alcohol dehydrogenase is encoded by a nucleic acid sequence set forth in SEQ ID NO: 11 and the transaminase is ω- transaminase encoded by a nucleic acid sequence set forth in SEQ ID NO: 12.

It may be advantageous to provide the styrene or substituted styrene from conversion of L-phenylalanine or substituted L-phenylalanine in the same one-pot reaction system. Accordingly, in some embodiments, the same genetically engineered cells or other genetically engineered cells produce styrene or substituted styrene from L-phenylalanine or substituted L-phenylalanine by a deamination reaction catalyzed by overexpression of an ammonia lyase and a decarboxylation reaction catalyzed by overexpression of a decarboxylase.

In preferred embodiments, the ammonia lyase is phenylalanine ammonia lyase and the decarboxylase is phenylacrylic acid decarboxylase.

In some embodiments, the phenylalanine ammonia lyase comprises an amino acid sequence set forth in SEQ ID NO: 13, variants, mutants, or fragments thereof and phenylacrylic acid decarboxylase comprises an amino acid sequence set forth in SEQ ID NO: 14, variants, mutants, or fragments thereof.

In some embodiments, the phenylalanine ammonia lyase is AtPAL2 from Arabidopsis thaliana, encoded by a nucleic acid sequence set forth in SEQ ID NO: 15 and wherein the phenylacrylic acid decarboxylase is AnPAD from Aspergillus niger, encoded by a nucleic acid sequence set forth in SEQ ID NO: 16.

It may be advantageous to provide the L-phenylalanine for production of styrene by catalysis of glucose in the same one-pot reaction system.

In some embodiments, the same genetically engineered cells or other genetically engineered cells produce L-phenylalanine from glucose by a reaction catalyzed by overexpression of at least one enzyme selected from a group comprising DAHP synthase (AroG), shikimate kinase (AroK), shikimate dehydrogenase (YdiB), chorismate mutase/prephenate dehydratase (PheA) and tyrosine aminotransferase (TyrB), or mutants thereof.

In some embodiments, AroG comprises an amino acid sequence set forth in SEQ ID NO: 17, variants, mutants, or fragments thereof; AroK comprises an amino acid sequence set forth in SEQ ID NO: 18, variants, mutants, or fragments thereof; YdiB comprises an amino acid sequence set forth in SEQ ID NO: 19, variants, mutants, or fragments thereof; PheA comprises an amino acid sequence set forth in SEQ ID NO: 20, variants, mutants, or fragments thereof and TyrB comprises an amino acid sequence set forth in SEQ ID NO: 21 , variants, mutants, or fragments thereof.

In some embodiments, AroG is encoded by a nucleic acid comprising SEQ ID NO: 22; AroK is encoded by a nucleic acid comprising SEQ ID NO: 23; YdiB is encoded by a nucleic acid comprising SEQ ID NO: 24; PheA is encoded by a nucleic acid comprising SEQ ID NO: 25 and TyrB is encoded by a nucleic acid comprising SEQ ID NO: 26.

According to an embodiment of the invention, glucose can be catalyzed to 2-PE in the one-pot reaction system. In some embodiments, the genetically engineered cells produce 2-PE from glucose by a reaction catalyzed by overexpression of DAHP synthase (AroG), shikimate kinase (AroK), shikimate dehydrogenase (YdiB), chorismate mutase/prephenate dehydratase (PheA) and tyrosine aminotransferase (TyrB), phenylalanine ammonia lyase (AtPAL2), phenylacrylic acid decarboxylase (AnPAD), styrene monooxygenase, styrene oxide isomerase, an aldehyde reductase and/ or an alcohol dehydrogenase, variants, mutants, or fragments thereof.

In some embodiments, AroG is replaced by a feedback inhibition resistant mutant AroG* encoded by a nucleic acid comprising SEQ ID NO: 27 and/or PheA is replaced by a feedback inhibition resistant mutant PheA* encoded by a nucleic acid comprising SEQ ID NO: 28.

In some embodiments, the method according to any aspect of the invention further comprises deletion or inactivation of err and/or prephenate dehydrogenase (tyrA) genes.

In some embodiments, the err gene and/or tyrA gene are/is deleted and replaced with a short 10-20 bp length double stranded DNA, an example of which is shown in SEQ ID NO: 52 and SEQ ID NO: 53, respectively. In some embodiments, the at least one overexpressed enzyme is located on one or more plasmids. An example of suitable plasmids are T7 expression plasmids.

In some embodiments of the method of the invention, the one-pot reaction system comprises use of an aqueous medium.

It was found that 2-PE production above a certain concentration becomes toxic to the genetically engineered cells and sequestration and/or removal of 2-PE from the fermentation medium is desired. This may be effected by use of a bi-phasic medium.

In some embodiments, the one-pot reaction system comprises the use of a bi-phasic medium.

In some embodiments, the bi-phasic medium is an aqueous: solid resin medium. In some embodiments, the bi-phasic medium is an aqueous: organic solvent medium. In some embodiments, the one-pot reaction system comprises the use of a tri-phasic medium comprising an aqueous: organic solvent: solid resin medium.

In some embodiments, the one-pot reaction system comprises the use of a tri-phasic medium comprising an aqueous: organic solvent: functionalized nanoparticles medium.

In one aspect of the invention there is provided one or more genetically engineered/ recombinant prokaryotic or eukaryotic cells selected from the group comprising bacterial cells, yeast cells, mammalian cells and insect cells, wherein said cells comprise at least one expression construct and/or heterologous nucleic acid molecule that encodes at least one catalytic enzyme required in the pathway from glucose to substituted or unsubstituted phenylacetaldehyde, 2-phenylethanol, phenylacetic acid or phenylethylamine. According to a second aspect of the invention there is provided an isolated strain of genetically engineered cells capable of increased bioproduction of substituted or unsubstituted phenylacetaldehyde, 2-phenylethanol, phenylacetic acid or phenylethylamine in a one-pot reaction system compared to wild type cells, wherein the cells overexpress a combination of enzymes selected from groups (i)-(iv), which comprise:

(i) styrene monooxygenase and styrene oxide isomerase for generating substituted or unsubstituted phenylacetaldehyde from styrene or substituted styrene;

(ii) styrene monooxygenase, styrene oxide isomerase and an aldehyde dehydrogenase for generating substituted or unsubstituted phenylacetic acid from styrene or substituted styrene;

(iii) styrene monooxygenase, styrene oxide isomerase, an aldehyde reductase and/ or an alcohol dehydrogenase for generating substituted or unsubstituted 2-phenylethanol from styrene or substituted styrene; and

(iv) styrene monooxygenase, styrene oxide isomerase and a transaminase for generating substituted or unsubstituted phenylethylamine from styrene or substituted styrene.

In some embodiments, the styrene monooxygenase is from Pseudomonas sp. VLB120 or its mutants, styrene oxide isomerase is from Pseudomonas sp. VLB120 or its mutants, the aldehyde dehydrogenase is from Escherichia coli or its mutants, the alcohol dehydrogenase is from Saccharomyces cerevisiae and the transaminase is ω-transaminase from Chromobacterium violaceum or its mutants.

In some embodiments, the styrene monooxygenase is encoded by a nucleic acid sequence set forth in SEQ ID NOs: 7 and 8; styrene oxide isomerase is encoded by a nucleic acid sequence set forth in SEQ ID NO: 9; the aldehyde dehydrogenase is encoded by a nucleic acid sequence set forth in SEQ ID NO: 10; the alcohol dehydrogenase is encoded by a nucleic acid sequence set forth in SEQ ID NO: 11 and the transaminase is ω- transaminase encoded by a nucleic acid sequence set forth in SEQ ID NO: 12.

It may be advantageous to provide the styrene or substituted styrene from conversion of L-phenylalanine or substituted L-phenylalanine in the same one-pot reaction system.

Accordingly, in some embodiments, the same or different genetically engineered cells produce styrene or substituted styrene from L-phenylalanine or substituted L- phenylalanine by a deamination reaction catalyzed by overexpression of an ammonia lyase and a decarboxylation reaction catalyzed by overexpression of a decarboxylase.

In preferred embodiments, the ammonia lyase is phenylalanine ammonia lyase and the decarboxylase is phenylacrylic acid decarboxylase. In some embodiments, the phenylalanine ammonia lyase is AtPAL2 from Arabidopsis thaliana, encoded by a nucleic acid sequence set forth in SEQ ID NO: 15 and wherein the phenylacrylic acid decarboxylase is AnPAD from Aspergillus niger, encoded by a nucleic acid sequence set forth in SEQ ID NO: 16.

It may be advantageous to provide the L-phenylalanine for production of styrene by catalysis of glucose in the same one-pot reaction system.

In some embodiments, the same or different genetically engineered cells produce L- phenylalanine from glucose by a reaction catalyzed by overexpression of at least one enzyme selected from a group comprising DAHP synthase (AroG), shikimate kinase (AroK), shikimate dehydrogenase (YdiB), chorismate mutase/prephenate dehydratase (PheA) and tyrosine aminotransferase (TyrB), or mutants thereof.

In some embodiments, AroG is encoded by a nucleic acid comprising SEQ ID NO: 22; AroK is encoded by a nucleic acid comprising SEQ ID NO: 23; YdiB is encoded by a nucleic acid comprising SEQ ID NO: 24; PheA is encoded by a nucleic acid comprising SEQ ID NO: 25 and TyrB is encoded by a nucleic acid comprising SEQ ID NO: 26.

According to an embodiment of the invention, glucose can be catalyzed to 2-PE in the one-pot reaction system.

In some embodiments, the genetically engineered cells produce 2-PE from glucose by a reaction catalyzed by overexpression of DAHP synthase (AroG), shikimate kinase (AroK), shikimate dehydrogenase (YdiB), chorismate mutase/prephenate dehydratase (PheA) and tyrosine aminotransferase (TyrB), phenylalanine ammonia lyase (AtPAL2), phenylacrylic acid decarboxylase (AnPAD), styrene monooxygenase, styrene oxide isomerase, an aldehyde reductase and/ or an alcohol dehydrogenase, variants, mutants, or fragments thereof.

In some embodiments, AroG is replaced by a feedback inhibition resistant mutant

AroG* encoded by a nucleic acid comprising SEQ ID NO: 27 and/or PheA is replaced by a feedback inhibition resistant mutant PheA* encoded by a nucleic acid comprising SEQ ID NO: 28.

In further embodiments, the isolated strain according to any aspect of the invention further comprises a deletion or otherwise inactivation of err and/or prephenate dehydrogenase (tyrA) genes. In some embodiments, the err gene and/or tyrA gene are/is deleted and replaced with a short 10-20 bp length double stranded DNA, an example of which is shown in SEQ ID NO: 52 and SEQ ID NO: 53, respectively.

In some embodiments, the isolated strain of genetically engineered cells of the invention are wild type strains containing the necessary enzymes. Preferably said cells are genetically engineered bacterial cells. More preferably said cells are Escherichia coli. In some embodiments, the isolated strain of genetically engineered cells are recombinant E. coli strains co-expressing multiple enzymes.

It will be appreciated that the method outlined above works by the combination of particular enzymes into a single reaction system.

According to an aspect of the present invention, there is provided an isolated nucleic acid molecule encoding at least one catalytic enzyme, according to any aspect of the present invention. More particularly, in some embodiments the present invention provides an isolated nucleic acid molecule encoding at least one heterologous catalytic enzyme selected from groups (i)-(iv), which comprise:

(i) a nucleic acid encoding styrene monooxygenase and styrene oxide isomerase for generating substituted or unsubstituted phenylacetaldehyde from styrene or substituted styrene;

(ii) a nucleic acid encoding styrene monooxygenase, styrene oxide isomerase and an aldehyde dehydrogenase for generating substituted or unsubstituted phenylacetic acid from styrene or substituted styrene;

(iii) a nucleic acid encoding styrene monooxygenase, styrene oxide isomerase, an aldehyde reductase and/ or an alcohol dehydrogenase for generating substituted or unsubstituted 2-phenylethanol from styrene or substituted styrene; and

(v) a nucleic acid encoding styrene monooxygenase, styrene oxide isomerase and a transaminase for generating substituted or unsubstituted phenylethylamine from styrene or substituted styrene.

In some embodiments the isolated nucleic acid molecule encodes at least one heterologous catalytic enzyme selected from phenylalanine ammonia lyase and phenylacrylic acid decarboxylase for generating styrene or substituted styrene from L- phenylalanine or substituted L-phenylalanine.

In some embodiments the isolated nucleic acid molecule encodes at least one heterologous catalytic enzyme selected from a group comprising DAHP synthase (AroG), shikimate kinase (AroK), shikimate dehydrogenase (YdiB), chorismate mutase/prephenate dehydratase (PheA) and tyrosine aminotransferase (TyrB), or mutants thereof for generating L-phenylalanine from glucose. In some embodiments AroG is replaced by a feedback inhibition resistant mutant AroG* encoded by a nucleic acid comprising SEQ ID NO: 27 and/or PheA is replaced by a feedback inhibition resistant mutant PheA* encoded by a nucleic acid comprising SEQ ID NO: 28.

It will be appreciated that the isolated nucleic acid of this aspect of the invention may encode a plurality of catalytic enzymes of which at least one is heterologous. For example, the plurality of catalytic enzymes is arranged as at least one module selected from the groups of modules (i)-(viii), which comprise: i) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms a styrene or substituted styrene to substituted or unsubstituted phenylacetaldehyde;

ii) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms a styrene or substituted styrene to substituted or unsubstituted phenylacetic acid;

iii) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms a styrene or substituted styrene to substituted or unsubstituted 2- phenylethanol;

iv) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms a styrene or substituted styrene to substituted or unsubstituted phenylethylamine;

v) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms a L-phenylalanine or substituted L-phenylalanine to a styrene or substituted styrene;

vi) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms a L-phenylalanine or substituted L-phenylalanine to a substituted or unsubstituted 2-phenylethanol;

vii) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms glucose to L-phenylalanine or substituted L-phenylalanine;

viii) a module comprising heterologous nucleic acid sequences that, when expressed, enzymatically transforms glucose to a substituted or unsubstituted 2-phenylethanol.

In particular, the isolated nucleic acid molecule may encode:

i) at least one of SEQ ID NOs: 1 , 2, 3 and 4, variants, mutants, or fragments thereof to transform a to a styrene or substituted styrene to substituted or unsubstituted phenylacetic acid; and/or

ii) at least one of SEQ ID NOs: 1 , 2 and 3, variants, mutants, or fragments thereof to transform a styrene or substituted styrene to substituted or unsubstituted phenylacetaldehyde; and/or

iii) at least one of SEQ ID NOs: 1 , 2, 3 and 5, variants, mutants, or fragments thereof to transform a styrene or substituted styrene to substituted or unsubstituted 2-phenylethanol; and/or

iv) at least one of SEQ ID NOs: 1 , 2, 3 and 6, variants, mutants, or fragments thereof to transform a styrene or substituted styrene to substituted or unsubstituted phenylethylamine and/or v) at least one of SEQ ID NOs: 13 and 14, variants, mutants, or fragments thereof to transform a L-phenylalanine or substituted L-phenylalanine to a styrene or substituted styrene; and/or

vi) at least one of SEQ ID NOs: 1 , 2, 3, 5, 13 and 14, variants, mutants, or fragments thereof to transform a L-phenylalanine or substituted L-phenylalanine to a substituted or unsubstituted 2-phenylethanol; or

vii) at least one of SEQ ID NOs: 17, 18, 19, 20, 21 , 50 and 51 , variants, mutants, or fragments thereof to transform glucose to L-phenylalanine; or

viii) at least one of SEQ ID NOs: 1 , 2, 3, 5, 13, 14, 17, 18, 19, 20, 21 , 50 and 51 , variants, mutants, or fragments thereof to transform glucose to a substituted or unsubstituted 2-phenylethanol.

According to another aspect of the invention there is provided a kit comprising at least one genetically engineered cell, expression construct or isolated nucleic acid according to any aspect of the invention.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows an overall novel artificial pathway to produce 2-phenylethanol (2-PE), phenylacetaldehyde (PA), phenylacetic acid (PAA), and phenylethylamine (PEA) from glucose.

Figure 2 shows a cascade transformation of L-Phe to styrene and genetic construction of midstream module 1 to co-express AtPAL and AnPAD. Figure 3 shows a cascade transformation of styrene to phenylacetaldehyde (PA) and genetic construction of downstream module 2-1 to co-express SMO and SOI.

Figure 4 shows a cascade transformation of styrene to 2-phenylethanol (2-PE) and genetic construction of downstream module 2-2 to co-express SMO, SOI, and ADH.

Figure 5 shows a cascade transformation of styrene to phenylethylamine (PEA) and genetic construction of downstream module 2-3 to co-express SMO, SOI, ω-ΤΑ, and AlaDH.

Figure 6 shows a cascade transformation of styrene to 2-phenylethanol (2-PE) and genetic construction of downstream module 2-4 to co-express SMO, SOI, and ALDH. Figures 7a and 7b show a SDS-PAGE analysis of whole-cell protein of (Fig. 7a) E. coli (StyABC-PAR) and (b) E. coli (StyABC-CvTA-AlaDH).

Figure 8 shows a time course of biocatalytic hydration of 60 mM Sty to 2-PE with E. coli (StyABC-PAR) cells (10 g cdw L ^"1 ) in KP buffer (200 mM, pH 8, 2 % glucose) and n- hexadecane (1 : 1).

Figure 9 shows a time course of biocatalytic hydroamination of 80 mM Sty to PEA with E. coli (StyABC-CvTA-AlaDH) cells (10 g cdw/L) in NaP buffer (200 mM, pH 8, 2 % glucose, 200 mM NH ₃ /NH ₄ CI) and n-hexadecane (1 : 1).

Figures 10a and 10b show (Fig. 10a) Genetic construct of plasmid pRSF-StyABC-EcALDH and (Fig. 10b) SDS-PAGE analysis of whole-cell protein of E. coli (StyABC-EcALDH) co- expressing SMO (StyA & StyB), SOI (StyC), and EcALDH.

Figures 11a to 11e show optimization of reaction conditions for biotransformation of Sty to PAA with E. coli (StyABC-EcALDH) cells (10 g cdw/L). (Fig. 11 a) biotransformation using different organic phase; (Fig. 1 1 b) biotransformation using different aqueous buffer; (Fig. 1 1c) biotransformation using different glucose concentration; (Fig. 11 d) biotransformation under different temperature; (Fig. 11 e) biotransformation using different ratio of organic phase: aqueous buffer.

Figure 12 shows a time course of biocatalytic hydroamination of 1300 mM Sty to PAA with E. coli (StyABC-EcALDH) cells (15 g cdw/L) in KP buffer (400 mM, pH 8, 0.5% glucose) and ethyl oleate (1 : 1).

Figures 13a and 13b show the (Fig. 13a) Genetic construct of plasmid pRSF-StyABC- ADH9v1 and (Fig. 13b) SDS-PAGE analysis of cell protein of E. coli (StyABC-EcALDH) (lane 1) and E. coli (StyABC-ADH9v1) (lane 2).

Figure 14 shows asymmetric cascade oxidation of a-methylstyrene (20 mM) to (S)-2- Phenylpropanoic acid using different amount of E. coli (StyABC-ADH9v1) resting cells in a two-liquid phase system of KP buffer (200 mM, pH 8.0) containing various amount of glucose and n-hexadecane (5: 1) at 30 °C. Figure 15 shows the screening of 12 constructed E.coli recombinant strains (10 g cdw/L for 2-PE production from .-phenylalanine (50 mM) in 2 h in biphasic system (n-hexadecane as organic solvent). Figure 16 shows the adsorption of 2-PE and .-phenylalanine by different resins. All resins were used in 10% (w/v). Initial concentration of 2-PE and /.-phenylalanine were each set in 50 mM inside the KP buffer (100 mM, pH 8.0) in 30°C for 2 h. Values are the mean of triplicate and the error bars indicate the standard deviation value. Figures 17a and 17b show (Fig. 17a) 2-PE product inhibition test with E.coli cells (R-PAL- PAD_E-SMO-SOI-PAR) treated with 2-PE in different concentrations for 3 hours before performing the reaction and (Fig. 17b) shows the Apparent Kinetics of the whole-cell E.coli (R-PAL-PAD_E-SMO-SOI-PAR). The reaction for Fig. 17b was done at 30°C and 250 rpm in 2 ml KP buffer (100 mM, pH 8.0) containing 0.5% (w/v) glucose, 5 g cdw/L of cell suspension, 10 mM /.-phenylalanine, and («) 0 or ( A) 3 mM of 2-PE in the aqueous phase, with n- hexadecane in the organic phase.

Figures 18a and 18b show (Fig. 18a) partition coefficient of 2-PE in aqueous-organic biphasic system. Partition coefficient measured at a phase ratio of 1 : 1. KP buffer (100 mM, pH 8.0) was utilized as the aqueous buffer. All data were obtained from the mean values of triplicate with standard deviations through HPLC analysis. K value for /.-phenylalanine was observed as ~0 in all the respective organic solvents mentioned and (Fig. 18b) screening of 7 different organic solvent for the one-pot production of 2-PE from /.-phenylalanine with the KP buffer (100 mM, pH 8.0) containing 0.5 % glucose and 50 mM /.-phenylalanine.

Figures 19a to 19e show (Fig. 19a) synthesis of OA-MNP-PS, (Fig. 19b and Fig. 19c) TEM and DLS, respectively, of OA-MNP and (Fig. 19d and Fig.19e) TEM and DLS, respectively, of OA-MNP-PS. Figure 20 shows the adsorption of 2-PE and /.-phenylalanine by different resins. All resins were used in 10% (w/v). Initial concentration of 2-PE and /.-phenylalanine were each set in 50 mM inside the KP buffer (100 mM, pH 8.0) in 30°C for 2 h. Values are the mean of triplicate and the error bars indicate the standard deviation value. Figures 21 a to 21 c show (Fig. 21 a) biotransformation of /.-phenylalanine (50 mM) to 2-PE with tri-phasic in-situ product removal via extraction and MNPs adsorption in 1 pot, (Fig. 21 b) biotransformation of /.-phenylalanine (50 mM) to 2-PE with tri-phasic in-situ product removal via extraction and XAD4 resins adsorption in 1 pot. Two ml aqueous phase containing 10 g cdw/L of resuspended cells, 50 mM of .-phenylalanine, and 0.5% glucose inside the KP Buffer (100 mM, pH 8) were reacted with 2 ml organic phase. Different type of resins (0.36 g) or MNPs (5 mg/ml) was added for the biotransformation, followed by the reaction for 24 h and subsequent separation of adsorbent, organic phase, and aqueous phase. (Fig. 21c) Repeated batch of 2-PE biotransformation from 100 mM .-phenylalanine with tri-phasic in- situ product removal via extraction and XAD4 resins adsorption in one pot. Cells (10 g/l) were resuspended in fresh buffer containing 0.5% glucose and 100 mM initial substrate concentration, then mixed with a new organic solvent and adsorbent to carry on the biotransformation.

Figure 22 shows a schematic representation of the approach for 2-phenylethanol production from glucose in E. coli. The genes highlighted in bold were overexpressed and the cross- marked pathways were deleted by deleting err or tyrA. Abbreviations: PTS, phosphotransferase system; EMP, Embden-Meyerhof-Parnas pathway; PPP, pentose phosphate pathway; G6P, glucose-6-phosphate; PEP, phosphoenolpyruvate; E4P, erythrose-4-phosphate; DAHP, 3-deoxy-d-arabino-heptulosonate-7-phosphate; DHQ, 5- dehydro-quinate; DHS, 5-dehydro-shikimate; SHIK, shikimate; S3P, shikimate-5-phosphate; EPSP, 3-enolpyruvylshikimate-5-phosphate; CHO, chorismate; PPA, prephenate; PPY, phenylpyruvate; L-Phe, /.-phenylalanine; C-acid, trans-cinnamic acid; Sty, styrene; StyO, styrene oxide; PA, phenylacetaldehyde.

Figures 23a and 23b show /.-phenylalanine production from glucose by E. coli mutants overexpressing /.-phenylalanine production pathway. The cell density and /.-phenylalanine production of (Fig. 23a) growing cells of all mutants at 24 h and (Fig. 23b) high cell density growing cells of T7-Phe and T7AA-Phe at 12 h is shown.

Figure 24 shows a time course profile of 2-PE production from glucose by T7AA-Phe-Sty. Figures 25a and 25b show 2-PE production from glucose in biphasic media by T7AA-Phe- Sty. (Fig. 25a) Cell growth profile with different ratio of M9 media and oleic acid in biphasic media and (Fig. 25b) the metabolites of the cultures at 24 h.

Figure 26 shows results from a bioreactor scale production of 2-PE from glucose in biphasic media by T7AA-Phe-Sty. 2-PE concentration in the organic phase was measured only at 40 h. DESCRIPTION

Bibliographic references mentioned in the present specification are for convenience listed in the form of a list of references and added at the end of the examples. The whole content of such bibliographic references is herein incorporated by reference.

Definitions

Certain terms employed in the specification, examples and appended claims are collected here for convenience.

The terms "amino acid" or "amino acid sequence," as used herein, refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence" is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.

As used herein, the term "comprising" or "including" is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. However, in context with the present disclosure, the term "comprising" or "including" also includes "consisting of". The variations of the word "comprising", such as "comprise" and "comprises", and "including", such as "include" and "includes", have correspondingly varied meanings.

The term "isolated" is herein defined as a biological component (such as a nucleic acid, peptide or protein) that has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins which have been isolated thus include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

The phrases "nucleic acid" or "nucleic acid sequence," as used herein, refer to an oligonucleotide, nucleotide, polynucleotide, or any fragment thereof, to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA- like or RNA-like material. In the context of the invention, "fragments" refers to those nucleic acid sequences which are greater than about 60 nucleotides in length, and most preferably are at least about 100 nucleotides, at least about 1000 nucleotides, or at least about 10,000 nucleotides in length which are not full-length native sequence but retain catalytic enzyme activity.

The term "oligonucleotide," as used herein, refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PCR amplification or in a hybridization assay or microarray. As used herein, the term "oligonucleotide" is substantially equivalent to the terms "amplimers," "primers," "oligomers," and "probes," as these terms are commonly defined in the art.

The terms 'variant' and 'mutant' are used interchangeably herein. The at least one nucleic acids encoding at least one catalytic enzyme may encode a variant or mutant of the exemplified catalytic enzyme which retains activity. A "variant" of a catalytic enzyme, as used herein, refers to an amino acid sequence that is altered by one or more amino acids. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "nonconservative" changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing catalytic activity may be found using computer programs well known in the art, for example, DNASTAR software. In some embodiments, variant enzymes are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, preferably at least 90%, homologous or identical at the amino acid level to an exemplary amino acid sequence described herein (e.g., alcohol dehydrogenase, ω-transaminase) or a functional fragment thereof— e.g., over a length of about: 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%, preferably at least 90%, of the length of the mature reference sequence, yet retain catalytic activity. Preferably said variant enzymes have at least 90% identity at the amino acid level and retain catalytic activity. An exemplary alcohol dehydrogenase is represented by SEQ ID NO: 5, and an exemplary ω-transaminase is represented by SEQ ID NO: 6.

The terms 'phenylacetaldehyde reductase' (PAR) and 'alcohol dehydrogenase' (ADH), as referred to herein, are used interchangeably.

A vector can include one or more catalytic enzyme nucleic acid(s) in a form suitable for expression of the nucleic acid(s) in a host cell. Preferably the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence(s) to be expressed. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences such as the T7 IPTG-inducible promoters disclosed in the Examples herein. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein (e.g., catalytic enzyme proteins, fusion proteins, and the like).

The recombinant expression vectors of the invention can be designed for expression of catalytic enzyme proteins in prokaryotic or eukaryotic cells. For example, polypeptides of the invention can be expressed in bacteria (e.g., E. coli), insect cells (e.g., using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. Alternatively, the recombinant expression vector(s) can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.

To maximize recombinant protein expression in E. coli is to express the protein in a host bacterium with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res. 20:21 11-2118). Such alteration of nucleic acid sequences of the invention can be carried out by known DNA synthesis techniques and is described in the Examples.

The catalytic enzyme expression vector can be a yeast expression vector, a vector for expression in insect cells, e.g., a baculovirus expression vector, a vector for expression in bacterial cells, e.g. a plasmid vector, or a vector suitable for expression in mammalian cells. When used in mammalian cells, the expression vector's control functions can be provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.

The methods described hereinbefore make use of enzymes to catalyse a sequence of reactions. While these reactions may be performed individually or, more particularly, two or more of them in combination, it is particularly preferred that all of the reactions are combined into a cascade reaction sequence that provides the product from the initial starting material in one pot, thereby eliminating the need for isolation of the intermediates and, potentially, increasing the overall yield of the reaction sequence. These cascade reactions may involve the use of one or more reactive components selected from the group consisting of cells, immobilized cells, cell extracts, isolated enzymes and immobilized enzymes in said reaction vessel.

In this invention, we proposed a novel biocatalytic route (pathway) to produce "natural" 2-PE, PA, PAA, and PEA from the easy available biobased .-phenylalanine and glucose (Fig. 1). The route (pathway) is novel and clearly different from natural pathway. More importantly, the enzymes in the new pathway are known to be very efficient. For example, we had demonstrated the deamination-decarboxylation of .-phenylalanine by lyase and decarboxylase to produce styrene (the key intermediate) with up to 15 g/L. The further conversion of styrene into 2-PE, PA, PAA, and PEA are similar to natural styrene degradation pathway, which is known to be high efficiency. Recombinant E. coli strains were engineered to express the enzymes in high amount and optimal ratio, thus being efficient whole-cell catalysts for bioproduction of 2-PE, PA, PAA, and PEA. In addition, further process engineering will reduce the product toxicity and increase the final product concentration. In summary, our method will be a cost-effective way to produce valuable natural 2-PE, PA, PAA, and PEA from biobased resources.

The whole route was divided into three parts: 1) upstream shikimate pathway to produce L-Phe from glucose (Fig. 1); 2) midstream deamination-decarboxylation module to convert L-Phe to styrene (Fig. 2); 3) downstream modules to functionalize styrene to 2-PE, PA, PAA, and PEA, respectively (Figs 3-6). The upstream shikimate pathway to produce L- Phe is well-known and has been engineered to produce L-Phe in high concentration (> 50 g/L). We recently demonstrated the midstream module of converting L-Phe to styrene (Sty) by co-expression of phenylalanine ammonia lyase (AtPAL2) from Arabidopsis thaliana and phenylacrylic acid decarboxylase (AnPAD) from Aspergillus niger [Zhou, Y., Wu, S., Li, Z. Angew. Chem. Int. Ed. 55: 1 1647-11650 (2016) ]. In the downstream modules, Sty was converted to styrene oxide (SO) by styrene monooxygenase (SMO) with consumption of oxygen. Styrene oxide isomerase (SOI) was employed for conversion of SO to give aldehydes PA. In the last step, PA could be oxidized to PAA by an aldehyde dehydrogenase (ALDH), reduced to 2-PE by aldehyde reductase (e.g. PAR) /alcohol dehydrogenase (ADH), or aminated to PEA by ω-transaminase (ω-ΤΑ). Preferably, these multiple reactions are performed simultaneously or sequentially in one reaction vessel, allowing for green, efficient, and economical production of 2-PE, PA, PAA, and PEA, directly from biobased L-Phe or glucose. Thus one-pot cascade reactions could avoid the expensive and energy-consuming isolation and purification of intermediates, minimize wastes generation, and overcome the possible thermodynamic hurdles in traditional multi-step synthesis. Preferably, multiple enzymes are co-expressed inside one recombinant microbe strain, and the whole cells of the strain are directly applied as catalysts for directly fermentative production of 2-PE, PA, PAA, and PEA. Alternatively, these enzymes or modules could be separately expressed in several cells, purified individually, or immobilized and the biocatalyst (enzymes, cells, immobilized enzymes, and immobilized cells) can be mixed together in one pot to carry out the reaction. For example, a recombinant strain for fermentative production of L-Phe, and other strains for directly convert L-Phe to 2-PE, PA, PAA, and PEA respectively through the designed pathway (midstream and downstream modules).

In an embodiment of the invention, all the enzymes responsible for the reactions are co-expressed in one recombinant E. coli strain. In this case, all the chemical reactions are taken place inside a single cell. To construct the recombinant biocatalyst, the enzymes are cloned as several artificial operons or separately on one plasmid or several compatible plasmids. After transforming the plasmids into the E. coli strain, the multiple enzymes are co- expressed and the whole recombinant cells are served as a biocatalyst for the cascade reactions. The expression level of multiple enzymes could be adjusted and optimized for efficient cascade transformation without significant accumulation of intermediates. There are many methods to achieve tuning the expression level of multiple enzymes: using different plasmids, inducer, promoters or ribosome binding sites with different strength.

In a preferred embodiment, the cascade transformations are better performed in aqueous phase. For low concentration biotransformation, aqueous one phase system fulfills the requirement and can achieve the final product easily. However, the intermediate Sty and SO are generally hydrophobic (limited solubility in aqueous phase) and toxic for the cell and enzyme (may have substrate inhibition). Thus, an organic: aqueous two-phase reaction system is a better choice for high-concentration biotransformation. The Sty and SO are better soluble in organic phase, while the diols, amino alcohols, amino acids, cells, and enzymes are mostly in the aqueous phase. By applying the two-phase reaction system, the problems of low solubility and inhibition of Sty and SO are solved.

Other forms of biocatalyst could also be applied to synthesize 2-PE, PA, PAA, and

PEA. They include isolated enzyme, enzymes immobilized on nano or micro size support (such as magnetic nano particles) to increase their stability and re-usability, wild type microbial cells, and recombinant cells immobilized on some carriers. By utilizing isolated enzymes, immobilized enzymes or immobilized cells, the cascade biocatalysis can be performed to produce 2-PE, PA, PAA, and PEA from biobased L-Phe or glucose. A mixture of different forms of biocatalyst is also a suitable system to carry out the cascade biocatalysis.

EXAMPLES

Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2012).

Strain, biochemicals, and culture medium

Escherichia coli T7 expression cells were purchased from New England Biolabs.

Primers (DNA oligos) were synthesized from IDT. Phusion DNA polymerase, fast digest restriction enzymes, and T4 DNA ligase were bought from Thermo Scientific. LB medium, tryptone, yeast extract, and agar were obtained from Biomed Diagnostics. Chloramphenicol, streptomycin, ampicillin, kanamycin, and glucose were purchased from Sigma-Aldrich. IPTG (Isopropyl β-D-l-thiogalactopyranoside) was obtained from Gold Biotechnology.

The culture medium used in this study is standard M9 medium supplemented with glucose (20 g/L), yeast extract (6 g/L). The M9 medium contains 6 g/L Na ₂ HP0 ₄ , 3.0 g/L KH ₂ P0 ₄ , 0.5 g/L NaCI, 1.0 g/L NH ₄ CI, 1 mM MgS0 ₄ , 0.1 mM CaCI ₂ , and 1 ml_/L I ^"1 trace metal solution. The trace metal solution contains 8.3 g/L FeCI ₃ ^« 6H ₂ 0, 0.84 g/L ZnCI ₂ , 0.13 g/L CuCI ₂ ^« 2H ₂ 0, 0.1 g/L CoCI ₂ ^« 2H ₂ 0, 0.1 g/L H ₃ B0 ₃ , 0.016 g/L MnCI ₂ ^« 4H ₂ 0, and 0.1 g/L Na ₂ Mo0 ₄ ^« 2H ₂ 0 in 1 M HCI.

SDS-PAGE Analysis and Quantification

Freshly prepared E. coli whole cells were centrifuged and resuspended in Dl water to a density of 8 g cdw/L (OD ₆ oo = 20). The cell suspension (60 μί) was mixed with 20 μΙ of SDS sample buffer (4x Laemmli Sample Buffer with DTT, Bio-Rad) and heated to 98°C for 15 min. 60 μΙ of 0.2 g/L, 0.1 g/L, and 0.05 g/L of BSA standards were also mixed with 20 of SDS sample buffer and heated to 98°C for 15 min. Then the mixture was centrifuged (13000 g) for 10 min. 10 μί of the supernatant was used to load into the sample well of 12 % SDS-PAGE gel (hand cast). The electrophoresis was run in a setup of Mini-Protean tetra cell at 100 V for 15 min and 150 V for 75 min. After running, the PAGE gel was washed with water and then stained with Bio-Safe Coomassie Stain (Bio-Rad) according to the instruction. The figure was obtained with GS-900 calibrated densitometer (Bio-Rad), and quantification analysis was done with the volume tools in the Image Lab software (Bio-Rad). General procedures for Culturing E. coli Cells for Biotransformation

E. coli strain was initially inoculated in LB medium (1 mL) containing appropriate antibiotics (50 mg/L kanamycin, 50 mg/L chloramphenicol, 50 mg/L streptomycin, 50 mg/L ampicillin) for 8-10 h (280 rpm) at 37°C and then were transferred to a 250 mL tribaffled flask with 50 mL of M9 medium supplemented with glucose (20 g L ^"1 ), yeast extract (6 g L ^"1 ), and appropriate antibiotics. The cells continued to grow at 37°C and 250 rpm for about 2 h to reach an OD ₆₀₀ of 0.6, and then IPTG (0.5 mM final concentration) was added to induce the enzyme expression. The cells were further grown at 22°C overnight (12-13 h), and harvested by centrifugation (4000 g, 10 min). Chemical and Materials

The following chemicals were purchased from Sigma-Aldrich:

Sty-m-OMe-Sty, a-Me-Sty, p-Me-a-Me-Sty, Sty oxide, PA, PAA- p-OMe-PAA, rac-2- Phenylpropanoic acid-a,4-Dimethylphenylacetic acid, (S) -2-Phenylpropanoic acid, acetic acid, ethyl oleate, n-hexadecane, kanamycin, glucose, NaCI, Na ₂ S0 ₄ , Na ₂ HP0 ₄ , NH ₄ CI, KH ₂ P0 ₄ , K ₂ HP0 ₄ , TFA, phenethylamine, and benzyl alcohol. p-OMe-Sty was from Alfa Asear. Oleic acid, p-F-a-Me-Sty and p-CI-a-Me-Sty were from TCI chemical. Acetonitrile, ethyl acetate, 2-propanol and n-hexane were purchased from Tedia. n- Heptane, silica gel 60 and TLC plates were purchased from Merck. LB medium, yeast extract, and agar were purchased from Biomed Diagnostics. DNA polymerase, ligase, and restriction enzymes were purchased from Thermo Fisher.

Analytical Methods

Cell growth was monitored by spectrophotometry (NanoDrop™, Thermo Fisher Scientific Inc., Massachusetts, USA) measurement of the optical density (OD ₆ oo) at 600 nm. Metabolites such as L-Phe and 2-PE were measured by high-performance liquid chromatography (Prominence, Shimadzu Corporation) equipped with photodiode array (DAD) detectors. The media samples were centrifuged and filtered, and eluted through Agilent Poroshell 120 SB-C18 column (150 χ 4.6 mm, 2.7 μηι) under reversed phase condition with 30 % acetonitrile and 70 % ultrapure water containing 0.1 % TFA. Flowrate: 0.4 ml/min, temperature: 25 °C. Detector: photodiode array detector. Wavelength: 210 nm. 2-PE extracted in the oleic acid was eluted through Agilent ZORBAX RX-SIL column (150 χ 4.6 mm, 5 μηι) with 2 % acetonitrile and hexane. Glucose levels during fed-batch fermentation was monitored by HPLC equipped with refractive index detector. The samples were eluted using Aminex-HPX87P column (Biorad, USA) with ultrapure water as mobile phase.

2-PE (organic phase) and styrene were analyzed using Agilent 7890A Gas Chromatography (GC). Column: Agilent HP-5 (30 m x 0.32 mm x 0.25 mm). Temperature programme: initial temperature at 70°C, increase 25 °C/min until it reached 200°C; subsequently increase to 250°C at 50°C/min, then hold for 1 minute; Lastly, increase to 270°C at 20°C/min.

Size and morphology of synthesized-MNPs were determined using JEOL JEM 2010 Transmission Electron Microscope (TEM - JEOL, USA). Hydrodynamic diameter and size distribution were characterized using zetasizer (Molvern).

HPLC analysis of Sty-p-OMe-Sty was performed on a Shimadzu prominence HPLC system with a photodiode array detector and a reverse-phase Agilent Poroshell 120 SB-C18 column (150 x 4.6 mm, 2.7 mm) at 25°C. Mobile phase: 50% water with 0.1 % TFA: 50 % acetonitrile. Flow rate: 0.5 mLmin ^"1 . The concentration was determined by comparison of peak areas at 210 nm to those on the calibration curve of the authentic compound. Retention times: phenethylamine (internal standard) 3.2 min, PAA 4.8 min, o-F-PAA 5.0 min, m-F-PAA 5.1 min, p-F-PAA 5.0 min, m-CI-PAA 6.0 min, p-CI-PAA 6.1 min, m- Br- PAA 6.4 min, p-Br- PAA 6.5 min, m- Me- PAA 5.7 min, p-Me-PAA 5.7 min, m-OMe-PAA 4.8 min, p-OMe-PAA 4.7 min.

GC-FID analysis of Sty - p-OMe-Sty, Sty oxide and PA was performed on an Agilent 7890A gas chromatograph system with an FID detector. Column: Agilent HP-5 (30 m x 0.32 mm x 0.25 mm). Temperature program: start at 70°C, increase to 200°C at 25°C min ^"1 , increase to 250°C at 50°Cmin ^"1 , hold for 1 min, and then increase to 270°C at 20°C min ^"1 . The concentration was determined by comparison of peak areas to those on the calibration curve of the authentic compound. Retention times: benzyl alcohol (internal standard) 2.8 min, Sty 2.2 min, o-F-Sty 2.2 min, m-F-Sty 2.2 min, p-F-Sty 2.2 min, m-CI-Sty 3.1 min, p-CI-Sty 3.1 min, m-Br-Sty 3.5 min, p-Br-Sty 3.5 min, m-Me-Sty 2.6 min, p-Me-Sty 2.6 min, m-OMe- Sty 3.4 min, p-OMe-Sty 3.4 min.

Chiral HPLC analysis of a-Me-Sty - p-Me-a-Me-Sty (concentration) was performed on the same HPLC system with a reverse-phase Daicel Chiralpak AD-3R column (150 x 4.6 mm, 3 mm) at 15°C. The concentration was determined by comparison of peak areas at 210 nm to those on the calibration curve of authentic compound.

Method A: mobile phase consisting of 80 % water with 0.1 % TFA: 20 % acetonitrile was delivered at 1.0 mLmin ^"1 . Retention times: benzyl alcohol (internal standard) 5.8 min, (R)-2- Phenylpropanoic acid 13.9 min, (S)-2-Phenylpropanoic acid 14.6 min, (R)-p-F-a-Me-PAA 19.2 min, (S)-p-F-a-Me-PAA 19.9 min, (R)-p-Me-a-Me-PAA 32.3 min, (S)-p-Me-a-Me-PAA 33.4 min.

Method B: mobile phase consisting of 70% water with 0.1 % TFA: 30% acetonitrile was delivered at 1.0 mLmin ^"1 . Retention times: benzyl alcohol (internal standard) 3.8 min, (R)-p- Me-a-Me-PAA 12.2 min, (S)-p-Me-a-Me-PAA 13.0 min.

The ee values of (S)-2-Phenylpropanoic acid-(S) -p-Me-a-Me-PAA were measured with another chiral HPLC analysis method using a Daicel Chiralpak ADH column (250 x 4.6 mm, 5 mm) at 25°C. Mobile phase consisting of 90% n-hexane with 0.1 % TFA: 10% 2- propanol was delivered at 1.0 mLmin ^"1 . Retention times: (R)-2-Phenylpropanoic acid 5.7 min, (S)-2-Phenylpropanoic acid 6.3 min, (R)-p-F-a-Me-PAA 5.7 min, (S)-p-F-a-Me-PAA 6.3 min, (R)-p-CI-a-Me-PAA 6.0 min, (S)-p-CI-a-Me-PAA 6.6 min, (R)-p-Me-a-Me-PAA 5.7 min, (S)-p- Me-a-Me-PAA 6.4 min.

Conversion of Styrene and Substituted Styrene to 2-phenylethanols (2-PEs), phenylacetaldehydes (PAs), phenylacetic acids (PAAs), and phenylethylamines (PEAs)

A representative example was demonstration of converting (substituted) styrene to (substituted) 2-PE. Previously, we had engineered E. coli co-expressing SMO from styrene degradation Pseudomonas sp. VLB120 [Wu, S., Chen, Y., et al., ACS Catal. 4: 409-420 (2014)]. The gene of SOI was amplified and constructed together with SMO to give an artificial operon as module 2-1 on plasmid pRSFduet-1 (Fig. 3). In addition, the gene of phenylacetaldehyde reductase (PAR) from tomato [Tieman, D. M., Loucas, H. M., et al., Phytochemistry 68: 2660 (2007)] was engineered together with SMO-SOI to give an artificial operon as module 2-2 in this project (Fig. 4). The plasmid containing module 2-2 was transformed to give E. coli (StyABC-PAR) strain, which was used as the whole-cell biocatalyst for converting Sty to 2-PE. The expression of the enzymes was examined by a SDS-PAGE analysis, and all the enzymes were clearly visible (Fig. 7a). By using 10 g cdw/L of resting cells of E. coli (StyABC-PAR), 60 mM of Sty was quickly converted to 2-PE in the initial 3 h, and only a very small amount of SO (0.3 %) and PA (3 %) were accumulated at initial 1 h (Fig. 8). At end of reaction (8 h), the desired 2-PE was formed in 93 % analytical yield with little Sty (2 %) was remained. Importantly, another isomer of 2-PE, 1- phenyethanol, was not produced in the biotransformation. The E. coli (StyABC-PAR) was employed for converting substituted Sty to substituted 2-PE (Table 1). Many substituted 2- PEs were produced in very high conversion (≥90 %): fluoro substituted 2-PEs (entries 2-4), methyl substituted 2-PEs (entries 9, 10), and methoxy substituted 2-PEs (entries 11 , 12). 2- PEs with a chloro (entries 5, 6) or bromo (entries 7, 8) substituent were formed in good conversion of 89-62 %. Importantly, 1-phenyethanols were not produced in the biotransformation. This demonstrated the efficient conversion of Sty to 2-PE via the novel pathway.

To achieve conversion of (substituted) Sty to (substituted) PEA (Fig. 5), an artificial operon containing ω-ΤΑ and AlaDH was constructed. The genes of CvTA from Chromobacterium violaceum [Kaulmann, U., Smithies, K., et al., Enzyme Microb. Technol. 41 : 628-637 (2007)] and AlaDH from Bacillus subtilis were combined to give CvTA-AlaDH on plasmid pCDFduet-1. The SMO-SOI on plasmid pACYCduet-1 was co-transformed with the above plasmid into E. coli (RARE) strain [1 1] to give the recombiant strain E. coli (StyABC- CvTA-AlaDH) co-expressing the four enzymes, SMO, SOI, CvTA, and AlaDH as module 2-3. Transformation of Sty at various concentration was performed with resting cells of E. coli (StyABC-CvTA-AlaDH), and high conversion to PEA was achieved for 75 mM Sty. 80 mM Sty was quickly converted to PEA in the first 4 h, and some SO (up to 9 %) and PA (up to 2 %) was accumulated and converted (Fig. 9). At end of reaction (10 h), the amine PEA was produced in 93 % conversion. Notably, alcohol byproduct 2-PE was detected in trivial amount (0.6 %), indicating a very high chemo selectivity. In addition, another isomer of PEA, 1-phenyethylamine, was not observed, demonstrating an excellent regioselectivity. The E. coli (StyABC-CvTA-AlaDH) was employed for converting substituted Sty to substituted PEA (Table 2). Fluoro-substituted PEAs (entries 2-4), methyl-substituted PEAs (entries 9, 10) and methoxy- substituted PEAs (entries 11 , 12) were produced in very high conversion of 94-99 %. Chloro-substituted PEAs (entries 5, 6) and bromo-substituted PEAs (entries 7, 8) were formed in good to moderate conversion of 86-45 %. The chemo-selectivity of amine over alcohol was also very high (all >20 : 1), and the regioselectivity was excellent. The current system could be improved by further optimization of reaction conditions or using more efficient enzymes.

Another representative example was demonstration of converting (substituted) styrene to (substituted) PAA. The gene of phenylacetaldehyde dehydrogenase (EcALDH) from E. coli [Ferrandez, A., Prieto, M. A., et al., FEBS Lett. 406: 23 (1997)] was engineered together with SMO-SOI to give an artificial operon as module 2-4 (Fig. 6). The plasmid containing module 2-4 was transformed to give E. coli (StyABC-EcALDH) strain, which was used as the whole-cell biocatalyst for converting Sty to PAA. The expression of the enzymes was examined by a SDS-PAGE analysis, and all the enzymes were clearly visible (Fig. 10 (b) and Fig. 13 (b)). The best biotransformation condition was found by exploring different organic phase, buffer, glucose concentration, temperature, and ratio of the two phases (Fig. 11). Under the optimal condition, 15 g cdw/L of resting cells of E. coli (StyABC-EcALDH) successfully converted 130 mM of Sty to 122 mM of PAA (94% overall yield) in 6 h, and no intermediate was accumulated during the reaction (Fig. 12). The E. coli (StyABC-EcALDH) was employed for converting substituted Sty to substituted 2-PE (Table 3). Many substituted PAAs were produced in very high conversion (≥90 %): fluoro-substituted PAAs (entries 2-4), methyl-substituted PAAs (entries 9, 10), and methoxy-substituted PAAs (entries 11 , 12). PAAs with a chloro (entries 5, 6) or bromo (entries 7, 8) substituent were formed in good conversion of 87-78 %. This demonstrated the efficient conversion of Sty to PAA by the recombinant E. coli.

To produce (S)-PAA in very high ee, alcohol dehydrogenase ADH9v1 , as reported in [P. Konst, H. Merkens, et al., Angew. Chem. Int. Ed. 51 : 9914-9917 (2012)] with S- selectivity for the enantioselective oxidation of racemic PA was cloned. The gene of ADH9v1 was synthesized and constructed together with styC and pRSF-StyAB to form a recombinant plasmid pRSF-StyABC-ADH9v1 (Fig. 13 (a)). This plasmid was transformed into E. coli T7 express strain to give E. coli (StyABC-ADH9v1). The strain was grown and induced under the same cultivation procedure for E. coli (StyABC-EcALDH). SDS-PAGE analysis of the cell protein confirmed the expression of StyA, StyB, StyC, and ADH9v1 (lane 2 in Fig. 13 (b)).

Further description of exemplary embodiments are provided below.

Example 1. Genetic engineering of E.coli containing Module 2-1 and expressing SMO and SOI The styC gene coding SOI (SEQ ID NO: 9) from Pseudomonas sp. VLB120 was first synthesized and codon optimized for E. coli according the published sequence. Then it was amplified using primers StyC- Kpnl- RBS-F

(CGGGTACCTAAGGAGATATATAATGTTACACGCGTTTGAACGTA AAATG; SEQ ID NO:

29) and StyC-Hindlll-Xhol-R (ACTGCTCGAGAAGCTTACTCGGCTGCCGCG TGTGGAACGGCTTTACG; SEQ ID NO: 30) and Phusion DNA polymerase (available from

Thermo). The PCR products were double-digested with Kpnl and Xhol, and then ligated to same digested pRSF-SMO plasmid [Wu, S., Chen, Y., et al., ACS Caial. 4: 409-420 (2014)] with T4 DNA ligase. The ligation product was transformed (heat shock) into E. coli T7 Expression competent cells (available from New England Biolabs) to give pRSF-SMO-SOI. This module 2-1 was sub-cloned to other three vectors by the following procedures. Module 2-1 operon was amplified with primers StyA-BspHI-F (ACTGTC ATGAAAAAGCGTATCGGTATTGTTGG; SEQ ID NO: 31) and StyC-Hindlll-Xhol-R (ACTGCTCGAG AAGCTTACTCGGCTGCCGCGTGTGGAACGGCTTTACG; SEQ ID NO:

30) , digested with BspHI and Xhol, and then ligated to double digested pACYCduet, pCDFduet, and pETduet (available from Novagen). The transformation of these products gave pACYC-SMO-SOI, pCDF-SMO-SOI, and pET-SMO-SOI respectively. Example 2. Genetic engineering of E. coli containing Module 2-2 and expressing SMO, SOI, and PAR

The pad gene coding ADH (alcohol dehydrogenase; SEQ ID NO: 11) from tomato Saccharomyces cerevisiae was first synthesized and codon optimized for E. coli according the published sequence. Then it was amplified using primers PAR-Hindlll-RBS-F (ACTGAAGCTTTAAGGAGATATATAATGAGCGTGAC CGCGAAAACCGTG; SEQ ID NO:

32) and PAR-Xhol-R (ACTGCTCGAGTCACATGCTTGAACTCCCG CCGAAA; SEQ ID NO:

33) and Phusion DNA polymerase (available from Thermo). The PCR products were double digested with Hindi 11 and Xhol, and then ligated to same digested pRSF-SMO-SOI plasmid

(see example 1) with T4 DNA ligase. The ligation product was transformed (heat shock) into E. coli T7 Expression competent cells (available from New England Biolabs) to give pRSF- SMO-SOI-PAR. This module 2-2 was sub-cloned to other three vectors by the following procedures. Module 2-2 operon was amplified with primers StyA-BspHI-F (ACTGTC ATGAAAAAGCGTATCGGTATTGTTGG; SEQ ID NO: 31) and PAR-Xhol-R (ACTGCTCGAGTCACAT GCTTGAACTCCCG CCGAAA; SEQ ID NO: 33), digested with BspHI and Xhol, and then ligated to double digested pACYCduet, pCDFduet, and pETduet (available from Novagen). The transformation of these products gave pACYC-SMO-SOI- PAR, pCDF-SMO-SOI-PAR, and pET-SMO-SOI-PAR respectively.

Example 3. Production of 2-PE from Sty via cascade biocatalysis by using E. coli containing Module 2-2 and expressing SMO, SOI, and PAR

The recombinant E.coli (StyABC-PAR) containing the plasmid pRSF-SMO-SOI-PAR was grown in 1 mL LB medium containing 50 mg/L kanamycin at 37°C and then inoculated into 50 mL M9 medium containing glucose (20 g/L), yeast extract (6 g/L), and 50 mg/L kanamycin. When OD ₆ oo reached 0.6, 0.5 mM IPTG was added to induce the expressing of enzymes. The cells continued to grow and expressed protein for 12 hours at 22°C before they were harvested by centrifuge (4000g, 10 mins). The cells were resuspended in 200 mM KPB buffer (pH=8.0) to 10 g cdw/L with 2% glucose (for cofactor regeneration). To a 2 mL of aqueous system, a 2 mL n-hexadecane containing 60 mM Sty was added to the reaction system to form a second phase. The reaction was conducted at 30°C and 300 rpm in a 100- mL flask for 8 hours. 100 L of aqueous phase samples were taken during the reaction and analyzed by reverse phase HPLC (Agilent poroshell 120 EC-C18 column, acetonitrile: water = 50: 50, and flow rate 0.5 mL/min) to quantify the production of 2-PE in aqueous phase. 100 \JL of organic phase samples were taken during the reaction and analyzed by GC-FID (Agilent HP-5 column, 70°C increase to 200°C at 25°C/min, increase to 250°C at 50°C/min, hold for 1 min, and then increase to 270°C at 20°C/min) to quantify Sty, SO, PA, 2-PE in the organic phase. 2-PE was produced from Sty, with a best result of about 56 mM (93% yield) obtained in 8 h (Fig. 8). This result showed that the constructed recombinant strain is a powerful catalyst for the cascade biotransformation of Sty to 2-PE.

Example 4. Efficient production of substituted 2-PE from substituted Sty via cascade biocatalysis by using E. coli (StyABC-PAR)

E. coli (StyABC-PAR) was grown in 1 mL LB medium containing 50 mg/L kanamycin at 37°C and then inoculated into 50 mL M9 medium containing glucose (20 g/L), yeast extract (6 g/L), and 50 mg/L kanamycin. When OD ₆ oo reached 0.6, 0.5 mM IPTG was added to induce the expressing of enzymes. The cells continued to grow and expressed protein for 12 h at 22°C before they were harvested by centrifuge (4000g, 10 mins). The cells were resuspended in 200 mM KPB buffer (pH=8.0) to 10 g cdw/L with 2 % glucose (for cofactor regeneration). To a 2 mL of aqueous system, a 2-mL n-hexadecane containing 20 mM of substituted Sty was added to the reaction system to form a second phase. The reaction was conducted at 30°C and 300 rpm in a 100-mL flask for 8 hours. 100 L of aqueous phase samples were taken during the reaction and analyzed by reverse phase HPLC (Agilent poroshell 120 EC-C18 column, acetonitrile: water = 50: 50, and flow rate 0.5 mL/min) to quantify the production of substituted 2-PE in aqueous phase. 100 of organic phase samples were taken during the reaction and analyzed by GC-FID (Agilent HP-5 column, 70°C increase to 200°C at 25°C/min, increase to 250°C at 50°C/min, hold for 1 min, and then increase to 270°C at 20°C/min) to quantify substituted Sty, SO, PA, 2-PE in organic phase. As shown in Table 1 , all the 12 substituted 2-PEs were produced in good to excellent yield 62-99 % in 8 h. This prove the relative broad scope of the cascade biotransformation system. Table 1. Conversion of substituted styrene to substituted 2-PEs with E. coli (StyABC- PAR).

Regioselectivity

Entry Substrate Product Conv. to 2-PE [%] ^[bl

2-OH : 1-OH ^[cl

1 Sty H > 99 > 99 : 1

2 o-F-Sty o-F-2-PE 90 > 99 : 1

3 m-F-Sty m-F-2-PE 94 > 99 : 1

4 p-F-Sty p-F-2-PE 98 > 99 : 1

5 m-CI-Sty m-CI-2-PE 89 > 99 : 1

6 p-CI-Sty p-CI-2-PE 80 98 : 2

7 m-Br-Sty m-Br-2-PE 83 99 : 1

8 p-Br-Sty p-Br-2-PE 62 98 : 2

9 m-Me-Sty m-Me-2-PE 99 > 99 : 1

10 p-Me-Sty p-Me-2-PE 96 > 99 : 1 m-OMe-2-

11 m-OMe-Sty > 99 > 99 : 1

PE

12 p-OMe-Sty p-OMe-2-PE 96 98 : 2

[a] Sty (20 mM in organic phase) was transformed with resting cells (10 g cdw L ^"1 ) in KP buffer (200 mM, pH 8, 2 % glucose) and n-hexadecane (1 : 1) at 30°C and 250 rpm for 8 h. [b] Determined by HPLC analysis of aqueous phase and GC-FID analysis of organic phase, [c] Measured by GC-FID analysis.

Example 5. Preparation of (substituted) 2-PE from (substituted) Sty by using E. coli (StyABC-PAR) in a 100 mL system

E. coli (StyABC-PAR) was grown in 1 mL LB medium containing 50 mg/L kanamycin at 37°C and then inoculated into 50 mL M9 medium containing glucose (20 g/L), yeast extract (6 g/L), and 50 mg/L kanamycin. After 5 h growth at 37°C, the 50 mL culture was expanded into 2 L M9 medium containing glucose (20 g/L), yeast extract (6 g/L), and 50 mg/L kanamycin and continue culture at 37°C. When OD ₆ oo reached 0.6, 0.5 mM IPTG was added to induce the expressing of enzymes. The cells continued to grow and expressed protein for 12 hours at 22°C before they were harvested by centrifuge (4000g, 10 mins). The cells were resuspended in 200 mM KPB buffer (pH=8.0) containing 2 % glucose to 10 g cdw/L. To a 50 mL of aqueous system, a 50-mL n-hexadecane containing 50 mM of Sty was added to the reaction system to form a second phase. The reaction was conducted at 30°C and 300 rpm in a 1-L flask for 24 hours. After reaction, the two phase was separated by centrifugation (4000g, 15 mins). The aqueous phase was extracted with 50 mL ethyl acetate for three times, and the hexadecane phase was wash with 50 mL water for three times. The wash water was combined and extracted with 100 mL ethyl acetate for two times. All the ethyl acetate was combined, dried over Na ₂ S0 ₄ and evaporated. The residue was further purified by flash chromatography with n-hexane: ethyl acetate = 5: 1. The fractions were combined and evaporated to get 253 mg of pure 2-PE with 83% isolated yield. Using the similar process, p-fluoro-2-PE, p-methyl-2-PE, and m-methyoxyl-2-PE were also isolated in 74 %, 66 %, and 78 % yield, respectively.

Example 6. Genetic engineering of E. coli containing Module 2-3 and expressing SMO, SOI, CvTA, and AlaDH

The cvTA gene (coding CvTA; SEQ ID NO: 12) and aid gene (coding for AlaDH; SEQ ID NO: 34) was amplified together from the previous template pRSF-AlkJ-CvTA-AlaDH [Wu, S., Zhou, Y., et al., Nat. Commun. 7: 1 1917 (2016)] using primers CvTA-BamHI-BspHI-F (ACTGGGATCCGATCATGATGCAAAAACAACGCACCACCTCAC; SEQ ID NO: 35) and AlaDH-Xhol-R (ACTGCTCGAGTTAAGCACCCGCCACAGATGATTCA; SEQ ID NO: 36). The PCR product was double digested with BspHI and Xhol, and then ligated to pCDF (digested with Ncol and Xhol) with T4 DNA ligase. The ligation product was transformed (heat shock) into E. coli T7 Expression competent cells to give pCDF-CvTA-AlaDH. The pCDF-CvTA-AlaDH and pACYC-SMO-SOI plasmids were co-transformed into competent cells of E. coli RARE strain [Kunjapur, A. M., Tarasova, Y., Prather, K. L. J. Am. Chem. Soc. 136: 1 1644-11654 (2014)] to give E. coli (StyABC-CvTA-AlaDH) co-expressing SMO, SOI, CvTA, and AlaDH. The competent cells of E. coli RARE strain were made according to the following protocol: it was grown in 1 mL LB media at 37°C for overnight; then 100 culture was inoculated into 5 mL fresh LB media containing appropriate antibiotic at 37°C until OD ₆₀₀ reached 0.5 (about 2 h); then the cells were harvested by centrifugation (2500 g, 10 min, 4°C) and resuspended in 1 mL cold CaCI ₂ solution (0.1 M) on ice. The cell suspension was kept on ice and shaken at 90 rpm for 2 h, and then harvested by centrifugation (2500 g, 8 min, 4°C) and resuspended in 0.2-0.5 mL cold CaCI ₂ solution (0.1 M) to obtain the competent cells. Example 7. Production of PEA from Sty via cascade biocatalysis by using E. coli containing Module 2-3 and expressing SMO, SOI, CvTA and AlaDH The recombinant E. coli (StyABC-CvTA-AlaDH) was grown in 1 ml_ LB medium containing 50 mg/L chloramphenicol and 50 mg/L streptomycin at 37°C and then inoculated into 50 ml_ M9 medium containing glucose (20 g/L), yeast extract (6 g/L), 50 mg/L chloramphenicol and 50 mg/L streptomycin. When OD ₆ oo reached 0.6, 0.5 mM IPTG was added to induce the expressing of enzymes. The cells continued to grow and expressed protein for 12 h at 22°C before they were harvested by centrifuge (4000g, 10 mins). The cells were resuspended in 200 mM NaPB buffer (pH=8.0) to 10 g cdw/L with 2 % glucose (for cofactor regeneration) and 200 mM NH ₃ -NH ₄ CI (pH 8.25). To a 2 mL of aqueous system, a 2-mL n-hexadecane containing 80 mM Sty was added to the reaction system to form a second phase. The reaction was conducted at 30°C and 300 rpm in a 100-mL flask for 10 h. 100 of aqueous phase samples were taken during the reaction and analyzed by reverse phase HPLC (Agilent Poroshell 120 EC-C18 column, acetonitrile: water: TFA = 30: 70: 0.1 , and flow rate 0.5 mL/min) to quantify the production of PEA in aqueous phase. 100 L of organic phase samples were taken during the reaction and analyzed by GC-FID (Agilent HP- 5 column, 70°C increase to 200°C at 25°C/min, increase to 250°C at 50°C/min, hold for 1 min, and then increase to 270°C at 20°C/min) to quantify Sty, SO, PA, 2-PE in organic phase. PEA was produced from Sty, and the best result is about 74 mM (93 % yield) obtained in 10 h (Fig. 9). This result showed that our constructed recombinant strain is powerful catalyst for the cascade biotransformation of Sty to PEA. Example 8. Efficient production of substituted PEA from substituted Sty via cascade biocatalysis by using E. coli (StyABC-CvTA-AlaDH) n-C ₁₆ H ₃₄ /NaP buffer

Sty 30 °C, 10-24 h ^PEA

E. coli (StyABC-CvTA-AlaDH) was grown in 1 mL LB medium containing 50 mg/L chloramphenicol and 50 mg/L streptomycin at 37°C and then inoculated into 50 mL M9 medium containing glucose (20 g/L), yeast extract (6 g/L), and 50 mg/L chloramphenicol and 50 mg/L streptomycin. When OD ₆₀₀ reached 0.6, 0.5 mM IPTG was added to induce the expressing of enzymes. The cells continued to grow and expressed protein for 12 h at 22°C before they were harvested by centrifuge (4000g, 10 mins). The cells were resuspended in 200 mM NaPB buffer (pH=8.0) to 10 g cdw/L with 2 % glucose (for cofactor regeneration) and 200 mM NH ₃ -NH ₄ CI (pH 8.25). To a 2 mL of aqueous system, a 2-mL n-hexadecane containing 20 mM of substituted Sty was added to the reaction system to form a second phase. The reaction was conducted at 30°C and 300 rpm in a 100-mL flask for 10-24 hours. 100 of aqueous phase samples were taken during the reaction and analyzed by reverse phase HPLC (Agilent poroshell 120 EC-C18 column, acetonitrile: water: TFA = 30: 70: 0.1 , and flow rate 0.5 mL/min) to quantify the production of substituted PEA in aqueous phase. 100 of organic phase samples were taken during the reaction and analyzed by GC-FID (Agilent HP-5 column, 70°C increase to 200°C at 25°C/min, increase to 250°C at 50°C/min, hold for 1 min, and then increase to 270°C at 20°C/min) to quantify substituted Sty, SO, and PA in organic phase. As shown in Table 2, all the 12 substituted PEAs were produced in good to excellent yield 45-99 % in 10-24 h. This prove the relative broad scope of the cascade biotransformation.

Table 2. Conversion of substituted styrene to substituted PEAs with E. coli (StyABC- CvTA-AlaDH).

Entry Substrate Product Conv. to Chemoselectivity Regioselectivity

PEA [%] ^[bl

NH ₂ : OH ^[bl 2-NH ₂ : 1-NH ₂ ^[cl

1 Sty H 98 96 : 4 > 99 : 1

2 o-F-Sty o-F-PEA 94 99 : 1 > 99 : 1

3 m-F-Sty m-F-PEA > 99 99 : 1 > 99 : 1

4 p-F-Sty p-F-PEA 96 98 : 2 > 99 : 1

5 m-CI-Sty m-CI-PEA 86 98 : 2 > 99 : 1

6 p-CI-Sty p-CI-PEA 76 98 : 2 > 99 : 1

7 m-Br-Sty m-Br-PEA 45 97 : 3 > 99 : 1

8 p-Br-Sty p- Br- PEA 60 98 : 2 > 99 : 1

9 m-Me-Sty m-Me-PEA 93 97 : 3 > 99 : 1

10 p-Me-Sty p-Me-PEA 99 98 : 2 > 99 : 1

1 1 m-OMe-Sty m-OMe-PEA > 99 99 : 1 > 99 : 1

12 p-OMe-Sty p-OMe-PEA 94 96 : 4 > 99 : 1

[a] Sty (20 mM in organic phase) was transformed wil h resting cells (10 g cdw/L) in NaP buffer (200 mM, pH 8, 2 % glucose) and n-hexadecane (1 : 1) at 30°C and 250 rpm for 10 h. [b] Determined by HPLC analysis of aqueous phase and GC-FID analysis of organic phase, [c] 1-phenylethylamines were not detected, [d] 24 h reaction time. Example 9. Preparation of (substituted) PEA from (substituted) Sty by using E. coli (StyABC-CvTA-AlaDH) in a 60 mL system

E. coli (StyABC-CvTA-AlaDH) was grown in 1 mL LB medium containing 50 mg/L chloramphenicol and 50 mg/L streptomycin at 37°C and then inoculated into 50 mL M9 medium containing glucose (20 g/L), yeast extract (6 g/L), and 50 mg/L chloramphenicol and 50 mg/L streptomycin. After 5 h growth at 37°C, the 50-mL culture was expanded into 2 L M9 medium containing glucose (20 g/L), yeast extract (6 g/L), and 50 mg/L chloramphenicol and 50 mg/L streptomycin and continue culture at 37°C. When OD ₆₀₀ reached 0.6, 0.5 mM IPTG was added to induce the expressing of enzymes. The cells continued to grow and expressed protein for 12 h at 22°C before they were harvested by centrifuge (4000g, 10 mins). The cells were resuspended in 200 mM NaPB buffer (pH=8.0) to 10 g cdw/L with 2% glucose (for cofactor regeneration) and 200 mM NH ₃ -NH ₄ CI (pH 8.25). To a 50 mL of aqueous system, a 10-mL n-hexadecane containing 50 mM Sty was added to the reaction system to form a second phase. The reaction was conducted at 30°C and 300 rpm in a 1-L flask for 24 h. After reaction, the two phase was separated by centrifugation (4000g, 15 mins). The aqueous phase was adjusted to pH = 13 with NaOH and extracted with 50 mL ethyl acetate for three times. All the ethyl acetate was combined, dried over Na ₂ S0 ₄ and evaporated. The residue was further purified by flash chromatography with dichloromethane: methanol: triethylamine = 100: 5: 1. The fractions were combined and evaporated to get 236 mg of pure PEA with 78 % isolated yield. Using the similar process, p-fluoro-PEA, p-methyl- PEA, and m-methyoxyl-PEA were also isolated in 68 %, 71 %, and 82 % yield, respectively.

Example 10. Genetic engineering of E. coli containing Module 2-4 and expressing SMO, SOI, and EcALDH

The padA gene coding EcALDH (phenylacetaldehyde reductase; SEQ ID NO: 10) from E. coli was amplified using primers EcALDH-Notl-RBS-F (ACTGCGGCCGCTAAGGAGATATATAATGAC AGAGCCGCATGTAGCAGTAT; SEQ ID NO: 37) and EcALDH-Xhol-R (ACTG CTCGAG TTAATACCGT ACACACACCGACTTAG; SEQ ID NO: 38) and Phusion DNA polymerase (available from Thermo). The PCR product were double digested with Notl and Xhol, and then ligated to same digested pRSF-SMO-SOI plasmid (see example 1) with T4 DNA ligase. The ligation product was transformed (heat shock) into E. coli T7 Expression competent cells (available from New England Biolabs) to give pRSF-SMO-SOI-EcALDH. This module 2-4 was sub-cloned to other three vectors by the following procedures. Module 2-4 operon was amplified with primers StyA-BspHI-F (ACTGTC ATGAAAAAGCGTATCGGTATTGTTGG; SEQ ID NO: 31) and EcALDH-Xhol-R (ACTGCTCGAGTTAATACCGTACACACACCGACTTAG; SEQ ID NO: 38), digested with BspHI and Xhol, and then ligated to double digested pACYCduet, pCDFduet, and pETduet (available from Novagen). The transformation of these products gave pACYC-SMO-SOI- EcALDH, pCDF-SMO-SOI-EcALDH, and pET-SMO-SOI-EcALDH respectively.

Example 11. Production of PAA from Sty via cascade biocatalysis by using E. coli containing Module 2-4 and expressing SMO, SOI, and EcALDH

The recombinant E. coli (StyABC-EcALDH) containing the plasmid pRSF-SMO-SOI- EcALDH was grown in 1 mL LB medium containing 50 mg/L kanamycin at 37°C and then inoculated into 50 mL M9 medium containing glucose (20 g/L), yeast extract (6 g/L), and 50 mg/L kanamycin. When OD ₆₀₀ reached 0.6, 0.5 mM IPTG was added to induce the expressing of enzymes. The cells continued to grow and expressed protein for 12 hr at 22°C before they were harvested by centrifuge (4000g, 10 mins). The cells were resuspended in 400 mM KPB buffer (pH=8.0) to 15 g cdw/L with 0.5 % glucose (for cofactor regeneration). To a 2 mL of aqueous system, a 2 mL ethyl oleate containing 130 mM Sty was added to the reaction system to form a second phase. The reaction was conducted at 30°C and 300 rpm in a 100-mL flask for 6 h. 100 L of aqueous phase samples were taken during the reaction and analyzed by reverse phase HPLC (Agilent poroshell 120 EC-C18 column, acetonitrile: water = 50: 50, and flow rate 0.5 mL/min) to quantify the production of PAA in aqueous phase. 100 L of organic phase samples were taken during the reaction and analyzed by GC-FID (Agilent HP-5 column, 70°C increase to 200°C at 25°C/min, increase to 250°C at 50°C/min, hold for 1 min, and then increase to 270°C at 20°C/min) to quantify Sty, SO, PA in organic phase. PAA was produced from Sty, and the best result is about 122 mM (94 % yield) obtained in 6 h (Fig. 12). This result showed that our constructed recombinant strain is powerful catalyst for the cascade biotransformation of Sty to PAA.

Example 12. Preparation of (substituted) PAA from (substituted) Sty by using E. coli (StyABC- EcALDH) in a 48 mL system

E. coli (StyABC-EcALDH) was grown in 1 mL LB medium containing 50 mg/L kanamycin at 37°C and then inoculated into 50 mL M9 medium containing glucose (20 g/L), yeast extract (6 g/L), and 50 mg/L kanamycin. After 5 h growth at 37°C, the 50-mL culture was expanded into 2 L M9 medium containing glucose (20 g/L), yeast extract (6 g/L), and 50 mg/L kanamycin and continue culture at 37°C. When OD ₆₀₀ reached 0.6, 0.5 mM IPTG was added to induce the expressing of enzymes. The cells continued to grow and expressed protein for 12 hours at 22°C before they were harvested by centrifuge (4000g, 10 mins). The cells were resuspended in 400 mM KPB buffer (pH=8.0) to 15 g cdw/L with 0.5 % glucose (for cofactor regeneration). To a 40-mL of aqueous system, an 8-mL ethyl oleate containing 25-100 mM of substituted Sty was added to the reaction system to form a second phase. The reaction was conducted at 30°C and 300 rpm in a 250-mL tri-baffled flask for 10 hours. 100 \JL of aqueous phase samples were taken during the reaction and analyzed by reverse phase HPLC (Agilent poroshell 120 EC-C18 column, acetonitrile: water = 50: 50, and flow rate 0.5 mL/min) to quantify the production of substituted PAA in aqueous phase. As shown in Table 3, all the 12 substituted 2-PEs were produced in good to excellent yield 85-99% in 10 h. This proved the relative broad scope of the cascade biotransformation. After reaction, the two phases were separated by centrifugation (4000g, 15 mins). The aqueous phase was adjusted to pH = 2 with HCI and extracted with 100 mL ethyl acetate for two times. The ethyl acetate fractions were combined, dried over Na ₂ S0 ₄ and evaporated. The PAAs were purified by flash chromatography. Pure PAA was obtained at 82 % isolated yield (Table 3).

Table 3. Conversion of substituted styrene to substituted PAAs with E. coli (StyABC- EcALDH).

Scale Substrate Conv. to Isolated

Entry Substrate Product

KPB + EO [mL] Cone. [mM] PAA [%] yield [%]

1 Sty 100 + 20 100 PAA 96 82

2 o-F-Sty 40 + 8 50 o-F-PAA 98 71

3 m-F-Sty 40 + 8 50 m-F-PAA >99 73

4 p-F-Sty 40 + 8 50 p-F-PAA 98 76

5 m-CI-Sty 40 + 8 50 m-CI-PAA 94 81

6 p-CI-Sty 40 + 8 50 p-CI-PAA 85 75

7 m-Br-Sty 40 + 8 25 m- Br- PAA 91 77

8 p-Br-Sty 40 + 8 25 p-Br-PAA 87 71 9 m-Me-Sty 40 + 8 50 m- Me- PA A 99 67

10 p-Me-Sty 40 + 8 50 p-Me-PAA 98 56 m-OMe- 40 + 8 50 >99 66

11 m-OMe-PAA

Sty p-OMe- 40 + 8 50 >99 52

12 p-OMe-PAA

Sty

Example 13. Genetic engineering of E. coli expressing SMO, SOI and ADH9v1 (StyABC-ADH9v1) Gene coding ADH9v1 (SEQ ID NO: 39), as reported in [P. Konst, H. Merkens, et al.,

Angew. Chem. Int. Ed. 51 : 9914-9917 (2012)], was amplified using the primers ADH9v1- Hindlll-RBS-F: ACTGAAGCTTTAAGGAGATATATCATGAAAAATCGTGTTGC CTTTGTTAC (SEQ ID NO: 40) and ADH9v1-Xhol-R: ACTGCTCGAGTTAGTTAAACACCATACCACCAT (SEQ ID NO: 41) and Phusion DNA polymerase (available from Thermo). The PCR product were double digested with Hindi 11 and Xhol, and then ligated to same digested pRSF-SMO- SOI plasmid (see Example 1) with T4 DNA ligase. The ligation product was transformed (heat shock) into E. coli T7 Expression competent cells (available from New England Biolabs) to give E. coli (StyABC-ADH9v1) or (pRSF-SMO-SOI- ADH9v1). Example 14. Preparation of (S)-2- Arylpropionic Acids from a-methylstyrene derivatives with E. coli (StyABC-ADH9v1)

Freshly prepared E. coli (StyABC-ADH9v1) cells were resuspended to 20 g cdw/L in KP buffer (200 mM, pH 8.0). 100 mL of the cell suspension were mixed with 10 mL of n- hexadecane in a tri-baffled flask (500 mL). a-Me-Sty (2 mmol), or p-F-a-Me-STy - p-Me-a- Me-Sty (0.5 mmol) was added to start the reaction at 300 rpm and 30 8°C for 24 h. Aqueous phase samples (100 mL) were separated by centrifugation (13000 g, 3 min), diluted with 400 mL TFA solution (0.5 %) and 500 mL acetonitrile (with 2 mM benzyl alcohol), and analyzed by chiral HPLC to quantify the concentration and ee of 2-Phenylpropanoic acid - p-Me-a-Me- paa. At the end of the reaction, the mixture was subjected to centrifugation (4000 g, 15 min) to collect the aqueous phase. The reaction flask, the cells and n-hexadecane were washed with water (20 mL). The aqueous phase and washed water were combined, adjusted to pH ≤2 with HCI, saturated with NaCI, and then extracted with ethyl acetate (3 x 100 mL). The ethyl acetate was collected, dried over Na ₂ S0 ₄ , and subjected to evaporation by using a rotary evaporator. The crude product was purified by flash chromatography on a silica gel column with an eluent consisting of n-hexane: ethyl acetate of 5: 1 and acetic acid (0.5 % as additive) (R _f =0.2-0.3). The collected fractions were subjected to GC-FID analysis to confirm the purity. The desired fractions were combined, subjected to evaporation (n-heptane was added to remove acetic acid by forming azeotrope), and dried overnight under vacuum.

Example 15. One-Pot Synthesis of (S)-Arylpropionic Acids from a-Methylstyrene Derivatives with E. coli (StyABC-ADH9v1 )

Asymmetric cascade oxidation of a-methylstyrene (20 mM) was examined with resting cells of E. coli (StyABC-ADH9v1) under different conditions on a small scale (Fig. 14). The ee of (S)-2-Phenylpropanoic acid was excellent (98-99 %) under all these conditions, and high conversion (80 %) was achieved with resting cells (20 g cdw/L) and no glucose.

To demonstrate the cascade oxidation for asymmetric synthesis of 2-arylpropionic acid, E. coli (StyABC-ADH9v1) resting cells (20 g cdw/L) were employed to transform a- Methylstyrene (20 mM) in a larger system consisting of 100 ml_ KP buffer and 10 mL /?- hexadecane. After reacting for 24 hours, (S)-2-Phenylpropanoic acid was produced in 82 % conversion (Table 4). Work-up, extraction with ethyl acetate, and purification by flash chromatography gave 195 mg of pure (S)-2-Phenylpropanoic acid in 65 % isolated yield. The ee of (S)-2-Phenylpropanoic acid is excellent (98 %). The cascade biooxidation was further applied to transform ring-substituted a-methylstyrenes (S)-p-F-a-Me-PAA - (S) -p-Me-a-Me- PAA (5 mM) in the same system of 100 mL KP buffer and 10 mL n-hexadecane. (S) -p-F-a- Me-PAA - (S)-a,4-Dimethylphenylacetic acid were successfully produced in 67-75 % conversion, and similar work-up, extraction, and purification afforded pure (S)-p-F-a-Me-PAA - (S) -p-Me-a-Me-PAA in 46-52 % isolated yield. The ees of (S)-4-F-a-Me-PAA and (S) -p- Me-a-Me-PAA were also excellent (97-98 %), while the ee of (S)-p-CI-a-Me-PAA is slightly lower (92 %). These results clearly demonstrated that the epoxidation-isomerization- oxidation cascade is highly regio- and stereo-selective for the conversion of 2-arylpropenes to give (S)-2-arylpropionic acids. This unique one-pot asymmetric oxidation has no chemical counterpart thus far.

Table 4. One-Pot Synthesis of (S)-Arylpropionic Acids from a-Methylstyrene Derivatives with E. coli (StyABC-ADH9v1) Entry Substrate Cone. Product Conversion ee Isolated

[mM] to Product [%] yield [%]

[%]

1 a-Me-Sty 20 (S)-2-Phenylpropanoic 82 98 65

acid

2 p-F-a-Me-Sty 5 (S)-p-F-a-Me-PAA 75 97 49

3 p-CI-a-Me-Sty 5 (S)-p-CI-a-Me-PAA 67 92 46

4 p-Me-a-Me- 5 (S)-p-Me-a-Me-PAA 73 98 52

Sty

Conversion to 4 [%] and ee [%] are determined by chiral HPLC analysis.

One-Pot Production of Natural 2-PE from L-Phe

Example 16. Genetic engineering of E. coli containing Module 1 and Module 2-2 and expressing PAL, PAD, SMO, SOI, and PAR

AtPAL2 from Arabidopsis thaliana was chosen for the deamination of L- phenylalanine, while AnPAD (fdd and padl) from Aspergillus niger was selected for decarboxylation of cinnamic acid. Both genes were subsequently cloned together in compatible plasmids to provide the first cascade containing Module 1.

The synthesized gene of AnFDC (fdd) with nucleic acid sequence (SEQ ID NO: 42) encoding AnPAD protein sequence (SEQ ID NO: 43) was amplified using primers "AnFDC- BspHI-F: ACTGTCATGAGCGCGCAACCTGCGCACCTG" (SEQ ID NO: 44) and "AnFDC- EcoRI-R: ACTGGAATTCTTAGTTACTGAAGCCCATTTTGGTC" (SEQ ID NO: 45) with Phusion DNA polymerase. The PCR product was double-digested with BspHI and EcoRI, and then ligated to the Ncol and EcoRI digested pRSFDuet-1 with T4 DNA ligase. The ligation product was transformed into E. coli T7 Expression competent cells to give pRSF- AnFDC. On the other hand, the synthesized gene of AnPAD (padl) with nucleic acid sequence (SEQ ID NO: 16) encoding AnPAD protein sequence (SEQ ID NO: 14) was amplified using primers "AnPAD-EcoRI-RBS-F:

ACTGGAATTCTAAGGAGATATATCATGTTCAACTCACTTCTGTCCGGC" (SEQ ID NO: 46) and "AnPAD-Pstl-R: ACTGCTGCAGTTATTTTTCCCAACCATTCCAACG" (SEQ ID NO: 47). The PCR product was double digested with EcoRI and PstI, and then ligated to the same digested pRSF-AnFDC with T4 DNA ligase. The ligation product was transformed into E. coli T7 Expression competent cells to give pRSF-PAD plasmid. Then, the gene of AtPAL2 with nucleic acid sequence (SEQ ID NO: 15) encoding PAL protein sequence (SEQ ID NO: 13) was amplified from the cDNA library of Arabidopsis thaliana (purchased from ATCC 77500) using primers "AtPAL-Ndel-F: ACTGCATATGGATCAAATCGAAGCAATGTTGTG" (SEQ ID NO: 48) and "AtPAL-Xhol-R: ACTGCTCGAGTTATTTTTCCCAACCATTCCAACG" (SEQ ID NO: 49). The PCR product was double digested with Ndel and Xhol, and then ligated to the same digested pRSF-PAD with T4 DNA ligase. The ligation product was transformed into E. coli T7 Expression competent cells to give pRSF-PAD-PAL plasmid. PAD-PAL was also sub-cloned to the other three vectors by the following procedure. PAD-PAL was amplified with primers "AnFDC-BspHI-F: ACTGTCATGAGCGCGCAACCTGCGCACCTG" (SEQ ID NO: 44) and "AtPAL-Xhol-R: ACTGCTCGAGTTATTTTTCCCAACCATTCCAACG" (SEQ ID NO: 49), digested with BspHI and Xhol, and then ligated to Ncol and Xhol digested pACYCDuet-1 , pCDFDuet-1 , and pETDuet-1. The transformation of these products gave pACYC-PAD-PAL, pCDF-PAD-PAL, and pET-PAD-PAL, respectively.

For the second cascade containing Module 2-2, pRSF-SMO-SOI-PAR, pACYC- SMO-SOI-PAR, pCDF-SMO-SOI-PAR, and pET-SMO-SOI-PAR were produced according to Example 2.

In order to achieve an equal enzyme expression in the E. coli strain, all 5 main enzymes were divided into 2 different modules, PAL-PAD and SMO-SOI-PAR using 4 different plasmids each, pACYC, pCDF, pET, and pRSF, respectively. The twelve recombinant plasmids were then transformed to E. coli T7 competent cells to provide 12 E. coli strains, each co-expressing PAL, PAD, SMO, SOI, and PAR. Plasmids for Module 1 and Module 2-2 combined plasmids for Module 1 and Module 2-2 are shown in Table 5.

Table 5. Engineering of recombinant E. coli expressing PAL, PAD, SMO, SOI, and PAR using two modules (PAL_PAD and SMO_SOI_PAR) with different plasmids

Plasmid for

Plasmid for PAL PAD Combined plasmids for Module 1

SMO SOI PAR

(M1) and Module 2-2

(M2-2)

pACYC (M1) pACYC (M2-2) pACYC (M1)_pCDF (M2-2) (AC) pCDF (M1) pCDF (M2-2) pACYC (M1)_pET (M2-2) (AE) pET (M1) pET (M2-2) pACYC (M1)_pRSF (M2-2) (AR) pRSF (M1) pRSF (M2-2) pCDF (M1)_pACYC (M2-2) (CA) pCDF (M1)_pET (M2-2) (CE)

pCDF (M1)_pRSF(M2-2) (CR) pET (M1)_pACYC (M2-2) (EA) pET (M1)_pCDF (M2-2) (EC)

pET (M1)_pRSF (M2-2) (ER)

pRSF (M1)_pACYC (M2-2) (RA) pRSF (M1)_pCDF (M2-2) (RC) pRSF (M1)_pET (M2-2) (RE)

Example 17. Screening of Recombinant E. coli strains for 2-PE Production

One-pot synthesis of 2-PE production via resting cells biotransformation was conducted in furtherance to test the activity of all those 12 strains (Table 5). KP buffer (potassium phosphate, 100 mM, pH 8) in 10 g cdw/L cell density containing 50 mM of L- phenylalanine was used as initial substrate, together with n-hexadecane as the organic phase, in a total volume of 4 ml (1 : 1). Glucose (0.5%) was added to the reaction mixture for the purpose of NADH regeneration via cellular metabolism. As shown in Figure 15, all 12 strains was successfully produced 2-PE with different concentrations due to the difference in gene dosage effect which will cause on the difference on the plasmid copy number during the replication within cell growth. The E. coli strains containing pRSF-PAL-PAD_pET-SMO- SOI-PAR gave the best conversion among all other 1 1 strains, hence it was selected for further investigation.

Example 18. 2-PE Product Inhibition

Apart from the achievements that have been reported so far, most of the 2-PE production is hindered by the product toxicity. Concentrations of 2-PE higher than 2-3 gr/L will inhibit the cells, causing a low conversion of the product in the end of the biotransformation. [Etschmann, M., Bluemke, W., et al., J. Appl. Microbiol. Biotechnol. 59: 1- 8; (2002); Hua, D., Xu, P. Biotechnol. Adv. 29: 654-660 (2011); Hua, D.L, Liang, X.H., et al., Asian J. Chem. 25(11): 5951-5954 (2013); Stark, D., Zala, D., et al., Enzyme Microb Technol. 32: 212-223 (2003)] 2-PE will aim the cell membrane once it is formed during the biotransformation, hence enlarging the membrane fluidity and deflating both glucose and substrate uptake [Seward, R., Willets, J.C., Dinsdale, M.G., and Lloyd, D. J Inst Brew. 102: 439-443 (1996)]. Protein and RNA inhibitions towards E. coli were also reported before due to the exceeding concentration of 2-PE [Luchini, J.J., Corre, J., and Cremieux, A. Res. Microbiol. 141 : 499-510 (1990)]. The product inhibition was investigated by adding different concentration of 2-PE to the cell suspension (10 g cdw/L) of E. coli (pRSF-PAL-PAD_pET-SMO-SOI-PAR) in KP buffer (100 mM, pH 8.0) containing 0.5 % glucose in aqueous phase with n-hexadecane as the organic phase, followed by the incubation at 30°C and 250 rpm for 3 h.

Referring to Figure 17(a), the results clearly demonstrated that 2-PE was toxic to the cells and hindered the production of 2-PE. 2-PE concentrations above 30 mM in the aqueous phase started to inhibit the biotransformation, while almost no activity was observed when the cells were pretreated with 90 mM 2-PE before performing the biotransformation. Biotransformation was done in aqueous phase with KP buffer (100 mM, pH 8.0) containing cell suspension (10 g cdw/l), 0.5% glucose, and 50 mM L-phenylalanine as initial substrate while n-hexadecane as the organic phase (250 rpm, 2h, 30°C)

To further investigate the product inhibition, the apparent kinetics of the whole-cells was measured and determined using Lineweaver-Burk plot. 2-PE concentration of 3 mM was used for 15 minutes in order to determine the kinetic, thus the product toxicity towards the cells could be neglected. Competitive inhibition was shown (Fig. 16), with the apparent Ki value of 4.8 mM for 2-PE. Apparent Vmax value was found to be 22.8 μΓηοΙ/Γηίη^ CDW, while the apparent Km value was 2.57.

Example 19. Screening and Selection of Organic Solvent for partitioning 2-PE

In-situ product removal technique via extraction have been conducted to remove the obtained 2-PE from the aqueous phase and make its concentration below the inhibitory level. We investigated different type of organic solvents and ionic liquids to perform the extractive biotransformation for 2-PE production via styrene-derived pathway from .-phenylalanine, starting from the analysis of their partition coefficients in the biphasic system. Product and substrate coefficient partitions in organic and aqueous phase were determined by adding different concentration of 2-PE and .-phenylalanine, respectively, into KP buffer (100 mM, pH 8.0) together with the respective organic solvents. Reaction mixtures were incubated for 1 h (280 rpm, 30°C).

Results in Figure 18 (a) show that most of the organic solvents used were able to extract 2-PE from the aqueous phase. It is shown through the K value which is »>1 , which indicated that most of the 2-PE was extracted to the organic phase. However, n-hexadecane appeared to have much lower partition coefficient, hence reducing the extraction efficiency.

Further investigation was conducted in order to determine the biocompatibility of the organic solvents towards the biocatalyst. The E. coli cell pellets (pRSF-PAL-PAD_pET-SMO- SOI-PAR, 10 g cdw/L) in KP buffer (100 mM, pH 8.0) containing 0.5 % glucose and 50 mM /.-Phe were used to perform the biotransformation by utilizing the respective organic solvents for 24 h (280 rpm, 30°C). From Figure 18(b), it is shown that all of organic solvents used in the biotransformation were biocompatible and could enhance 2-PE production up to 150 %. Biodiesel was proven as the best organic solvent and this can clearly be seen from the highest 2-PE obtained after 24 h of biotransformation. Apart from n-hexadecane, all organic solvents used were also still be able to extract 2-PE while the biocatalysts performed the reaction. This clearly demonstrated that extractive biotransformation enables to significantly improve the 2-PE productivity.

Example 20. Preparation and Characterization of Nano-Solid Adsorbent

The use of a magnetic adsorbent consisting of iron oxide core and benzene ring functional groups on the surface was investigated. Polystyrene was employed to coat the OA-MNP in order to protect the iron oxide cores.

The synthesis of the OA-MNP-PS is shown in Figure 19a. The OA-MNP and OA- MNP-PS were found to have a diameter of 12 nm and 1 18 nm, respectively, as well as a hydrodynamic size of 23 nm and 180 nm, respectively (Figure 19c and 19e). Separation was done by magnetic force or centrifugation (30 min, 13 000 g). The investigation was performed by analysing the product adsorption towards the synthesized MNP through the hydrophobic interactions.

Example 21. Adsorbent Screening

In-situ product adsorption (ISPA) can be applied as an in-situ product removal (ISPR) alternative technique, where resins or other adsorption media are implemented to minimize the 2-PE product inhibition. Product concentration increased up to 6.2 g/l when macroporous resin D101 was applied during the biotransformation. However, ISPA also suffers from limitation, such as a low specificity and adsorption capacity for 2-PE [Mei, J., Min, H., and Lu, Z. Process Biochemistry. 44: 886-890 (2009)].

Seven different adsorbents were used, including OA-MNP-PS. As shown in Figure 20, the 2-PE concentration in the aqueous phase remains low below the inhibitory level, which clearly demonstrated that most of the 2-PE was adsorbed into the adsorbent surface through hydrophobic interactions. The adsorption capacity for L~phenylalanine was significantly lower than 2-PE due to its hydrophilic structure. XAD4 resins gave the best performance among all the other 5 micro-size resins tested due to its cross-linked hydrophobic polystyrene structure, higher surface area, low porosity, as well as its application to remove hydrophobic compounds in pharmaceuticals. Surprisingly, the use of MNPs enables good selectivity towards 2-PE as a nano-scale adsorbent. Example 22. Tri-phasic Cascade Biotransformation of /.-Phenylalanine to 2-PE with In- Situ Product Removal (ISPR) via Extraction and Adsorption in 1 Pot XAD4 resin (0.36 g) or OA-MNP-PS (5mg/ml) was selected as the adsorbent, together with oleic acid, which was proven to give the best extractive biotransformation in the biphasic system (Example 18). Tri-phasic biotransformation was carried out with resting cells E. coli (pRSF-PAL-PAD_pET-SMO-SOI-PAR, 10 g cdw/L) resuspended in KP buffer (100mM, pH 8.0) containing 0.5% glucose and 50 mM .-phenylalanine with aqueous to organic ratio of 1 : 1 in a total volume of 4 ml. As shown in Figure 21 (a) and 21 (b), oleic acid gave the highest product conversion among all the other organic solvents tested after 24 h of biotransformation. Addition of XAD4 resin and OA-MNP-PS to the system further enhanced the 2-PE productivity, where 45 mM ( ^« 5.5 g/l) and 40 mM (-4.9 g/l) of 2-PE were obtained, respectively. When the initial substrate was increased to 100 mM, 70 mM (-8.6 g/l) and 65 mM (-8 g/l) of 2-PE was produced, respectively, using the same system within a single batch (not shown). These results were 2-fold times higher than the previous biotransformation performed without the addition of adsorbent. The existence of oleic acid was also proven to improve the MNPs-tri-phasic system although the specificity of OA-MNP- PS towards 2-PE was not as high as XAD4.

In order to perform the repeated batch biotransformation, cells were resuspended in fresh buffer containing 0.5 % glucose and same initial substrate concentration, and mixed with a new organic solvent and adsorbent to carry on the biotransformation. As shown in Figure 21 (c), the system could retain up to 83 % from its previous batch to produce 2-PE (12 h per-batch). Cumulatively, 250 mM of 2-PE (-31 g/l) was obtained within 7 batches (84 h), marking one of the highest 2-PE production that has been achieved so far.

Microbial production of 2-PE from glucose

Example 23. Engineering of Native Biosynthetic Pathway for L-Phe Overproduction

A scheme for the production of 2-PE from glucose is shown in Figure 22. The key enzymes for improving L-Phe production were identified as DAHP synthase (AroG), shikimate kinase (AroK), shikimate dehydrogenase (YdiB), chorismate mutase/prephenate dehydratase (PheA) and tyrosine aminotransferase (TyrB). The activities of AroG and PheA were reported to be inhibited in the presence of L-Phe and/or L-tyrosine and feedback inhibition resistant mutants (AroG15 renamed as AroG*, PheA"" ^" renamed as PheA*) were reported for both enzymes [ Liu, S.-P., et al. Process Biochemistry. 48(3): 413-419 (2013)]. Also, the expression of all these key enzymes are feedback regulated.

AroG* (SEQ ID NO: 27), aroK, ydiB, pheA* (SEQ ID NO: 28), and tyrB were cloned and overexpressed using strong promoter in T7 strain (T7-Phe).

The genes for overexpression and deletion for overproduction of L-Phe were amplified by PCR from E. coli MG1655 genomic DNA. pCDFDuet was used for overexpression of aroG*, aroK, ydiB, pheA* and tyrB genes. The genes aroG*, aroK, ydiB were cloned in multiple cloning site-1 and pheA* and tyrB were cloned in multiple cloning site-2. Two overexpression plasmids were used for the styrene-mediated pathway genes. pRSFDuet was used for the overexpression of PAL, FDC and PAD and pETDuet was used for the overexpression of styABC and PAR, as described in Example 15.

Gene deletion methodology:

The err and tyrA chromosomal deletions were performed using homologous recombination and the pKOV vector invented by Link et al. [Link, A. J., Phillips, D., & Church, G. M. Journal of bacteriology, 179(20): 6228-6237 (1997)]. The pKOV plasmid was obtained as a gift from George Church (Addgene plasmid # 25769). Briefly, -600 bp of upstream and downstream DNA base pairs of the target gene were used to provide sufficient homology for gene replacement. The target genes were replaced by random 10-20 bp length double stranded DNA. The err gene and tyrA gene deletion sequences, comprising the short double stranded DNA flanked by the 600 bp upstream and downstream gene nucleotide sequences are shown in SEQ ID NO: 52 and SEQ ID NO: 53, respectively. The replacement '10-20 bp' length double stranded DNA inserts are at nucleotide positions 619-635 and 598-610, respectively of SEQ ID NO: 52 and 53.

The integration of the recombinant pKOV plasmid into chromosome was performed by growing the E. coli T7 strain containing recombinant pKOV plasmid at 42°C using chloramphenicol as selection marker. After successful replacement of target DNA with insertion fragment, the deletion was confirmed using PCR. Further, the plasmid sequence was removed from the chromosome using sucrose as selection pressure and the deletion was confirmed by PCR and DNA sequencing. Similar approach was performed in single mutants to delete additional gene and create double mutants.

Table 6. List of strains and plasmids constructed in Example 23 to Example 26

Strain/plasmid Description

Strain

T7 Escherichia coli T7 express Τ7ΔΟ T7Acrr

Τ7ΔΤ lAtyrA

Τ7ΔΔ J7AcrrAtyrA

Plasmid

Phe pC D FDuet-aroG*-aroK-ydiB-pheA *-tyrB

Sty pRSFDuet-PAL-FDC-PAD, pETDuet-styABC-AR

pKOV-crr pKOV plasmid for err deletion

pKOV-tyrA pKOV plasmid for tyrA deletion

Overexpression of the key enzymes resulted in increased L-Phe production by T7- Phe strain (Fig. 23(a)). ~2 mM L-Phe was produced in 24 h whereas T7 strain containing empty plasmid (T7) did not produce any detectable amounts of L-Phe. Also, no significant difference in growth was observed.

To further improve L-Phe production, efforts to improve precursor availability was attempted. One approach was limiting the usage of PEP in PTS system by the deletion of err. As shown in Figure 22 (a), deletion of err significantly improved the cell growth but decreased the L-Phe production. It was believed that decreased glucose uptake rate is the reason for reduced L-Phe production.

The second target to improve precursor availability was prephenate dehydrogenase (TyrA). TyrA converts prephenate the precursor of L-Phe to tyrosine. Therefore, tyrA was deleted (Τ7ΔΤ) and L-Phe production was studied in T7AT-Phe. As shown in Figure 23 (a), there was significant increase in L-Phe production up to 5 mM was observed even with decrease in cell growth.

Surprisingly, the double mutant T7AA-Phe could produce -13 mM L-Phe which is ~6- fold higher than T7-Phe (Fig. 23 (a)).

As T7AA-Phe cell growth was relatively low, a brief experiment was performed by growing T7-Phe and T7AA-Phe in rich media (LB broth) for 6 h to increase the cell density and shifted to M9 media with a start OD ₆₀₀ of ~5 (Fig. 23 (b)). Cell density of 20 OD ₆₀₀ could be reached within 12 h in this experiment which is ~4 fold higher than the previous experiment and 16 mM of L-Phe could be obtained within 12 h of fermentation. This promising result also suggests that optimization of media and culture conditions to increase cell growth could improve the L-Phe productivity.

Example 24. Overexpression of Styrene-mediated 2-PE Production Pathway in L-Phe producer After the successful overproduction of L-Phe from glucose, the conversion of L-Phe to 2-PE was attempted using the styrene-mediated pathway enzymes (Fig. 22). Recombinant plasmids pRSF ^: Duet- PAL- F DC- PAD and pETDuet-styABC-PAR, prepared according to Example 2, were transformed into the L-Phe producer strain (T7AA-Phe) and the resultant recombinant strain was named as T7AA-Phe-Sty.

2-PE production from glucose by T7AA-Phe-Sty was conducted in a shake flask for 24 h. T7AA-Phe-Sty could produce 1.3 mM 2-PE directly from glucose and 2.5 mM of unconverted L-Phe was also present at the end of fermentation (Fig. 24). Example 25. Improvement of 2-PE Production using In-Situ Product Removal

The low production of 2-PE and L-Phe and accumulation of L-Phe could be also due to the toxicity of 2-PE seen in Example 23. Therefore, 2-PE production from glucose by T7AA-Phe-Sty in biphasic media with different ratios of M9 media and oleic acid (v:v) such as 1 :0.25, 1 :0.5 and 1 : 1 were tested for in-situ 2-PE removal.

As shown in Figure 24, introduction of biphasic media showed increased growth rate confirming the inhibitory effects of 2-PE during cell growth. All three cultures with biphasic media showed increased growth rate during the exponential phase compared to the control (M9 media only) (Fig. 24 (a)). However, only 1 :0.25 showed increased cell density at the end of fermentation, suggesting that oleic acid could also inhibit the cell growth probably due to its surfactant properties or by interfering in oxygen transfer.

As shown in Figure 25 (b), metabolites analysis revealed that all four cultures produced similar amounts of L-Phe and 2-PE but the distribution of 2-PE in the aqueous and organic phases were different. The 1 : 1 culture showed complete extraction of 2-PE from media whereas in 1 :0.25 and 1 :0.5 cultures there was 0.4 and 0.2 mM 2-PE present in the aqueous phase. This result suggests that a trade-off between cell growth and 2-PE extraction should be considered while choosing the ratio of organic phase in a biphasic system. Addition of small volumes of organic phase at different time points could be a better strategy to remove 2-PE without affecting the cell growth.

Example 26. Evaluation of Engineered Strains for 2-PE Production in Bioreactor Scale

To understand the potential of T7AA-Phe-Sty for 2-PE production from glucose, a bioreactor-scale fermentation with biphasic media was performed. The bioreactor could facilitate a high cell density necessary for reaching higher titers.

A bioreactor-scale fermentation was performed with 1 L of 2x M9 media in a 3L bioreactor. Glucose was maintained between 50-200 mM by intermittent feeding of 500 g L ^"1 glucose solution. 0.1 mM IPTG was added at 6 h and 100 ml_ oleic acid was added every 8 h starting from 10 h of fermentation. The growth temperature was shifted to 22°C from 30°C after induction.

As shown in Figure 26, cell growth increased linearly and continued to grow until the end of fermentation probably due to the continuous removal of 2-PE from the media. L-Phe concentration started to increase after induction and maintained at ~3 mM from 18 h whereas 2-PE concentration started to increase from the same time point. The 2-PE concentration in the aqueous and organic phase were 2 and 17 mM, respectively, at 40 h of fermentation. The presence of only 2 mM 2-PE in the aqueous phase shows the efficient in- situ 2-PE removal by oleic acid. A total of 19 mM 2-PE (2.3 g/L) was produced from glucose at the end of 40 h fermentation under non-optimized conditions.

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Any listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that such document is part of the state of the art or is common general knowledge.

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